CA2164274A1 - Transferrin binding proteins of pasteurella haemolytica and vaccines containing same - Google Patents

Abstract

Novel transferrin binding proteins from Pasteurella haemolytica, and nucleic acid molecules encoding the novel proteins are disclosed. Antibodies against the novel proteins are disclosed. The invention also relates to vaccines containing the novel proteins of the invention. The invention also provides methods for identifying substances which affect the binding of transferrin to the proteins and methods for screening for agonists or antagonists of the binding of the proteins and transferrin.

Description

Title: Transferrin Binding Pr~tei"s of Pasteurella haemolytica and Vaccines Cont~inin~ Same FIELD OF THE INVENTION
The invention relates to novel transferrin binding proteins of Pasteurella haemolytica, truncations, analogs, homologs and isoforms thereof;
nucleic acid molecules encoding the proteins and truncations, analogs, and homologs of the protei.~s; vaccines containing the ~roLeins; antibodies against the proteins and uses of the protein and nucleic acid molecules.

BACKGROUND OF THE INVENTION
Members of the genus Pasteurella comprise a group of related bacterial species that are important pathogens of ruminants. This group includes the species Pasteurella haemolytica which has been classified into ~wo biotypes, A and T, on the basis of sugar utilization, and into 16 serotypes which are recognized on the basis of their somatic antigens (6,12). The T-type strainsof P. haemolytica, characterized by utilization of trehalose, have been recentlyreclassified as a new species P. trehalosi (34).
Pneumonic pasteurellosis caused by Pasteurella haemolytica is a major economic problem to the cattle, sheep and goat industries world-wide.
Shipping fever, a variation of this disease, is a major problem in the cattle industry in North America and is almost exclusively caused by type A1 strains of this species (3). Serotype A2 is the most prevalent disease-causing type in sheep but other serotypes may be important in sheep and goats (16). The related species, Pasteurella trehalosi (formerly know as T-type P. haemolytica) is the causitive agent of septicemia in lambs, a problem plaguing the sheep industry particularly in the United Kingdom. Similarly, strains of the related species Pasteurella multocida, are responsible for haemorrhagic septicemia, a serious infection in cattle and water buffallo, which is particularly serious inSouth East Asia.
Vaccination is a desired method of control for pasteurellosis in ruminants but success has been limited by the lack of immunizing preparations that induce protection against all disease-causing serotypes, particularly if a vaccine effective for all ruminants is considered. Killed whole cell vaccines elicited inconsistent levels of protection and antibody response in calves (36). Homologous vaccines containing sodium salicylate extracts (SSEs) 5 protected sheep against diseases due to serotypes A1, A6 and A9 (17) but not against the more epidemic serotype A2 (12). An exotoxin produced by P.
haemolytica which is specifically lethal to leucocytes and alveolar macrophages from ruminants (4) has shown a lot of promise as a vaccine candidate in protection experiments in calves and sheep (13,35) but there is 10 limited protection against heterologous serotypes (33).The inclusion of proteins induced under iron-limited growth conditions into a vaccine for pasteurellosis in lambs has been implicated in enhanced protection (15).
Previous studies have established that the ability of pathogenic bacteria to acquire iron in vivo is a critical factor in their pathobiology (7,11).
15 One mechanism of iron retrieval from the host iron-binding glycoprotein, transferrin, involves direct binding of transferrin by surface receptors on the bacteria and the removal of iron from transferrin and uptake into the cell (21).The transferrin receptor has been shown to consist of two proteins, called transferrin binding protein 1 or A (Tbpl or TbpA) and transferrin binding 20 protein 2 or B (Tbp2 or TbpB). Receptor-mediated type of iron uptake has beendemonstrated to operate in type A bovine strains of P. haemolytica (26). Cells of P. haemolytica growing in vitro under iron-limited conditions express a number of iron-repressible outer membrane proteins (IROMPs) identical to those produced by cells recovered in vivo from infected sites in animals with 25 pasteurellosis (9,10). Especially prominent among these proteins were those of molecular sizes 100, 77, 70 and 60 Kda (9,10). The 100 Kda protein has been identified as one of the host specific transferrin receptors in bovine isolates (26) while some of the other IROMPs had been suggested as possibly associated with the 100 Kda protein in an iron acquisition receptor complex (26). The role 30 of the IROMPs expressed by P. haemolytica from lambs (10) in iron acquisitionhas not been elucidated, neither is it known if similar proleins are expressed by goat isolates.

P. haemolytica acquires iron from bovine host transferrin by a receptor-mediated type of mechanism. The proposal that bacteria with this type of iron acquisition mechanism may be solely dependent upon their surface receptor for iron acquisition in vivo (29) implies that they can only 5 cause disease in those hosts whose transferrin is recognized by their surface receptors. P. haemolytica has been reported to cause disease in cattle, sheep and goats and accordingly their surface receptors would be expected to recognize these hosts' transferrins. Therefore it is important to determine whether sheep and goat isolates also possessed transferrin receptors involved in iron 10 acquisition, to evaluate their specificities for difrerel-t ruminant transferrins and to determine if there is antigenic relatedness amongst the surface rece~toLs from the different strains causing pneumonic pasteurellosis in cattle,sheep and goats.

SUMMARY OF THE INVENTION
Transferrin receptors were identified in a collection of Pasteurella haemolytica (and P. trehalosi) strains of various serotypes and biotypes (A and T) from cattle, sheep and goats. Growth studies (Table 2), binding studies (Figure 26) and affinity isolation experiments (Figure 27) demonstrated that these receptors had identical specificities which recognized transferrins from cattle, sheep and goats. This suggests that there are conserved regions on the receptor proteins, involved in ligand binding, which are accessible at the cell surface.
Antisera prepared against the individual purified receptor proteins (TbpA and TbpB) from a type A1 strain of P. haemolytica demonstrated considerable crossreactivity against receptor proteins from a representative selection of strains ( Figure 28). The cross-reactivity was also observed against intact cells (Figure 29) indicating that there are conserved immunological epitopes at the cell surface which could serve as targets for the host's immune effector mechanisms.
The genes encoding the TbpA (tbpA) and TbpB (tbpB) receptor proteins from a type A1 P. haemolytica strain have been cloned and sequenced (Figures 30-33). Recombinant receptor ~roLeins produced in E. coli have been purified directly from intact cells by a simple affinity isolation procedure andthe purified recombinant receptor proteins have been shown to retain their functional (binding) and immunological properties.
The tbpA and tbpB genes have been isolated from a collection of P.
haemolytica (and P. trehalosi) isolates by PCR amplification with primers based on the sequence of the 3' and 5' ends of the type Al gene. Comparative analysis of the amplified genes by restriction endonuclease cleavage (with Sau3Al) indicated that the genes are highly conserved amongst most of the isolates.
Since transferrin receptors are accessible at the cell surface and mediate a critical in vivo function, they are ideal vaccine candidates. The recognition of conserved surface epitopes by anti-receptor antibody, suggests recombinant receptor protein from a type Al strain of P. haemolytica will be an effective vaccine antigen for shipping fever in North America and possibly for prevention of pasteurellosis in ruminants worldwide.
The present inventors have identified and characterized two transferrin binding proteins from P. haemolytica, herein referred to as TbpA
and TbpB (also know as Tbpl and Tbp2), which are iron regulated proteins.
The present inventors have cloned and sequenced the genes encoding TbpA
and TbpB, herein refeL~ed to as tbpA and tbpB respectively (also known as tbpl and tbp2). The present invention therefore provides a purified and isolated nucleic acid molecule comprising a sequence encoding a TbpA protein and a purified and isolated nucleic acid molecule comprising a sequence encoding a TbpB protein. The TbpA and TbpB proteins bind ruminant transferrins and function in receptor-mediated iron acquisition by P.
haemolytica in its ruminant hosts. The TbpA protein is approximately lOOkDa and TbpB is approximately 60 kDa in size.
The term "isolated and purified" refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An "isolated and purified" nucleic acid is also free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3'ends of the nucleic acid) from which the nucleic acid is derived. The term "nucleic acid" is intended to include DNA and RNA and can be either double stranded or single stranded.
In an embodiment of the invention, the purified and isolated nucleic acid molecules comprise a sequence encoding a TbpA having the amino acid sequence as shown in Figure 31 and a sequence encoding a TbpB having the amino acid sequence as shown in Figure 33. In a preferred embodiment of the invention, the purified and isolated nucleic acid molecules comprise a sequence encoding a TbpA and having the nucleic acid sequence as shown in Figure 30 and a sequence encoding a TbpB having the nucleic acid sequence as shown in Figure 32.
The invention also contemplates (a)nucleic acid molecules comprising a sequence encoding a truncation of TbpA or TbpB which is unique to the protein, an analog or homolog of the TbpA or TbpB or a truncation thereof, (herein collectively referred to as "TbpA related protein" or "TbpB related proteins"); (b) a nucleic acid molecule comprising a sequence which hybridizes under high stringency conditions to the full length nucleic acid encoding the TbpA or TbpB having the amino acid sequence as shown in Figures 31 and 33 respectively, or to a TbpA or TbpB related protein; (c) a nucleic acid molecule comprising a sequence which hybridizes under high stringency conditions to the full length nucleic acid sequence of the tbpA or tbpB genes having the sequences as shown in Figures 30 and 32 respectively.
Appropriate high stringency conditions which promote DNA
hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, 6.0 x sodium chloride/sodium citrate (SSC) at about 45C, followed by a wash of 2.0 x SSC at 50C may be employed. The stringency may be selected based on the conditions used in the wash step. By way of example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50C. In addition, the temperature in the wash step can be athigh stringency conditions, at about 65C.

The invention further contemplates a purified and isolated double stranded nucleic acid molecule containing a nucleic acid molecule of the invention, hydrogen bonded to a complementary nucleic acid base sequence.
The nucleic acid molecules of the invention may be inserted into an 5 appropriate expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
Accordingly, recombinant expression vectors adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the invention and one or more transcription and translation elements operatively 10 linked to the nucleic acid molecule.
The recombinant expression vector can be used to prepare transformed host cells expressing TbpA or TbpB, or a TbpA or a TbpB related protein. Therefore, the invention further provides host cells containing a recombinant molecule of the invention. The invention also contemplates 15 transgenic non-human mammals whose germ cells and somatic cells contain a recombinant molecule comprising a nucleic acid molecule of the invention which encodes an analog of TbpA or TbpB, i.e. the pro~e~,l with an insertion, substitution or deletion mutation.
The invention further provides a method for preparing a novel 20 TbpA or TbpB, and TbpA or TbpB related proteins, utilizing the purified and isolated nucleic acid molecules of the invention. In an embodiment a method for preparing TbpA or - TbpB is provided comprising (a) transferring a recombinant expression vector of the invention into a host cell; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected 25 transformed host cell under conditions which allow expression of TbpA or TbpB; and (d) isolating the recombinant TbpA or TbpB.
The invention further broadly contemplates a purified and isolated TbpA or TbpB which binds to ruminant transferrin. In an embodiment of the invention, a purified TbpA or TbpB is provided which has the amino acid 30 sequence as shown in Figure 31 or Figure 33 respectively. The invention also includes truncations of the protein and analogs, homologs, and isoforms of the protein and truncations thereof (i.e., "TbpA or TbpB related proteins").

The TbpA and TbpB, or TbpA and TbpB related proteins of the invention may be conjugated with other molecules, such as proteins to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins.
The invention further contemplates antibodies having specificity against an epitope of TbpA or TbpB, or TbpA or TbpB related proteins of the invention. Antibodies may be labelled with a detectable substance and they may be used to detect the TbpA or TbpB, or TbpA or TbpB related proteins of the invention in tissues and cells.
The invention also permits the construction of nucleotide probes which are unique to the nucleic acid molecules of the invention and accordingly to TbpA or TbpB, or TbpA or TbpB related proteins of the invention. Thus, the invention also relates to a probe comprising a sequence encoding TbpA or TbpB, or TbpA or TbpB related proteins. The probe may be labelled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleotide sequence coding for a protein which displays one or more of the properties of TbpA or TbpB.
The invention still further provides a method for identifying a substance which is capable of binding to TbpA or TbpB, or TbpA or TbpB
related proteins, or an activated form thereof, comprising reacting TbpA or TbpB, or TbpA or TbpB related proLeills, or an activated form thereof, with at least one substance which potentially can bind with TbpA or TbpB, or TbpA or TbpB related proteins, or an activated form thereof, under conditions which permit the formation of complexes between the substance and TbpA or TbpB, or TbpA or TbpB related ~roleil,s, or an activated form thereof, and assaying for complexes, for free substance, for non-complexed TbpA or TbpB or a TbpA
or TbpB related proteins or an activated form thereof. Substances which potentially can bind TbpA or TbpB, or TbpA or TbpB related ~roleills, include transferrins, particularly ruminant transferrins, analogs and derivatives of transferrins and antibodies against TbpA and TbpB, or TbpA or TbpB related proteins.

Still further, the invention provides a method for assaying a medium for the presence of an agonist or antagonist of the interaction of TbpA or TbpB, or TbpA or TbpB related proteins, and a substance which binds to TbpA or TbpB, or TbpA or TbpB related proteins or an activated form thereof. In an embodiment, the method comprises providing a known concentration of TbpA or TbpB, or TbpA or TbpB related proteins, with a substance which is capable of binding to TbpA or TbpB, or TbpA or TbpB
related proteins and a suspected agonist or antagonist substance under conditions which permit the formation of complexes between the substance and TbpA or TbpB, or TbpA or TbpB related proteins, and assaying for complexes, for free substance, for non-complexed TbpA or TbpB, or TbpA or TbpB related proteins. In a preferred embodiment of the invention, the substance is a ruminant transferrin, analog, derivative or part thereof or an antibody against TbpA or TbpB, or TbpA or TbpB related proteins.
Substances which affect expression of TbpA or TbpB, or TbpA or TbpB related proteins, may also be identified using the methods of the invention by comparing the pattern and level of expression of TbpA or TbpB, or TbpA or TbpB related proteins of the invention, in cells in the presence, and in the absence of the substance.
The substances identified using the methods of the invention may be used in the treatment of animals, particularly ruminants infected with P.
haemolytica and accordingly they may be formulated into pharmaceutical compositions for adminstration to ruminants, such as cattle, sheep and goats suffering from infection with P. haemolytica or exposed to infection by P.
haemolytica.
The present inventors have demonstrated that the TbpA or TbpB, or TbpA or TbpB related proteins of the invention, are immunogenic. The invention also relates to antibodies against the TbpA or TbpB, or TbpA or TbpB related proteins of the invention. In an embodiment, the antibodies are cross reactive against TbpA or TbpB or TbpA, or TbpB related ~roleh~s, from a wide range of serotypes of P. haemolytica. The antibodies may be used in the diagnosis and treatment of P. haemloytica infection and may be used, for example, in passive immunization to treat or prevent diseases in ruminants caused by P. haemolytica.
Conventional methods can be used to prepare the antibodies.
To produce monoclonal antibodies, antibody producing cells (lymphocytes) 5 can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, [e.g., the hybridoma technique originally developed by Kohler and Milstein (Nature 256, 495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al.
Monoclonal Antibodies in Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-96), and screening of combinatorial antibody libraries (Huse et al., Science 246, 1275 (1989)]. Hybridoma cells can be screened immunochemically for 15 production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated. Therefore, the invention also contemplates hybridoma cells secreting monoclonal antibodies with specificity for TbpA or TbpB, or TbpA or TbpB related proteins, as described herein.
The term "antibody" as used herein is intended to include fragments 20 thereof which also specifically react with a protein, or peptide thereof, having the activity of TbpA or TbpB. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated 25 to reduce disulfide bridges to produce Fab' fragments.
Chimeric antibody derivatives, i.e., antibody molecules that combine a non-ruminant animal variable region and a ruminant constant region are also contemplated within the scope of the invention. Chimeric antibody molecules can include, for example, the antigen binding domain from an 30 antibody of a mouse, rat, or other species, with bovine constant regions.
Conventional methods may be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes the gene product of novel Tbp genes of the invention (See, for example, Morrison et al., Proc.
Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397;
Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494.
The invention further includes vaccine compositions comprising the TbpA or TbpB, or TbpA or TbpB related proteins of the invention, either alone, or in combination. In a preferred embodiment, one or more of recombinant TbpA or TbpB, or TbpA or TbpB related proteins of the invention, are used in the vaccine compositions. The invention still further includes methods of immunizing a host, preferably a ruminant host against infection by P. haemolytica by administering therapeutically effective amounts of such vaccines. The present inventors have demonstrated that different strains of P. haemolytica, from a range of ruminants, are able to bindand utilize a range of ruminant transferrins. Thus it is contemplated that the vaccine compositions of the invention will be useful as broad spectrum vaccines suitable for immunizing a range of ruminants, such as sheep, cows and goats against infection with a wide range of P. haemolytica biotypes and serotypes.
The vaccine compositions may be prepared from TbpA or TbpB or related proteins isolated and purified from P. haemolytica, preferably P.
haemolytica A1, by the methods described herein. It is also contemplated that immunogenic truncations or fragments of the TbpA or TbpB or related proteins may be used in the compositions. Such compositions may contain any combination of the described prolei~s or immunogenic fragments thereof.
Further, the composition may contain proteins isolated from one or more biotypes or serotypes of P. haemolytica. Recombinant proteins comprising Tbpa or TbpB, or TbpA or TbpB related proleills of the invention, may also be employed in vaccine compositions.
The vaccine compositions can be prepared by ~ se known methods for the preparation of pharmaceutically acceptable vaccines which can be administered to animals. The vaccine composition may be in an oral or injectable form or in a form for administration intranasally, transdermally or any other suitable route of administration and may include pharmaceutically acceptable vehicles. On this basis, the vaccine compositions include, albeit notexclusively, solutions of the isolated and purified or recombinant proteins of the invention in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH
and iso-osmotic with the physiological fluids. Suitable pharmaceutically acceptable vehicles or diluents are described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, PA, U.S.A., 1985). It is contemplated that the vaccine may be used to augment the immune response of animals, preferably ruminants, such as bovines, ovines and caprines, most preferably bovines, to infection with a wide range of biotypes and serotypes of P. haemolytica., preferably P. haemolytica A1.
The invention also contemplates that the nucleic acid molecules of the invention encoding TbpA or TbpB, or TbpA or TbpB related proteins, may be incorporated into recombinant viral vector vaccines for use in augmenting the immune response of a ruminant to P. haemolytica or for treating P.
haemolytica infection. Recombinant viral vectors may be constructed using techniques known in the art .
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating prefelled embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

2~ 64274 Example 1 TABLE OF CONTEN'rS

LIST OF FIGURES AND TABL~ ......................................... vi LIST OF ABBREVIATIONS .............................................viii CHAPI'ER 1- LITERATURE REVIEW
I. P~teurel~ haem~nca a) Bovine pneumonic pasteurellosis b) Vaccine trials ........................................ 3 c) Iron uptake in P. haemo~ca ........................... 4 II. Iron in the Mammalian Host ................................ 5 a) Iron metabolism ....................................... 6 b) Distrlbution of host iron ............................. 6 III. Siderophore-Mediated Iron Uptake ......................... 10 a) Classification ........................................ 10 b) Mechqnism of siderophore uptake ....................... 16 IV. Nonsiderophor~-MeAiq~ Iron Uptake ........................ 17 a) Neisseria spp.........-........................................... 18 b) Haemophil~ luenzae ............................................... 20 c) Acnnobacillus pleuropneumoniac ....................... 21 V. Regulation of Iron Uptalce Systems ........................22 VI. Potential Vaccine Col~,ponent~ ................................ 23 VII. Thesis Objectivc . ................. ......... ..... 24 CHAPrER 2 - MATERIALS AND ME~HODS
1. Bacterial Strains and Cloning Vectors .......................... 2S
II. Enzymes, Chemicals and Antisera ..... .................. 26 III. DNA Methods .................................................. 27 a) Chromosomal DNA isolation ................................. 27 b) Restriction endon~lcle~ce digestion and ligation .......... 28 c) ~epalalion of c~ nl E. co~ cells .......................... 28 d) Large-scale plasmid isolation ............................. 29 e) Radiolabelling of DNA probes by random priming ............ 30 f) Agarose gel ele~llophoresis and Southern hybriidi7~tion ... 31 g) Southern colony blot ...................................... 33 h) Pol~ e.a_c chain reaction (PCR) . .................... 34 i) Pwif~cation of DNA fr~P,rn~-~tc from agarose gels ......... 35 j) DNA dideo~y se.~ ncin~ ................................... 3S
IV. Protein Methods ............................................... 38 a) Isolation of inner andy outer membranes ................... 38 b) Bradford determin~tion of protein concentralioll .......... 39 c) Sodium dodec~l sulfate polyacrylamide gel ele~o~horesis . . . 40 d) Weste~ immunnblot ~ysis ................................... 40 e) 1-7 protein e~pression ............................... 42 I. Cloning the tbpA,tbpB genes ...............................43 II. Sequence An~ysis .........................................50 III. Predicted Protein Topology .... .......... . . . . . .... 51 IV. Distribution of tbpA in P. haemo~tica and Related Species .... 61 V. Homology Studies ................. .;....................... 74 VI.T7 Protein E~pression ....................... .............. 83 VII. Western Immunoblot An~ysis ................ .............. 93 I. Se~e~ analysis ........................... .............. 101 II. Predicted protein topology ................. .............. 103 III. Distribution of tbpA in P. haemo~ytica and Related Species .. 105 IV. Homology Studies ............................................. 107 a) Neissena spp.............................................. 108 b) ~. pku~p~mo~............................................ 109 c) TonB depe~ent-r~xptor proteins ...........................109 V. Proposed Model for P. haemofynca Iron Uptake .................. 110 VI. 17 Protein E~pression .......................................114 VII. Western Immunoblot Analysis ................................. 114 CHAPTER 5 - SUMMARY AND CONCLUSIONS ................ 117 REFERENCES ......................................... 119 APPENDIX A. MEDIA RECIPES .......................... 136 LIS~ OF ~lGURES AND TABLES
Flgure page 1. Uptake of iron from transferrin in m~mm~lian cells.................. .9 2. Schematic diagram of two me~ods of iron acquisition by pathogenic bacteria........................................... l2 3. The structure of prototypical hydro~m~te and c~hol sideropho.~s.................................................. 15 4. ResultofPCRanalysis................................................. 47 5. Restriction endonuclease map of plasmids 9 and 10................... 49 6. Nucleotide sequence of P. haerno ~y n ca tbp A , tbp B ............. 5 3 7. The promoter region oftbp B ........................................ 5 9 8. HydropathyplotofTbplprotein......................................... 63 9. Hydropathy plot of Tbp2 protein..................................... 65 10. Emini surface probabilityplotofTbpl ............................... 67 11. Emini surface probability plotofTbp2............................... 69 12. Chou-FasmanpredictionofTbpl........................................ 71 13. Chou-Fasman prediction of Tbp2..................................... 73 14. Southern hybridization aslalysis of the si~cteen serotypes of P. haemo~yticagenomic DNAdig~sted with Clal................... 76 15. Southern hybridization analysis of the si~teen serotypes of P. haemo~icagewmicDNAdigestedwithHindllIlBamHI................ 78 16. Southern hybridization analysis of A. p k urop n eu m o nia e and A. suis ger~",ic DNA........................................... 80 17. Restriction endonuclease map of the tbpA, tbpB regions in P. haemo~tica, A. suis, and A. pleuropn~umoniae............... 82 r 2164274 18. Amino acid ali~nmerl~ of P. haemohy~ica Tbpl and the Tbpl proteins of N. gonor~hoeae and N. rneningitidis................ 85 19. Amino acid alignme~ of P. haerno~nca Tbpl and A. pleuropncumoniae serotype 1 and 7 TfbA proteins............. 88 20. Dendrogram showing the genetic relatedness between P. haernoly~ica N. gonorrhoeae, N. meningitidis Tbpl, A. pleuropneumoniaeserotypes 1 and 7TfbA proteins.............. 90 21. Amino acid alignmell- of P. haemo~tica Tbpl and E. coli TonB
dependent outer Illelllbl~ r~e?t~s............................92 22. T7 e~cpression of tbpA in E. coli IM 109 (DE3)...................... 96 23. Western immunoblot of P. haemolynca, E. col~ membrane plepatations using anti-autologous sera from rabbits...........98 24. Western immunoblot of P. haemo~ica, E. coli membrane plep~ations using sera from F~yonse vact~inat xl calves.......100 25. ~pose~ model of iron uptalce in Neisseria..........................112 Table page 1. Oligonucle~tides used in pol~,-.. e.~se chain reaction................ 45 LIST OF ABBREVIATIONS

P C R...................................................................... Polymerase C~Lain Reaction LT.................................................................................... Luria Thymidine kb.......................................................................................... kilo-base kDa....................................................................................... kiio-dalton BCIP................................................................ 5-bromo~chloro-3-indoyl phosphate NBT............................................................................ nitro-blue tetrazolium SDS ........................................................................... sodium dodecyl sulfate PAGE............................................................... polya~rylamide gel electrophoresis TE buffer.................................................................... Tris-EDTA buffer (pH7.5) TAE buffer.................................................................. Tris-ætate buffer ~pH7.9) TBE buffer.......................................................Tris-boric acid-EDTA buffer (pH 8.3) r CHAPrER 1 - LI l ~;RATURE REVIEW
I. Pasteurella haemolytica Pas~eurella haemotynca is a Gram negative coceobacillus which is found as a mucosal commerl~q-l in healthy rumin-q-ntc (Newsome and Cross, 1932). This bacterial species is character~z~d by fermentative carbohydrate metabolism and by a positive oxidase test (Biberstein, 1978). P. haemo~ca is divided into sixteen distinct serological types on the basis of an indirect hemagglutin test (Frank and Smith, 1983).
The si~teen serotypes are also divided into two biotypec, A and T, based on their fermentation of arabinose and trehalose, res~ctirely (Smith, 1961). The A biotype includes serotyp~s 1, 2, 5-9, 11-14, and 16 and the remqining serotypes 3, 4, 10 and 15 belong to biotype T. The two biotypes share only 3-13% DNA homology and it has been suggested that each biotype should be classified as a separate species (Bingharn et al., 1990; Sneath and Stevens, 1990). In addition, severaJ untypable strains of P. haemo~ytica have been desc. l~cd (Frank, 1988). P. kaerno~tica is the causative agent of respiratory and septicemic infec~ions in cattle, sheep and goats (Yates, 1982).

a) Borine ~)n~-- non* yz,~ c.llosls Bovine pneumonic pa~ellosis is the most economically significant disease of cattle in North America (Panciera and Corstvet, 1984; Yates, 1982). This disease affects 20 to 80% of all shipped cattle, causing death in up to 10% of those infected (Frank and Smith, 1983; Martin, 1983). In l9M, the estim~t~d loss due to pneumonic pas~ llosis was over S9.6 million dollars in Alberta feedlots alone (Church and Radostits, 1981).
Pneumonic pasteurellosis is also known as shipping fever, bovine re~spiratory disease and transit fever (Rehmtulla and Thomson, 1981).
As the name implies, ~shipping fever~ commonly occurs in feedlot operations where cattle are transported to and from distant locations (Martin el al., 1980; Panciera and Corstvet, 1984). Stress factors such as crowding, prolonged transport, starvation and e~treme temperature change~s preAispose cattle to the disease. (Blood et al., 1979;
Carter, 1973; Jensen et al., 1976; Jensen and Mackey, 1979). A variety of etiological agents have been isolated from the respiratory tract of infected cattle, but the predominant isolate is the bacterium P. h4emo~nca (Panciera and Corstvet, 1984).
Biotype A, serotype 1 strains are commonly ~soci~ted with shipping fever but the disease may also be caused by biotype A serotype 2 strains ¢l'homson et al., 1969).
In sl-~,ssed cattle, the number of P. hacmob~tica in the nasopharynx increase rapidly and eventually reach the lungs. Healthy cattle have the ability to clear P. haemo~nca from the lung effectively but stressed cattle which have depressed clearance n-ech~nisrm cannot (Lillie and Thomson, 1972). After P. haemolynca has colonized the bovine lung, tissue rl~rn~ge occurs resulting in blockage of the bronchioles.
The disease may also spread to the alveolar spaces, where fibrin and macrophages accumulate and result in IIl~ OSiS (Friend a al., 1g77~. Clinical s~ ~"-s of pasteurellosis include incleased respiratory rate, mucopurulent nasal discharge, loss of weight and diarrhoea (Hoerlein and Marsh, 1957; Adams a al., 1959; Sinha and Abinanti, 1962).

Some of thc virulenc~ factors of P. haernolynca include a leukotoxin, fimbriae, capsular polysaccharide, and lipopolysaccharide (Confer et al., 1990). In addition, a neuraminidase (Frank and T~b~t~h~i, 1983; Straus ct al., 1993), a sialoglycoprotease (Otulakowski et al., 1983; Abdullah et al., 1991), a serotype specific antigen (Gonzalez-Rayos e,t al., 1986; Lo et al., 1991) and three lipoproteins (Cooney and Lo, 1993) have been characterized but their role in the disease process is not kno vn.

b) Vac~ine trials Experimental and commercial vaccines against bovine pneumonic pasteurellosis, including both live and killed b~ ia, as well as various e~ctracts have had only limited succcss. Early vaccines were prepar~d from an inactivated whole cell preparation of P.
haernolynca A1. These bac~in vaccines had little effect on controlling disease and often induced a higher incidence and severity of pneumonia in vaccinated cattle (Confer et al., 1984; Confer ct al., 1985b; Confer a al., 1987; Mosier ct al., 1989). Vaccines prepared from P. haemoly~ica Al oell surface components have been shown to reduce the e~tent of lesions and in~"~ase the antibody titre (Confer et al., 1985a). Recently, a commercial vaccine has been developed, based on the soluble antigens present in the logarithmic phase culture ~llpe~ of P. ha~ ~ica A1. This vaccine, Presponse~, has been found to be > 70% eff~eiollc for pro~lion against bovine pneumonic pasteurellosis in both laboratory and field trials. (Shewen et al., 1988; Shewen and Wilkie, 1988; Jim et al., 1988). Although this vaccine is known to contain P.
haernoly~ica leukoto~in, many of the other co,llponents have not yet been identified.

r Other researchers have shown protection against pasteurellosis in lambs using other co~..ponent~ of P. haemobnca such as iron-regulated outer membrane proteins.
Ovine pasteurellosis is similar to shipping fever and may be caused by several different serotypes of P. haemo~ynca biotype A (Confer et al., 1990). Iron-regulated proteins are produced when the bacterium is grown under conditions of iron r~~ ;lion and have been shown to be present in natural infections (Donachie and Gilmour, 1988; Gilmour e~ al., 1991).

c) Iron uptake in P. haomolytica LittJe is known about iron acquisition by P. h~crno.~y~ica during infection, however, studies have suggested that iron plays an il--po- ~nt role in the disease process.
For e~ample, Al-Sultan and Aitken (1984), demonstrated that mice injected with ferric ammonium citrate before infeclion with T serotypes of P. haemo~nca had significantly lowered LD50 values and were therefore more susceptible to infection than untreated mice. A similar e,~liment by Chengappa c~ al. (1983) demor~l a~ that a 2%
haemoglobin p~p~ation injected in mice before e~-~sure to P. haemo~nca also e~h~ d virulence. Gentry ct al. (1986) found that ~ sr.,"in, lactoferrin, and conalbumin all stimL~l~t~ ~;~otoAi-- pr~s~clion in P. haerr~o~nca Al.
Iron-regulated outer ule.~b-ane proteins were first de3elilJcd in P. haemo~nca A2, which causes ovine pasteurellosis. Donachie and Gilmour (1988) r~po. ~d that cells collected from the pleural fluid of infect~ sheep had two additional outer membrane proteins of 71 IcDa and 100 kOa. Serum from convalescent sheep contained antibodies r to these iron-repressible proteins, which suggested that these proteins were expressed in natural infections and were antigenic. Further studies de,l,onakdled that a vaccine cont~ining iron-regulated proteins significantly enh~nee~ protection against e,~[i",ental pasteurellosis in lambs (Gilmour e~ al., 1991). A third iron-regulated protein of 35 kDa was identified in the periplasm of P. haerno~nca A2 but its function in iron-uptake is not known (Lainson et al., 1991).
Three novel outer membrane proteins were observed in P. haemo~t~ca Al cells grown under iron-limiting conditions. The proteins were 71, 77 and 100 kDa in molecular weight and reacted with convalescent antisera from P. hae no~ynca Al infected calves, suggesting that they were e~pressed in viw (Deneer and Potter, 1989).
Ogunnariwo and Schryvers (1990) found that P. haemo~tica Al cells could specifically utilize bovine transfellin as an iron source and proposed that the three iron-regulated proteins formed an iron acquisition comple~. In this iron acquisition comple~, the 100 kDa protein is the main transfellin binding protein or Tbpl.

II. IRON IN THE MAMM~L~N HOST
This section deals with the general aspects of iron uptake and metabolism in order to give background knowledge to the P. fulerno~ica A1 lran~rellin-binding proteins characterized in the thesis.

r 2164274 a) Iron metP~2';~
Iron is an essellti~l growth factor for virnlally all living cells. With the e~ception of certain strains of lactobacilli, (Archibald, 1983) all biological systems have an absolute requirement for iron in range of 0.4 ~M to 4 ~M (Weinberg, 1978). Two stable valencies (Fe 2+, Fe 3~) confer a wide o~tidation-reduction potential which allows iron to be useful in many chemical and enzymatic reactions. For example, iron is an esse~ cofactor for many enzymes such as RNA polymerase III (Shoji and Ozawa, 1986), ribonucleotide reductase (Reichard and Ehrenber, 1983), c~t~l~ce and peroxidase (Payne, 1988). In the electron transport chain, iron is required for cytochrome activity (Neilands, 1981) and succinate dehydrogenase (Davis and Hatefi, 1971). In m~mm~ls, iron is required for neutrophil function, T- and B-lymphocyte activity and o~ygen binding by haemoglobin and myoglobin (Woolridge and Williams, 1993).
Free iron is also polenl;~lly to~cic if lt pallicipa~s in a series of reactions known as the Haber-Weiss-Fenton chemistry (Flitter et al., 1983, Griffiths, 1987). In these reactions, free iron will catalyze the production of hydro~cide radicals and hydrogen pero~ide, which have been implicated in destroying biological membrane~s and breaking up of DNA (Weinberg, 1989). There is, therefore, a need to limit the amount of free iron within the m~mm~lian system.

b) Distr~bution of host iron Under aerobic conditions, iron is found in the Fe3~ form, which has very low solubility at physiological conditions. In order to com~ cate for this low solubility, and 2 1 64~74 to limit the amount of free iron available, m~mmqlc have evolved ~h~icms for m~int~injng iron in a soluble and non-to~ic form ~Weinberg, 1978). The majority of iron is intracellular where it is either located in the iron-storage protein ferritin or in heme-compounds such as haemoglobin, myoglobin, pero~idase and cytochrome c (Chricton and Charloteau~-Wauters, 1987).
E~tracelluar iron is bound to the iron-binding glycoproteins transferrin and lactoferrin. Transferrin is found in serum and Iymph and is responsible for the transport and recycling of iron. Each tran ferrin molecule has a molecular mass of 76 to 81 kDa and the capacity to bind reversibly with two atoms of ferric iron. The association constant of transr~ in is appro~cimately 10-2~ M which decleases when the pH is lower than 6.0 (Aisen and Listowsky, 1980; Chricton and Charloteau~-Wauters, 1987). Under normal physiological conditions, human serum contains 30% iron-saturated transferrin (Evans and Williams, 1980). The tran~f~llin-blound iron is delivered to the cells via a [cceptor mediated endocytosis process.
The transferrin rec~ptor in m~mm~lian cells is a tr~ncmembrane protein comprised of two 90 kDa disulphide-linked subunits. Each subunit contains a transferrin binding site which has an association constant of 10-9M for diferric ll~nsfe,.in and a much lower affinity for apo~ansfe.lin (Schneider and Williarns, 1983). Figure 1 shows thesequential steps in transfe~ t uptake in m~mm~lian cells. After the initial binding of transferrin to the r~p~or, the entire comple~c is endocytosed to forrn a clathrin-coated vesicle. The clathrin is shed and the vesicle becomes an endosome. A proton pump acidifies the pH
of the endocytic vesicle to pH 5Ø At this pH, the iron is released and the igure 1. Model for the uptake of iron from ~ansferrin in m~mm~lian cells (Williams and Griffiths, 1992).

' 2 ~ 64274 EA5E ~ I ¦ CO)~PA.~r~T f~ El~; Of ~POrll~6f Ell.RW

==~ UE~E~ WE 1 ~=

I CO.~TEO ~ESCLE
co~nA~;
~ COWI~ET of S~t-~; A O PECE~' PEC~G ~ ~ ~
/~ fEcErToA A~O C~ ll - ~ /

~ fELEASE ~ ~ l ~ ~lTllW E~COSOIE
r hEl~E ~ '5 5 - ~ RO~ T~A' ~ILI~ TO ~OO~IA
~0~ ~EU Sr~lTI~ESlS oP TO t1U~T-~ ¦

a~--l 1 21 64274 apotransferrin-r~ceptor comple~ is ftturJIed to the cell surface and is recycled (Romslo and Thorstenscn, 1990).
Lactoferrin is a protein which is found in mucosal secretions such as saliva, tears, milk, nasal mucus, and hepatic bile (Griffiths, 1987). This protein has no known role in iron transport and functions mainly as a bacteriostatic agent. Lactoferrin has a higher affinity for Fe3~ than transferrin, which allows it to withhold iron at reduced pH levels which often occur at sites of infection. Unsaturated lactoferrin is also a major component of the specific granules of neutrophils and is released during degranulation, which suggests that the protein may prevent e~ctracelluar bacterial growth, particularly at the site of an infl~mm~tory lesion (Mietzner and Morse, 1994).
Tlhe free iron conce.lllation in the e~-l,acelluar enviroh,llent is 1~'~M and i often reduced further during infection (Weinberg, 1978). In order to overcome the iron-r~s~i~t~d enviror~nelll of the ho t and establish infoclion, bacterial pathogens have evolved a number of different ~ t~g;~S to obtain sl~fr,cient ql~ntitiPs of iron. These strategies fall into two broad categories: i) mP~h~nicmc which involve the secretion of iron chPl~tors (siderophores) which bind to iron and transport it back to the bacterial cell and ii) those which involve direct utili77~tion of host iron proteins (Otto et al., 1992).
A sch~Pm~ diagram for both iron uptake me-ho lc is shown in Figure 2.

III. SIDEROPHORE-MEDIATED IRON UPrAKE

a) Cls~;fi~tion Siderophore-medi~P~ iron uptake is utilized by a wide range of bacterial and igure 2. Schematic diagram of the two m.o~ho~s of iron acquisition by pathogenic bacteria (Otto et al., 1992).
a) Siderophore-m~i~tç~ uptake b) Direct utili7~tion of host ~ar~r~llin and lactoferrin l ~ 216~274 Bacterial cell .~ Specific receptor ~ / -Fe3-0 0 Siderophore Transferrin or Lactoferrin ~ ~`
~ . - .
. ~ 3~
~9 ~v~- Bacterial cell Specific recepto Fe3- ~
~' ~ \
_ Transferrin or .- _ Fe3- Lactoferrin b) G~

_~
'~ ~
. ~
., . ~
,~ .~ .
.
,~ ~
. - 30-E~ `
-F~ 2164274 fungal species including pathogens such as Salmonella, Enterobacter, Pseu~'omonas and Eschcnchia coli. Siderophores are low molecular weight iron~hel~qtQrs which are secreted by microor~qnicmC into the e~tracellular medium (Neilands, 1981). Generally siderophores are of two chemical types, catecholates and hydrox~m. -q-tçs (Neilands, 1981), .¦ although siderophores with other chemical structures have been identified (Russel et al., 1984). The prototypical catecholate siderophore is enterobactin (enterochelin), which is produced by all enteric bacteria and which has a formation constant of 1052 at neutral pH
(Neilands, 1981). Aerobactin is hydroxamate siderophore produced by Salmonella (Colonna a al., 1985) and Enterobacter (Crosa ct al., 1988) spe ies and has a formation constant of 1023 at neutral pH (Neilands, 1981). As shown in Figure 3, catecholate . sidcrophor~s contain 2,3 dihydro~ybenzoic acid moieties as part of the binding ligand whereas the hydro~-q-m-q--e siderol.hole acrobacli" is derived from citric acid.
. Many bacteria secrete more than one type of siderophore in order to mal~imize iron uptake. For e1~ample, Pseudomonas ae~uginosa produces two siderophores, pyoverdin and pyochelin (Co~ and Adams, 1985; Cox a al., 1981) and E. col~ will produce both aerobaclin and en~lob~c~ (Der Vartanian, 1988). In addition, bacteria d such as Salrnonclla, can utilize siderophores which are produced by other microorg~nicmc .. - (Neilands, 1981; Luckcy a al., 1972). The m~hqnicrn for siderophore-mediated iron acquicitio~ has been extensively studied in E. col~ (Neilqn~lc~ 1981). l~erefole, enterobactin uptake in E. coli will be used as the example of siderophore-m~iq~ted iron uptake system.

Figure 3. The sl~uclure of prototypical hydro~m~tP- and catechol siderophores (Payne, 1988).
a) catechol siderophore el~roba ;Lin b) hydro~mate siderophore ae,ub~(i..

r 2l~4274 ~H
C = O

~C--c--C~

OH l1 ,NH C o_C~
o o"C~

b CH3 CH3 C=O C=O

N- OH N - OH

( CH2) 4 (CH2 )4 O COOH O

-r b) Me~hDnicm of siderophore uptake Iron-siderophore comple~es are 500 kDa to 1000 kDa in size and arc too large to diffuse through the porins of the bacterial outer membrane (Luckey et al., 1972).
Transport of these compleses requires the assist~nce of specific receptor proteins on the outer membrane. These receptor proteins are produc~d only under iron-limitin~
conditions and range in size from 70 to 100 kl~a. In E. coli, the enterobactin r~xp~or is the FepA protein (Hollifield and Neilands, lg78). Four other iron-regulated outer membrane proteins (Cir, FhuA, FhuE, Fiu) have also been identified (Wooldridge and Williams, 1993).
Topological models have been proposed for FepA and the other E. col~ r~eptor proteins and all havc similar structural elemen~ (Moeck ct al., 1994). l~ese proteins have amphip~thic regions which form antiparallel ,B-sheets which traverse the outer membrane. The intervening regions are thought to constiluLe loops which are eSpose~
to either the cell surface or in the periplasm. Rutz ct al. (1992) suggested that these loops~ may constitute both the ligand binding domain and a plug for the transport ch~nnel.
Transport of the iron-siderophore comple~ across the outer membrane also requires a second protein, TonB (Frost and R~enherg, 1975). This protein is anchored in the c~plas.,lic membrane and spans the periplasm. The f~J~ ;nn of TonB is to couple the proton motive force of the cytoplasmic membrane to the rece~tor protein, thereby providing the energy needed to carry t~e iron comple~ across the membrane (Postle, 1990). Two additional proteins E~bB, E~bD stabilize TonB in the cytoplasmic -i ~ 2 1 64274 membrane (Hantke and Zimmerman, 1981; Guterman and Dann, 1973). Studies in E.
cok have demor~s~ated that TonB inte.~b with the amino-terminal regions of the r~ptor proteins at a region known as the TonB~ bo~. (Bell ct al., 1990). The exact mechanism of TonB is not known, but Rutz et al. (1992) found that internal deletions within the FepA receptor converted the high-affinity receptor into a TonB-independent diffusion channel. This implies that recepto- proteins act as gated porins which move the iron-siderophore comple~c into an underlying membrane chqnnPI and into the periplasm. Liu ~ al. (1993) has confirmed this gated-porin model by demonstrating the presence of a large hydrophilic ch~nnel within FepA.
Transport of the iron-en~rob~lin comple~ across the cytoplasmic membrane requires a periplasmic binding protein transport system. The first element of the transport system is a periplasmic binding protein, FepB, which binds the iron comple~
(Elkins and Earhart, 1989; Shea and Mclntosh, 1991). FepC is a nucleotide binding protein which supplies the energy needed for ~ l. A third protein, FepD, is a cytoplasmic membrane-assooiat~d permease (Chenault and Earhart, 1991; Shea and Mclntosh, 1991). After the iron-siderophore comple~ enters the bacterial cytoplasm, the iron must be removed. Enter~b~tin utilizes an esterase to hydrolyse the ester bond in the sideropholc, thereby perrnitting iron relpqce (Coderre and Earhart, 1984; Fleming c~
al., 1985).

IV. NONSII)EROPHORE MEDIATED IRON U~AKE

Nonsiderophore mP~ ted iron uptake involves direct utili~tion of host llansf~llin -r~- 2164274 and lactoferrin as an iron source. This method of iron uptake requires direct contact between specific r~eptor proteins on the bacterial surface and the host iron proteins.
The best studied e~amples of this iron uptalce system are the human pathogens Neisseria gonorrhoeae and Neisseria meningitidis. Other pathogenic bacteria such as Haemophilus influenzae and Ac~inoboeill~ pkuropnuernoniac also utilize this mPch~nicm of iron uptake (Williams and Griffiths, 1992).

a) Ncissena spp.
Neisseria gonorrhoeae and N. meningi~is are human pathogens which cause localized infections on the mu~l surfaces of the urogenital tract and nasopharyn~
resp~c~ ely. Early studies de,l,ons~lated that transl~..in enh~ d meningoc4c~1 infection in mice (Holbein, 1981). Neither species produces siderophores (Archibald and DeVoe, 1980; West and Sparling, 1985; MjCL-PI~n ~t al., 1982) but both possess the ability to acquire iron directly from human transferrin (~ic~el~n and Sparling, 1981) and lactoferrin (Mic~e!cer~ et al., 1982). Co,l,~tili~e binding assays have demonstrated that pathogenic Neisseria species are able to utilize only human transferrin as an iron source, but not rabbit, horse, chi~n or bovlne trans~llin (Schryvers and Gonzalez, 1989; Mic~elcen and Sparling, 1981). In conLI~l, commensal Neisseria species cannot utilize transfe..in (Mic~lco~ and Sparling, 1981; Sil"onson ~t al., 1982) and many are unable to use l~clofe,lin (Schryvers and Lee, 1989).
Direct contact b~ n the host protein and the bac~lial cell surface has been shown to be essenlial for iron uptake in pathogenic Neisseria spp. (Archibald and DeVoe, -~ 2164274 1979). The binding of host iron proteins to the cell surface is mediated by specific saturable receptols (Schryvers and Morris, 1988; Lee and Bryan, 1989; Schrvyers and Lee, 1989). The transferrin and lactoferrin re~ptor proteins in N. gonorrhoeae and N.
meningitidis are believed to be distinct since neither protein is capable of blocking the binding of the other in competitive binding assays (Schryvers and Morris, 1988; Tsai a al., 1988). It is known that iron uptake occurs in an iron-regulated, energy-dependent manner and that the transferrin itself is not internalized in the process (Simonson et al., 1982; McKenna et al., 1988).
Under iron-limiting conditions, both N. gonorrhoeae and N. meningindis produce several novel outer membrane proteins (Dyer ct al., 1987; West and Sparling, 1985).
Affinity purification has identified two transr~lin-binding proteins in the Neisseria species: a 9~100 kDa protein and a 64-85 Icl~a protein termed transferrin binding proteins 1 and 2 (Ibpl and Tbp2), resye~ ely (Schryvers and Morris, 1988; Schryvers and Lee, 1989; Ferr6n et al., 1993). While Ibpl seems to be highly conserved among clinical N. meningitidis isolates, ~bp2 displays c~nsiderable heterogeneity among the meningococcal strains e~min~ (Schryvers and Lee, 1989). Analysis of the N.
meningitidis ~bp2 both ~nti~ni~lly and genomic or~ni7~tion of the genes has separa~ed the lbp2 into two classes (Rokbi et al., 1993).
The genes c~ing for Tbpl and Tbp2 in N. gonorrhoeae have been cloned and sequenced and are now refe.r~ to as tbpA and tbpB, r~ti~ely (Cornelissen ~ al., 1992; Anderson a al., 1994). The predicted amino acid sequence of Tbpl reveals homology with the TonB dependen~ outer membrane receptors in E. col, which implies ~, ~, that a TonB analog e~ists in Ncisseria spp. Mutants deficient in Tbpl could no longer bind nor utilize transfe,lin as their sole iron source (Cornelissen a al., 1992), which suggests that this protein is essential for transferrin utili7~tion. Mutants deficient in Tbp2 e~hibited only reduced transferrin blnding which indicates that the protein is not essential for iron acquisition in vitro (Anderson et al., 1994).
. Lactoferrin binding in both N. gonorrhoeae and N. meningindis is specific for human lactoferrin (Schrvyers and Lee, 1989). The lactoferrin-binding protein (Lbp) in both Neisseria spp. is a single 10~ kDa protein (Schryvers and Morris, 1988; Schryvers and Lee, 1989).
Under iron-limiting conditions, all pathogenic Neisseria species also pr~luce a 37 kDa ferric binding protein, Pbp (Mietzner et al., 1984; Mietzner a al., 1986; Genco et al., 1994). The Fbp protein is localized in the periplasm of the bacterial cell (Berish et al., 19191) and has been found to transiently accept one mol~ of ferric iron from transferrin (Chen et al., 1993). It has been proposed that Fbp is a functional analog of a Gram-negative periplasmic-binding protein component, such as PepB, which transports iron across the cytoplasmic membrane (Chen a al., 1993).

b) Hacmophilus in,p r~r~
Haemophilus ir~en~ae type b is an iu~po,~nt human p~,ogen which causes sepsis and m~ingi~i~ in children (Otto et al., 1gg2). Pathogenic strains of H. ir~luenzae type b do not I~I'~lUCe siderophor~s (Pidc~ck et al., 1988) but can utilize bovine, human and rabbit transfe,lin as a source of iron (Pidcock a al, 1988; Morton and Williams, ~ ~ 21 64274 1990). Nonpathogenic strains of Naemophilus such as H. parainfluenzae, cannot utilize human transferrin as an iron source (Morton and Williams, 1989).
Thc transferrin receptor comple% in H. influenzae consists of two transferrin binding proteins: a 100 kDa protein (lbpl) and a 70 to 90 kDa protein (lbp2) (Schryvers, 1989; Stevenson et al., 1992; Holland et al., 1992). The H. influenzae tranafe" in reoeptor proteins have been chardcter~ed and found to be analogous to those reportiedinNeisseriaspp.(Schryvers, 1989;0gunnariwoandSchryvers, l990;Schryvers and Gray-Owen, 19~2). An iron-regulated periplasmic protein with amino acid sequenoe homology with the Neisserial Fbp has also been described in H. influenzae (Harkness et al., 1992). In addition, three hit genes have been sequenced and were found to be homologous to the periplasmic binding protein transport system in Serratia marcescens (Sanders a al., 1994).

i v ~
c) Ac~nob~ciJ~s plcuropncumoni4c Actinobaoill~ pleuropneumoniae is the causative agent of porcine pleuropneumoniae and is able to utilize host transfellin as its sole iron source (Gonzales et al., 1990). Two iron-binding proteins of 56 and 105 kDa have been isolated from A. pkuropneumoniae serotypes 1,2 and 7. The gene enc~in~ for the transre~l h~-binding protein TfbA has been cloned and sc4ue~ from A. pleuropneumoniae serotype 7 (Gerlarch et al., 1992a) and serotype 1 (Ge.larch et al., 1992b). Both genes encode an outer membrane-localized li~opro~ein which also has si~nific~n~ homology to the N.
meningitidis Tbp2 N-terminus (Gerlach et al., 1992b).

~ ~ 21 64274 V. REGULATION OF IRON UPrAKE SYSTEMS
Siderophore and nonsiderophore iron-uptake systems may be encoded chromosomally or carried by plasmids, phage or transposons. In E. coli and S.
typhimuril~m, regulation of siderophore and siderophore receptor biosynthesis is mediated by a negative regulator designated fur (ferric uptake regulation) (Ernst et al., 1978). A
similar system has also been identified in other siderophore producing bac~.ia including - gPsclldomonas aeruginosa (~rince ct al., 1991), Kbrio chokrac (~itwin et al., l 9~2) and Yersinia pestis (Staggs and Perry, 1991). Many non-siderophore iron uptalce systems are also regulated by a Fur-like ~n~.h~nicm. Fur homologs have been identified and cloned from both N. gonorrhoeae and N. meningindis (Berish et al., 1993; Thomas and Sparling, 1994). H. influenzae iron uptake is regulated by cellular haemin concer~ tions in a Fur-like manner (Morton and Williarns, 1990; Morton et al., 1993).
In the presence of iron, Fur comple~es with ferrous iron and binds to the conserved sequen~s in the pro.l,ot~l region of the genes and bloc~s trans~ tion. Under iron limiting conditionc, Fur does not bind to these conserved regions or ~Fur bo~ces~, allowing transcription to occur (Bagg and Neilands, 1987). The fur gene itself is autogenously regulated and contains a Fur consensus se~ue~ (DeLorenzo ct al., 1987;
DeLorenzo ct al., 1988). Transcription offur is also subject to catabolite repression ~;(~eLorenzo ct al., 1988). The signifu~nee of such dual control is not clear, but it suggests a relationship between iron regulation and metabolic status of cells (~Nooldridge and Williams, 1993).
The e~pression of some virulence genes are also regulated by a fi~r or fur-like ..~..

loci. For e%ample, the c~pression of many toxins including the diphtheria toxin in Coryncbactenurn diptheriae (Boyd et al., 19~0), Shiga-like to~in in E. coli (Calderwood and Mek~13-1os, 1987), and the alpha-haemolysin in E. coli (Fasano, et al., 1990) are all derepressed in low iron conditions. In addition, the pilin gene in Aeromonas hydrophila (Ho et al., 1990), and supero~ide dismutase in E. coli (Niederhoffer et al., 1990) are also regulated by a Fur-like me~h~nicm.
.
VI. POTENTIAL VACC~E CO~fPONENTS

There is a growing interest in the utili7~tion of bact~ial iron-regulated outer membrane proteins as va~cine c~ o~Knts. There is considerable evidence to suggest that these proteins are produc~d in uvo and are important for the survival of pathogenic bac~. ~ (Otto et al., 19g2; Wooldridge and Williams, 1993; Mietzner and Morse, 1994).
In addition, iron-regulated proteins are often immunogcnic and in many cases show a significant degree of immunological cross-reactivity between different serogroups of one species (Black e~ ol., 1986; Fohn ct al., 1987). Studies with E. coli (Bolin and Jensen, 1987), ~. meningitidis (Ala'aldeen e~ al. j 1994), and P. haemolynca A2 (Gilmour~et al., 1991) iron-regulated proteins have suggested and these proteins may induce antibodies which block iron uptake or promote phagocytosis ~ F~- 21 64274 CH~l ~;K 2 - MATERIALS AND METHODS

I. BACTERIAL STRAINS AND CLON~G VECTORS
P. haemolytica strains were provided by Dr. P. Shewen, Department of Veterinary Microbiology and Immunology (VMI), University of Guelph, and were originally obtained from Dr. E. Biberstein, University of California, Davis, Dr. G.
Frank, USDA, Ames, Iowa, and Dr. W. Donachie, Moredun Research Institute, Edinburgh, U.K. Acnnobacilllls suis strain 3714, A. pleuropneumoniae strains CM5 and Shope 4074 were provided by Dr. S. Rosendql, VMI. E. coli strains HB101 and TG-1 were provided by Dr. R. Lo, Department of Microbiology, University of Guelph, and were used as recipient strains for cloning e~per~-erlt~. E. coli strain JM109 (DE3) was provided by Dr. C. Whitfield, D~pal~..ent of Microbiology, University of Guelph.
Pastcurella and ~c~inobaciU~s strains were n~qin~qin~d on she~p's blood agar and cultured in brain heart infusion broth (BHIB)-, (Difco L. bs, Detroit, ~irlligqn). E. coli HB101 was grown on Luria-Bertani plus thymidine (L~ (see Appendi~ A) supplemented with ampicillin (Sigma Chemio-ql Co., St. Louis, Missouri) at 100 mg/L for selection of recombinant plqcmi~ls. Similarly, E. coli TG-1 ard JM109 (DE3) werc grown on Davis minimal medium (see Appendi~ A) with ampicillin. Iron-depleted conditions were ~r~parcd by adding the iron ehrl~lQr ethyle~umin~-di(o-hy.l,~Ay~,henylacetic) acid (EDDA) (Sigma) to a final concerl-dtion of 100 ~LM. Iron-repleted conditions were .~pared by adding FeCI3 to 1 mM.
The plasmid pBR322, bac~,iophage vector~s M13/mpl8 and M13/mpl9 were used J I,~,,t~ I ~

as previously described (Lo and Cameron,1986; Lo etal., 1985; and Lo etal,, 1987).
The pBluescript vector was obtained from Stratagene (La Jolla, California). The recombinant clonc 482 was provided by Dr. A. Schryvers, Department of Microbiology, University of Calgary.

Il ENZYMES, CHEMICALS AND ANTISERA
Restriction endonucleases and DNA modifying enzymes were purchased from Bethesda Research Laboratories (BRL) (Burlington, Ontario) or Pharmacia Chemicals Incorporated (Dorval, Quebec) and were used as described by the manufacturers.
Radioisotopes were purchased from ICN Biomedical (Montreal, Quebec) or Amersham Laboratories (Oakvilie, Ontario).
Goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate and immunodetection reagents were purchased from Bio-Rad Laboratories (Mississauga, Ontario). Goat anti-bovine immunoglobulin G-alkaline phosphatase conjugate was purchased from Jackson Immunoresearch (West Grove, PA). Rabbit anti-autologous antiserum and bovine anti-Presponse antisera were obtained from Dr. P. Shewen, Department of Veterinary Microbiology and Immunology, University of Guelph. The rabbit "anti-autologous" antiserum was raised against the soluble antigens of P.haemolytica Al cultured in RPMI 1640 supplemented with that rabbit's own serum.
It is important to note that RMPI 1640 is an iron poor medium.

~ F~ 21 64274 m. DNA ~1 ~ODS
a) Chromosomal DNA isolation Chromosomal DNA was isolated from bacterial cells according to the method of Marmer (1961). Bacteria were inoculated into 250 ml of the appropriate medium and grown overnight at 37C with 150 rpm sh~king. The following day, the cells were pelleted by centrifugation at 4,000 % g in a GSA rotor in a Sorvall RC5-B refrigerated centrifuge (Dupont Instruments, Missi~-lga Ontario) for 10 min. The pellet was resuspended in 8 ml of a 0.6 M sorbitol, 0.05 mM Tris-HCI (pH 8.0), 0.05 M EDTA
solution. Lysozyme (Sigrna) was added to a final concentration of 3 mg/ml and the sample was in~ub~te~ for 30 min on ice. Two ml of Iytic solution (0.5 % SDS, 0.05 M
EDTA, 0.05 M Tris-Cl [pH 8.0]) and 3 mg/ml of proteinase K (Sigma) solution were added to the sample, which was then incub~ted for 4 h in a 37C water bath, followed by il~uba~ion at 56C.
The s~l,cnsion was eAI-aeled with an equa~ volume of phenol (Gibco/BRL) saturated with TE buffer (0.05 M Tris-HCI [pH 7.5], 0.001 M EDTA) and shaken at 3~
50 rpm for 45 min. The phenol and aqueous phas~s were separated by centrifugation at 12,000 % g at 5C for 10 min in a SS34 rotor. The slu~.-~lant was collected by a cut off wide mouth p~tlc~ pipette and the DNA was precipitated with 2-3 volumes of ice-cold 95 % ethanol. l~e strands of DNA were spooled onto a glass rod and dissolved in a small volume of 0.1 X SSC (lX SSC contains 0.15 M NaCI, 0.015 M sodium citrate).
The DNA was then ~eated with RNase to a final c4~ tion of 10 ~g/ml and inc ~b?~ at 37C for 30 min. The DNA was again precipitated by 2-3 volumes of cold .

~ ~ 2164274 gS % ethanol, spooled onto a glass rod and dissolved in lX SSC. Samples were stored at4C.

b) Restriction endonuclease dig~ion and ligation Plasmid and bacteriophage vectors were digested with the approp-iate reslriction endonuele~s according to m~n~ ctl-rer's instructions. Vector and insert DNA were mi~ed to a final volume of 5 ~1 and were ligated with 0.5 units of T4 DNA ligase.
Ligation mi~tures were either incubated for 34 hours at room temperature, or overnight at 14C prior to transformation into E. col~ cells.

c) Preparation of c~ et~ -' E. col~ oel~s Trar~l"~tion was used to introduce plasmid and bacteriophage DNA into c~.-,pcten~ E. coli cells (Mandel and Higa, 1970; Lcd~,l~r~ and Cohen et al., 1972).
E. coli strains to be transfol~llcd were grown overnight in LT broth at 37C with 150 rpm sh~king. The following day, a 1/40 subcult~re in 20 ml of the same medillm was pr~alcd and grown for an additional 60 min at 37C with 7S rpm sh~king. The cells were coll~ by ce,lll ir~g~tic!n alt 3,000 ~c g in an SS34 rotor and resu~nded in 10 ml stcrile ice-cold 50 mM CaC12. lbe s~ls~nsion was in~ b~t~ for 30 min on ice, then the cells were coll~ by celllrifug?tiQn and rcsu~n,ded in 2 ml sterile ice-cold 50 mM
CaC12. Tl~e co...l~t~n~ cells could then be stored at 4C and used for up to 3 days.
For trarL,fo~ ion, 0.2 ml of the co.u~t~nl cells were mLsed with the DNA
sample and i-d~"b~,d for 30 min on ice. llle cells were heat-choc~çd for 2 min at 42C

1~ 21642,4 and thcn 0.2 ml of LT broth was added. The oells were incubated at 37C for 15 min then platcd onto LT plates cont~ining appropriate antibiotics and incuba-ed overnight at 37C.

d) Large scale p~9crn~ so'~tion Large-scale plasmid isolation was performed according to the procedure of Clewell and Helinski (1969) with modifications. E. coli carrying the plasmid was inoculated into 250 ml LT broth containing ampicillin and grown overnight at 37C with 150 rpm s~ ing. The following day, chloramphenicol (Sigma) was added to a final c~ncenl~ation of 25 mgA and the culture was grown for a further 4-6 h. The cells were collected by centrifugation at 4,000 ~ g for 10 min in a GSA rotor. The cell pellet was resuspended in 4 ml of an ice-cold solution cont~ ining 25 % sucrose and 0.05 M Tris-HCI
(pH 8.0), then 1 ml of a fresh 10 mg/ml Iysozyme (Sigma) solution was added. Thc mi~ture was in~Ibq~ in a 37C ~at~,~t}, for 30 min, placed on ice for 5 min and then 2 ml of 0.25 M EDTA (pH 8.0) was then added. After a further 5 min ~ U~tiQn on icc, 5 ml of a Iytic solution (0.05 M Tris-HCI ~pH 8.0], 0.0625 M EDTA and 2 %
Triton X-100) were added. Thc mi~cture was relu.n~d to the 37C ~a~.l,a~ for 5-15 min until cell Iysis was complete. The mi~turc was then cu~ uged for 30 min at 27,000 ~ g and thc clear Iysate was transfelled to a clean test tube. The Iysate was mi~ced with solid CsCI (Boehringer Mannheim, Laval, Quebec) at 1 g/ml to a total of 4.5 ml. One hundred ~l of ethidillm bromide (10 mg/ml) were then added in 4.5 ml of the sample. The centrifuge tube was heat sealed and the sample was ce~ iruged at 240,000 i F 21 64274 g for a minimum of 9 h at 15C in a Rec~m~n VTi6S vertical rotor.
Plasmid DNA was recovered by puncturing the top and bottom of the centrifuge tube and collecting the lower of two bands in the tube. To extract the ethidium bromide from the sample, the plasmid DNA solution was mixed with an equal volume of CsCI
saturated n-butanol. After allowing the phases to separate, the upper layer cont~ining n-butanol and ethidium bromide was removed. This process was repeated three times.
Following ethidium bromide extraction, the lower aqueous phase was dialyzed to remove the CsCI. Dialysis tubing (Fisher) with a molecular cutoff of 10 kDa was prepared by boiling 2 ~ 15 min in 0.1 M Na bicarbonate and 1 ~ 15 min in 0.25 M EDTA (pH 7.5) and was stored at 4C in 50% ethanol and 1 mM EDTA. Prior to dialysis, the tubing was rinsed in dH20 and then filled with the plasmid DNA solution. The DNA was dialyzed for 24 h in 4 ~c lL of dialysis buffer (0.01 M Tris-HCI [pH 7.S at 4C], and 0.001 M EDTA) at 4C. The sample was stored at -20C.
Alternatively, the Fle~ci-prep kit from Pharmacia (Quebec City, Quebec) was used for small-scale plasmid preparation. This method involved a st~ndard alkaline cell Iysis, including RNase treA-tment and isopr~panol preoipitation (Birnboim and Doly, 1979; Isch-Horowicz and Burke, 1981). The plasmid DNA was purified and concenllat~ using a silica matri~ (Sephaglas FP~) in gn~nidinP hydrochloride.

e) Radio'Ahe~ g of DNA probes b~ random priming DNA fr~men~ were labelled with [a-32P]dATP (3,000 Ci/mmol, ICN) using the random primer DNA l~belling system of Gibco/BRL. This labelling system is based on F~ 21 64274 the method of Feinberg and Vogelstein (1983), with modifications (Feinberg and Vogelstein, 1984). Thc sample (25 ng of DNA in 10 111 of H20) was denatured by boiling for 5 min, then imm~i~tely cooled on ice. While still on ice, the following reagents were added: 2 ~1 of each of dCTP, dGTP and dTTP, 15 ~l of random primer buffer, 4 ~l [-32P]dATP and H20 to 49 ~l. The sample was mi~ced briefly and 3 units of Klenow Fragment was added. The reaction mi~cture was inrub~t~d for 1 h at 25 C and termin~ted by the addition of 5 ~1 of stop buffer.
The radiolabelled DNA was separated from unincorporated radionucleotides by gel filtration through a mini Sephade~c G-50 column. The column was prepared in a Pasteur pipette plugged with glass wool and was equilibrated with TE buffer prior to addition of the radiolabelled sample. The migration of DNA through the column was monitored using a Geiger counter (Mini-Instruments L~d., Esse~, Fngl~nd) The fu~t peak of r~ rtivity CCjllCi~ i to the Iq~elled DNA, while the second peak cGllesl)onded to the unincol~iatod [~2Pl-dATP. The DNA probe was denatured by boiling for 5 min before being added to the hybridization solution.

f~ Agarose gel electrophoresis and Southern l~.;di~tion Agarose gels were ~,~d by adding TAE buffer (40 mM Tris [pH 7.9], 1 mM
EDTA) to ele~t,ophoresis grade agarose powder (regular or low-n~el~ing point; Sigma) to a final concen~lation of 0.7% to 1%. The agarose gel was ele~ ophoresed in a h~ l flatbed gel dpp~lUS (Tyler Research, Edulon~on, Alberta).
DNA samples were mi~ced with 1/2 volume of tracking dye (50% glycerol, 0.1%

~ ~ 2~64~4 ladder (Gibco/BRI) or lambda DNA (Pharmacia) digested with Hindlll was used as a molecular standard. A running buffer of TAE supplemented with 1 ~g/ml of ethidium bromide was used. Samples were initially electrophoresed at 100V for S min, then the voltage was reduced to 10-12V for overnight electrophoresis. After electrophoresis, the samples were viewed with a medium range ultraviolet tranSi~ min~tor and photographed using Polaroid type 57 black and white film (Sharp ~ al., 1973; Hayward, 1972).
For Southern hybridization, the agarose gel was immersed in 0.25 M HCI for 15 min to depurinate the DNA. The gel was transferred to an alkaline solution consisting of 0.5 M NaOH and 1.5 M NaCI for 15 min, then neutralized in a solution of 0.5 M
Tris-HCl (pH 7.5), 1.5 M NaCI for 30 min. The DNA was trans~ d to a _ l nitrocellulose membrane (Schleicher and Shuell, Willowdale, Ontario) by electrophoretic transfer in a semi dry blotting apparatus (Tyler Research) in 20X SSPE buffer (3.6 M
NaCI, 0.2 M Na2PO~ [pH 7.0], 0.02 M Na2EDTA, 0.16 M NaOH) at a cona~nl current of 150 mA for 30 minut~s (Wahl ct al., 1979; Southern, 1~7S).
After ele~;~ophoretic transfer, the nitroeellulose membrane was washed in 2X
SSPE buffer for 10 min, and the DNA was cross-linked by a UV Cross-linker (Stratagene). The llelllblane was prehybridized in a sealed plastic bag con~ining a solution of 2S% (low ~ enc)~) or 50% (high ~illgenc~) forTn~mide (Gibco/BRL) in 0.1 % glycine, 5X BFP (100X BFP contains 2% w/v bovine serum ~lbumin~ Ficoll and polyvinyl pyrrolidine40), 5X SSPE buffer and 0.1 mg/ml sonic~t~ boiled salmon sperm carrier DNA. The sealed bags were placed in a 42C sh~king Wat~lb;illl where the membranes were allowed to prehybridize for at least an hour. The prehybridization buffer was then discarded and replace~ with hybridization buffer (10% dextran sulphate, 5X SSPE, SX BFP, 0.1% SDS, 0.1 mg/ml carrier DNA and 25 % or 50% formamide) containing the boiled, radiolabelled DNA probe The bags were placed in a 42C
shaking waterbath where the membranes were hybridized overnight.
After hybridization, the nitrocellulose membrane waC removed from the pla tic bag and washed 4 ~c 10 min in either high stringency (SX SSPE, 0.1% SDS) or low stringency (2X SSPE, 0.1~ SDS) wash buffer in a 42C ch~kin~ waterbath. The membrane was air-dried, placed on Whatman filter paper, covered with pla tic wrap and exposed to X-ray film (Cronex, Willingmington, Delaware) at -20C for 14 days until the desired e~posure was obtained. The e~posure time was determined by meacuring the intensity of the rY~io~ tive signal ~cing a Geiger counter. Autoradiographs were developed in Kodak GBX rapid developer (F~stm~n Kodak, Rochej~r, New York) for 2 min. The developing reaction was stopped by immersing the film in 2.5 % acetic acid for 1 min and fulced for 2 min in Kodak GBX fLl~er (Eastman Kodak).

g) Southern colony blot A master template of bacterial colonies grown on LT plus ampicillin was grown overnight at 37C. The colonies on the master plate were duplicated onto a nitrocellulose membrane overlaid on an LT plus ampicillin plate and grown for 2-3 h at 37C. The membrane was then overlaid on Whatman filter paper soaked in a 0.5 M
NaOH, 1.5 M NaCI solution and incub~t~J at room ~.~ alule (Rl~ for 5 min to Iyse the cells. The nitrocellulose membrane was then transferred to Whatman filter paper soaked in a 0.5 M Tris-HCI (pH 7.5), 1.5 M NaCI solution and incub~te~ for 5 min at RT to neutralize the membrane. The membrane was transr~ d to Wh~rn~n filter paper soaked in g5 % ethanol and sprayed with 95 % ethanol to precipitate the DNA. The DNA
on the membrane was cross-linked in a UV cross-linker (Stratagene), then prehybridized and hybridized as des.;,ibed above.

-h) Polgmerase chain reaction (PCR) PCR reactions were carried out in thin-walled 500 ~I GeneAmp microfuge tubes in a Perkin-Elmer Cetus 480 DNA Thermal Cycler, using the Perkin-Elmer Cetus PCR
core reagent kit which included deo~cynucleotides triphosphates, MgCI2, reaction buffer and Ampli-Taq DNA polymerase (Perkin-Elmer Cetus). Amplification reactions were ~, fiol",ed according to the method of Saiki et al. (1988), with modifications by Perkin-Elmer Cetus. PCR reactions were pel rOI ",ed in 100 f~l mi~tures containing lX reaction buffer (0.5 M KCI, 0.1 M Tris-HCI [pH 9.01). 0.2 mM of each of dNTP, 0.4 ~M
primer, 5 ~Lg of template, 15 mM MgC12 and 2.5 units of Ampli-Taq enzyme. The reaction mi~ture was heated at 95C for 2 minutes to denature the template DNA. Then 30 cycles of denalulatio,l, ~nnP~ling and e~ctension followed with telllpe~atures and times of 95C (1 min) 52C (1 min) and 72C (2 min) l~pecti~ely. The fastest available transitions between tell~ lur~s (ramp time of O.Ols) were used. A negative control which did not contain template DNA was included in each PCR run.
After amplification, the PCR pr~lucb were e~mined by agarose gel electrophoresis. PCR products were purified by elec~ophoretic separation through a I F~ ~164274 low-mclting point agarose gel followed by excision of required DNA fragments. The DNA products were purified from the agarose using a glass-bead matri~c purification kit (GENECLEAN).

i) Purifi~tiQn of DNA fra~nents from agarose gels DNA fragments were purified from agarose gels using the GENECLEAN kit from Bio/Can Scientific (Missi~ug~, Ontario). The GENECLEAN purification process is based on the procedure by Vogelstein and Gillespie (1979). The gel slice cont~ining the fragment was excised from the gel using a razor blade and placed in a 1.5 ml Eppendorf ifuge tube. An equal volume of stock NaI solution was added and the sample was inrub~ted for 5 min in a 55C waterbath until the agarose had completely melted.
GLASSMILK (Bio/Can Scientific) was added to the sample at a volume of 5 ~l for S ~g or less of DNA and the mi~cture was in--u~d on ice for S min. The silica matri~ was then collected by centrifugation at 16,000 ~ g for 10 sec and r~us~nded in 600 ~1 of NEW wash buffer (Bio/Can Scientific), The pellet was washed with NEW
buffer a total of three times. After the final wash, the silica matri~ was resuspended in 10 ILI of TE buffer and inoubq~P~ for 5 min at 55C. The sample was then cen~ ged and the TE recovered, avoiding the silica matri~ pellet. Samples were stored at -20C.

~' j) DNA dideoxy sequenci~g DNA fr~gmen~ were sequenced either by cloning into M13 mpl8/mpl9 bacteriophage vectors (single-stranded sequencing) or directly from recombinant plasmids ~ 2164274 (double-stranded sequencing) using the Pharmacia T7-sequencing kit as described by the ~ nuf~ rer~ The Pharmacia T7-se~ucncing kit procedure is based on the method outlined by Sanger ct al. (19~7).
For single stranded sequencing, DNA fragments were cloned into the M13 mpl8/mpl9 bacteriophage vector and transfol,llcd into coll.petent E. coli TG-l cells.
Recombinant phage ~plaques~, which appeared white due to the loss of B-g~l~ctosidase production, were selected. Each plaque was inocul~ted into 10 ml of LT broth seeded with 0.1 ml overnight culture of E. coli TG-1 grown in Davis minimal medium (see Appendi~ A) and incub~ 4-5 h at 37C with 75 rpm sh~king. The sample was cen~ ged at 12,000 ~ g for 10 min to remove the E. coli cells. The phage were precipitated from the culture sul)c(natant by the addition of 1/4 volume of 20%
polyethylene glycol (8,000 MW; Sigma), 2.5 M NaCl and in~uba~ed for 30 min on ice.
Precipitated phage were recovered by centrifugation in a microfuge at 12,000 ~ g for 10 min. The pellet was then resuspended in 0.6 ml of phage buffer (0.1 M Tris-HCI tpH
8.0], 0.001 M EDTA, 0.3 M NaCl).
The phage DNA was e~,acled wi~ 0.5 ml of phenol (Gibco/BRL) saturated with TE buffer. The phenol and aqueous phases were s~pafatod by cenllirugation at 14,000 ~c g for 10 min. The aqueous phase was eA~ d with 1:1 phenol:chloroform and finally with chloroform. The phage DNA was then precipitated with 1/10 volume 3 M sodium acetate (pH 7.0) and 2 volumes of cold 95 % ethanol and i~-cub~Pd at -20C overnight.
Precipitated DNA was collected by centrifugation at 14,000 ~ g for 10 min in a 4C
microfuge. The pellet was air-dried and resuspended in 50 ~1 of TE buffer and was used ~- ~ 2164274 for sequencing. The DNA was ~nn~le~ to either the universal M13 primer or specific primers in the presence of annealing buffer (Pharmacia T7 sequencing kit) by incubation at 65C for 10 min, then room temperature for 10 min prior to the sequencing reactions For double-stranded sequencing, the plasmid template ~as prepared using the procedure outlined in the Pharmacia T7 sequencing kit protocol, with modifications.
Plasmid DNA was adjusted to 1.5-2.0 ~g/32 ~l and denatured by the addition of 12 ~l of 2M NaOH for 1 min. Denaturation was terminated by the addition of 11 ~l of 3 M
sodium acetate (pH 5.0). The DNA was precipitated with 7 ~l of dH~O and 120 ~l of ice-cold absolute ethanol and incubated at -20C overnight.
The DNA was collected by centrifugation at 14,000 ~c g in a 4C microfuge for 10 min. The pellet was washed with 100 ~l of ice-cold 70% ethanol, centrifuged and dried under vacuum. The sarnple was resuspended in 5 ~Ll of dH20 and mixed with 5 ~1 of primer and 2 ~l of ~nnl~ling buffer. The mi~cture was ineub~te~ at 65C for 5 min, 37C for 10 min and 5 min at RT prior to sequencing.
Either [32PIdATP or [35S]dATP (specific activity of 3000 Ci/mmol) were used in the sequencing reactions. For short autoradiography exposure time, ~32P]dATP was used.
For superior resolution, ~35S]dATP was used. Oligonucl~ti~ p~ e.~ were synthe-cized on an Applied Biosystems In~l,.ational 391 PCR-Mate DNA synthesizer and purified according to the m~nuf~5tllrer's instruGti-)nc. The primers were qll~ntit~t*~ by I~ illg the optical density at 260 nrn prior to use.
For ~32P]dATP se~en~ing~ the sequencing gel consisted of 18 g of urea (ICN, Montreal, Quebec), 3.75 ml of 10X TBE buffer (1 M Tris [pH 8.3]. 0.02 M EDTA, and 21 64;274 .`
0.865 M boric acid) and 7.5 ml of 40% acrylamide (19:1 ratio of acrylamide:
bisacrylamide, Bio-Rad) made up to 38 ml with dH2O. The solution was stirred until thc urea was dissolved and then 0.23 ml of 10% ammonium persulfate (Sigma) and 10 ~1 of TEMED (N,N,N'N'-Tetramethylethylenediamine; Sigma) (0.01% final concentration) were added for polymerization.
For [3'S]dATP sequencing the gel consisted of 16.8 g of urea (ICN), 4.8 ml of 10X TBE buffer and 4 ml of a modified acrylamide solution (~Long Ranger-, l.T.
Baker, Phillipsburg, New Jersey). The solution was made up to 40 ml with dH2O and polymerized with 200 ~ul of ammonium persulfate (Sigma) and 20 ~1 of TEMED (Sigma).
The ruMing buffer consisted of lX TBE. The sequencing gel w~as run at 40 W/gel constant power for 2-6 hr. [35S]dATP sequencing gels were transferred onto a sheet of Whatman filter paper and dried under vacuum at 80C for 45 min. Both types of sequencing gels were e~posed to Crone~c 4 X-ray film (Crone~) for 1848 h at -20C.

. _ IV. PROTEIN METHODS
a) Isolation of inner and outer membranes Inner and outer membrane preparations were p~pared from E. coli and P.
hacmo~ca Al by the procedure of H~nc~cL and Carey (1979), with modifications (~o et al., 1991). Bacteria were grown in 250 ml of the ~ppropriale m~~ m at 37C
overnight. The cells were collected by cen~irugation at 4,000 ~ g, washed twice in 0.01 M Tris-HCl (pH 6.8) and resuspended in 7.5 ml of a cold Sucrose-Tris solution containing 20% su~ose, 0.01 M Tris-HCI (pH 6.8), Iysozyme (Imglml), DNæ (50 -~ ~ 2 t 64274 ~g/ml), and RNase (100 ~lg/ml). The oells were Iysed by French pressure cell three times at 16,000 - 18,000 psi at 4C. The sample was then oentrifuged at 1,085 % g for 5 min to remove unlysed oells.
The supernatant was layered onto a 70:52:sample:12 ~ sucrose gradient which consisted of 14 ml of the 70 and 52 % sucrose followed by 5 ml of the sample Iysate and 4-5 ml of 12 % sucrose. The gradient was centrifuged in a swinging bucket rotor at 80,000 ~ g for 1~18 h at 4C. The inner and outer membrane fractions were collected by aspiration. The inner membrane fraction was located between the 12~ and 52%
sucrose regions and had a yellowish-brown colour. The outer membrane fraction, which was white, was located near the 70% sucrose region. The collected fractions were loaded into ccn~ ge tubes, topped up with dH20 and cer,~ uged in a fL~ed-angle Ti80 rotor at 225,000 ~c g for 1 h at 4C. The pellet was then air-dried and resuspended in a Tris-HCI (pH 6.8), 0.001 M dithiothreitol buffer. Samples were stored at -20C.

b) Bradford det~ n of protein conc~.-tr~tion The protein concenLrations of the inner and outer membrane fractions were deterrnined using the method of Bradford (1976). Dilutions of each membrane fraction werc prepared and dH20 was added to a final sample volume of 0. 8 ml. The sample was then mi~ced with 0.2 ml of Bradford reagent (Biorad, Mi~si~sang~. Ontario) and incub~
at room temperature for 5 min to allow colour development to occur. The optical density (OD) of each sample was measured in a s~ ropho~ et~r at a wavelength of 595 nm.
A standard curve was plotted using bovine serum albumin (BSA; Sigma) in the 1-25 ~g F~- 21 64274 .
range. The protein concentration of each sample was e~trapolated from the BSA

standard curve.
.

c) Sodium dodecyl sulfate polyacrylamide gel electrophor~sis Proteins were analyzed using sodium dodecyl sulphate polyacrylamide elccl~ophoresis (SDS-PAGE) with 4% (w/v) s~Ling and 7.5% (w/v) sep~ating gels (Laemelli, 1970). The gels were polymerized by the addition of 0.1% ammonium persulfate and TEMED to 0.01%. The samples were solubilized at 100C for five minutes in an equal volume of 2X sample buffer (see Appendi~ A). A high molecular weight standard was also boiled and loaded onto the gel. A discontinuous buffer system was used (0.192 M glycine, 0.02 M Tris-HCI tpH 8.4~, O. l X SDS).
The gels were run at lOOV until the samples entered the separating gel, when the voltage was increased to 150V. The samples were run until the dye front ran off the bottom of the gel. The ~!~rL in~ gel was removed and the separating gel was either stained with Coomassie Brilliant Blue or electrophoretically transferred to nitrocellulose for Western immunoblotting. Gels were stained in Coomassie Brilliant Blue R250 (0.05% in 40% methanol, 10% ~r~tic acid) (F~stm~n Kodak) overnight and then dest~in~ in a rneth~nol: acetic acid solution.

d) Western i~ uoblot analysis The proteins on the acrylamide gel were transferred to a nitrocellulose membrane according to the method of Burnette (1981). The gel was s~alced in blotting buffer ~ 21 6~274 (0.192 M glycine, 0.025 M Tris-Cl [pH 8.4], 20% methanol) for 10 minutes to remove the SDS. A piece of nitrocellulose membrane (Schleicher and Shuell, Willowdale, Ontario) cut to fit the gel was also soaked in blotting buffer. The proteins were transferred to the nitrocellulose membrane in a Bio-Rad Transblot apparatus at 450 mA
for 3 h. A water~ooling system was used to prevent heating and breakdown of the blotting buffer.
After electrophoretic transfer, the nitrocellulose membrane was soaked in 3%
gelatin in lTBS buffer (0.02 M Tris-CI tpH 7.5], 0.5 M NaCI, 0.05% Tween-20) for 30 min to block the membrane. The nitrocellulose membrane was transferred to a 1/500 dilution of the first antl~ody in 1% gelatin and irrub~t~ overnight at room temperature with gentle sh~king. The membrane was then washed twice in TTBS buffer (15 min per wash) and placed in the second antlbody solution (1/2000 dilution) for an hour. The second antibody was g~at anti-rabbit or goat anti-bovine IgG-alkaline phosphatase conjugate (Bio-Rad) in 1% gelatin. The l.e.nb.dne was washed twice in TTBS buffer (15 min per wash) and then twice (5 min per wash) in NBT buffer (0.1 M Tris-CI ~pH
9.5], 0.1 M NaCl, 50 mM MgCI2). The membrane was then placed in the developing solution of 100 ~1 of each of the reagents 5-bromo~chloro-3-indolyl phosphate (BCIP, 25 mg/ml in dil.leLllylfo,...~mide; Sigma) and nitro-blue-tetrazolium (NBT, 50 mg/ml in 70~ dimethylform~mide; Sigma). Colour development was allowed to proc~d until the desired visibility of the bands was obtained. The colour reaction was stopped by rinsing the llle,llb.~ne in H20. The l.,e...blane was air dried.

'-I
e) 17 protein ex~,.~;;cn Proteins encoded by a recombinant plasmid were analyzed using the method of Tabor and Richardson (1985). The tbpA gene was cloned into the plasmid vector pBluescipt. The recombinant plasmid was transformed into E. coli JM109 (DE3), which is a strain of E. coli JM109 with the T7 RNA pol~ e,ase gene integrated into the chromosome and T7 polymerase gene under the control of the lac promoter (Yaninsch-Perron et al., 1985).
After transformation, the cells were grown overnight at 37C in Davis minimal medium containing 1.0% c~c~mino acids, 0.4% glucose and the appropriate antibiotics.
A 1/50 subculture into 20 ml of the same medium was prepar~ and inrubat~d at 37C
for an additional 3~ h until the OD5,a=0.6. The cells were collected by centrifugation at 14,000 ~ g for 5 min and the pellet was resuspended in Davis minim~l medium with 0.4% gluc~se. The sample was in. ub~t~d for 90 min at 37C, then 100 ~l of 5 mM
isopropyl-B-D-thiogalactopyranoside (IPTG) were added. After the cells were incllbated at 37C for 20 min, rifampicin was added (final concenllation of 400 llg/ml). The sample was incubated at 37C for 30 min and then labelled for 60 min with 5 ~Ci 135S]-methionine (-Trans-Label-, ICN Biom~i~l, Quebec). The cells were washed twice in ice-cold PBS and collected by centrifugation at 14,000 ~ g for 5 min in a microfuge.
The pellet was ~ pcn~l~d in 2X SDS-PAGE sample buffer.
The proteins were separated using SDS-PAGE and the gel was stained with Coomassie blue R250. The gel was then soaked in Amplifyn' (Amersham, Oakville, Ontario) for 30 min and dried under vacuum. Autoradiographs were e~posed 18~8 h.

-. CHAPTER 3 - PRELIMINARY RESULTS
~' I. Preliminary Cloning the tbpA, tbpB genes The first stage in cloning the tbpA gene was to screen a P. haemolytica Al gene library by polymerase chain reaction. An oligo primer specific for the N-terminal amino acid sequence of the Tbpl protein (Schryvers, personal communication) was synthesized.
A P. haerno~ynca A1 codon table (Lo, 1992) was used to optimize the primer sequence.
The Tbpl primer was used in conjunction with primers based on the junction sequences of the cloning vector pBR322 (Table 1). A 0.8 kbp PCR product was obtained (Figure 4) and cloned into the M13 vector and sequenced. Sequence analysis of this PCR
product demonstrated that the first twenty amino acids m~tched the sequence obtained by N-terminal amino acid sequencing of Tbpl.
The 0.8 kb PCR product was t~hen radiolabelled and used as a specific probe to screen the E. col~ clones c~ont~ining P. haerno~nca A1 gene library by Southern hybridization. Two recombinant clones, 9 and 10, hybridized strongly with the tbpA
probe. The plasmid DNA from each recombinant clone was analyzed by restriction endonucle~ce mapping (Figure 5). The insert of P. h~erno~ynca A1 was deterrnined to be appro~imately 8.7 kb and 2.3 kb for pl~cmi~lc 9 and 10, r~Li~ely. Initial se~uence analysis of the pl~$mi~c confirmed that the insert DNA from both plasmids share an overlapping region. Plasmid 9 eontained the entire tbpA gene but the region dire~tly upt~.~n of tbpA was different than the upstream region in plasmid 10. It is possible that the insert DNA in plasmid 9 was formed &om two DNA fragments from Table 1. Oligonucleotide primers used in PCR.
* - the underlined se~u~nee is the HindIlI site ** - the underlined se~uen~e is the E~;oRI site ; Primer Primer Sequcr~c Size .,.

lbpl thr- glu- as~ Iys- Iys- ilc- glu- glu 32 5'GGAAGCI~ACT-GAA-AAT-AAA-AAA-ATC-GAA-GAA * mer primer S' GGA~l~CCGTCCTGTGGATC ** 22 Ich mcr prim 5' GTGA~TTCCGGCGI'AGAGGAlY~ ** 22 right mer Figure 4. Result of PCR analysis a) Schemqtic diagram of the PCR procedurc Each circle represents a recombinant pBR322 plasmid and a possible PCR
reaction. llle Ibpl primer, primer left and primcr right are rcprcsentcd by the letters t, I and r r~~ i./ely The EcoR~ sitc on thc pBR322 plasmid is denoted by the letter, E.
In the upper plasmid, the lbpl primcr and primer left would amplify a PCR product cch.~ponding to the hcavy linc. Similarly in thc lowcr plasmid, thc PCR product amplificd by lbpl primcr and primcr right is represented by the heavy line.

b) Thc 0.8 kb PCR prol~c~ amplificd by Tbpl primcr and primcr left.

\~

E

b ,, y Figure 5. Restriction endonucle~e map of tbp plasmids 9, 10 and 482.
Open bo~c - pBR322 presented linearly. Crossh~tche~d bo~ - PCRII
presented linearly. The positions and the orientations of tbpA, tbpB are -as shown by the dark arrows. ~S

Abbreviations:
A -AvaI B - Bam Hl Bg - BglII C - ClaI
E~ - EcoRV H - Hind~ll Hc - HincII
S - PstI V - PvuII ., ,~

-~

-~ 2164274 ,, m _u~ -m _ r m m _ ,~
>
--CD --~ --S
a ~ 1 _ ~

~ -- ~

~ 21 64274 separate regions of the genome. In plasmid 10, the region directly upstream of tbpA
contained an additional open reading frame which corresponded to the tbpB gene. This plasmid, therefore, contained not only the S' region of tbpA but also part of the tbpB
gene, directly upstream from the tbpA gene.

A third recombinant clone, 482, contained the entire tbpB gene. This plasmid shares an overlapping region with plasmid 10 (Figure 5). The insert DNA in plasmid 482 is a PCR product obtained from P. haemolytica A 1 genomic DNA using primers specific for the amino acid sequence of Tbp2 protein. This PCR product was then cloned into the vector PCRII.

;!i;
~, ., The 3.0 kbp tbpA gene was sequenced from plasmid 9 (starting from the BglII
site). The 2.1 kbp tbpB gene and the 91 bp sequence between tbpA and tbpB was se~quenced from plasmids 482 and 10.

II. Sequenoe Analysis The 5.2 kbp of DNA was sequenced and shown to contain two open reading frames arldnged in tandem, with tbpB upstream of tbpA (Figure 6). This genetic org~ni7~tion is conaialent with other iron uptake systems in other bacteria where the genes often a[langed in an operon (Payne, 1988). Upon s~uer~e analysis of the cleduGed lbp 1 protein, a putative 28 amino acid leader peptide was observed. A putative cleavage sequence for lipoproteirls was observed in the deduced lbp2 protein. The close pro~cimity of tbpA and tbpB and the absenc~ of a promoter region in tbpA suggests that ~h 21 64274 the two proteins may be coordinately e~pressed. A Fur consensus sequence in the promoter region of tbpB suggests that the proteins may be regulated in a Fur-like manner. The Fur consensus sequence in P. haemo~tica tbpB is similar to the consensus sequence found in N. gonorrhoeae and N. meningitidis tbpB (Figure 7). The isoelectric point of Tbpl and Tbp2 was calculated by PCGene (Chargpro) to be 9.16 and 9.71 respectively, making them basic proteins.
' m. Pre~ d Protein Topology The sequence analysis program Gene Runner (Hastings Software) was used to analyze the physical ch~dc~eristics and to predict the secondary structure of the P.
hacmo~ca Tbpl and Tbp2 proteins. The hydropallly plot of Tbpl and Tbp2 (Figures 8 and 9) were generated using the method of Kyte and I~oolittle (1982). The positive peaks represent hydrophobic regions whereas hydrophilic areas of the protein appear as negative values. The f~st 28 amino acids of Tbpl form a hydrophobic region, which is characteristic of all signal sequences (Figure &). There are six other hydrophobic regions in the protein, which may be transmembrane domains of the protein.
Hydrophilic areas of the protein may be either exposed at the cell surface or in the periplasm. The hydropathy plot of N. gonorrhoeae was also gen.,~dled (Figure 8b). The N. gonorrhoeae Tbpl protein seems to be less hydrophobic than P. haemo~y~ca Tbpl but the location of some of the hydrophobic regions are similar. For example, both proteins have hydrophobic regions around the 200, 400, and 780 amino acid residues. The similarity in hydrophobic regions suggests that the two proteins share a significant degree 2~ 64274 1-igure 6. Nucleotide sequence of P. haenu~ytica tbpA and tbpB. Putative signal s~quence cleavage sites are indic~ted by an arrow. The start codon (ATG) is underlined. -tbpB

1 CGCTTGCAGA l-l-l~lAAAAA ATTTAGCTAA AATCAGACCT GGCTTGTATT

101 AAGCATCGTT l-l~ GCTA TTTACCGCTT GTGATTGATG TGGAACAGAG
151 GCTTAAATGC~CCAAACTGTG CCTTATTGGA ATTGGCCGGA ATTACTTTAA

351 GCGGTTTAGA GAATATTGCC CAATTCCCGG AAAl~l-l~AA AATGGTTCGC

- 451 AATGCCAGCG TTCGATCAGG G~ l-l-l-lAC AG&GCTGCGA TTAAACGGAA
SO1 T&CCAAGCGA GAGATCCCTT TCACCCTTTI GGCGATGTTT GACTACCGCC
SS1 GCCACCCTTT GCA~-l-l-l-lA TGTTATGGGC AAACC~l~l-l CGTTAAAAGC
601 CTGCCAGTGC AAAAl~l-l CACGTAGCAG CACTATAGGG CGAAl-l~l 751 Al~G~ l TGG~TGATGG TGTCATTTTG CATAAGAAAA ATGATGCGAT
801 TTCAGG-l-~-lA CCCACTGCGG CGATTATTTG GG~ ~-l~CG GGGATCGGTA
851 TTGCT&CAGG GGA~l-l-l~ ~-l~l-l-l~ATG C~l~ATCGC CA~-l~l~ATT
901 Al-~-l-l~Gl~l CTATICGATT Al~l~l-l~ GTTCAACGTT GGGTTCATCG

1051 ATACAAGTCA AAGATCAAAG TAGTGGAGAA GTTGCCGGTT ACAAAl-l~l , -70-~ .

~ 2 1 642 74 1201 l~ lATTT TTTATTTAAT TTCTTTCCAC AAAAGATCAT TTTCAATTAT

1301 GCACAACAAA CTATGGAACA ACAATCAAAA TGTACGCTTA TCGGCTGCC`G
1351 ATGATTTCGA TAATGATCGA TGTGCAGAAA TATTTGAACT TACGAl-l-l-l~

1501 CCCCTTCAAA TTGTATTTAT ATCAGCTACC GTGCCACCAT TCGTA~-l-l-l-l lSSl CGGATCAAGA TTAAAACAGA ATCCCTGCAT GCACAGCGAA ATCAGCTGCG

1651 AGTGCTAGAT l-l~l~AATAA AAAATTAGTG ACCAAGCTTG GGTGCATAAT
1701 GATGGTGATG AAAGAACGCT CAATGCTTGA CACGTTGCAG GCTAlrl~lA
1751 AGGGTATGGT AGTTACAGGC ACAGCCCAAA CGGCCAATTG CTGGl-l-l-l-l-l 1951 GGAAAACGTC AAATAGAAAA ATAATCATAA TTCCCCTTTG CTGGl-l~lAG
2001 ATAGCAGCGG GCAAl-l-l-l-l-l ATAAAAATTT GCAAAATTTA AATAA

~ ` 21 64274 ;bpA
'1 AGACCCTATC TAATGATAAT GAAATATCAT CATTTTCGCT ATTCACCTGT
51 TGC~TTAACA GTGTTATTTG CTCTTTCTCA TTCATACGGT GCTGCG~AC~
101 AAAATAAAAA AATCGAAGAA AATAACGATC TAGC-l~'l'l'Cl GGATGAAGTT
151 ATTGTGACAG AGAGCCATTA TGCTCACGAA CGTCAAAACG AAGTAAC~GG
;201 CTrGGGGAAA GTAGTGAAAA ATTATCACGA AATGAGTAAA AATCAAATTC

351 CCGTGTCAGC TTA~ ~ ATGGGCTACC ACCAGCGCAC AGTTATCATA

451 ACATTCGTTC AATTGAGTTA AGCAAAGGAG CAA~ C GGAATATGGC

551 TATTATTAAA GAGGGGCAGC ATTGGGGCTT ACATAGTAAG ACC~ -lATG

651 GTG~ -lCA AGCAC-l-l~l-l ATTGCAACTC ACCGACACGG TAAAGAGACC
'-701 AAAATTCATT CCGAGGCAAA TCAATTACAT ATTATTCGGC GTATAACCGG

801 AGGAT~-l-l-l-l- TTTAl~ ~ AAGATACTTG CCCAACATTA GATTGTACTC
851 Cl~l~CAAG GGTTAAGTIG AACGCGATAA TTTCCCAGTC AGAACATTTC
901 CGAATATACG CCTGGAAGAG GCGAAACAGC TT&AGATTCC TTATCGCACT

1001 CTTTAGATTA CAAGAGTAAT l~-l~-l-l-l-l-lA TGAAGTTTGG CTATCACTTT
-~

1151 TACTTATCAC TTTGGAACTA l~l-l-lATCAA GGGGATATTA TTTAGATGGC
1201 TTAGl~-l-lCA AGCCAAGGAT CCCTTATGGG TTGCGCATAT GCCATGTGAA

-~ 21 64274 1301 TAATCGCTGG TTGGATAGCA TTAACl~YG-l~ CGTACGTGCT TTGCGCTCTC
1351 GCTGCTGTGC TCTGAGTAAA CAAGATATTG AACTATATAG CCG~GCTACAT

1501 AAAGCATCGT GTCATTCATT TAGAATTTGA TAAAGCGCTA AAT~ GlC
1551 AAGGCGTATT TAAGCAAACC CACAAACTGA ATTTAGGCTT GGGC-l-l-l~AA
1601 TCGATTAATC GCTTATGATC ATG&GGATAT GACTGCCCAA TATACCAAAG
1651 GCCGGTTATA CCAGCTACCG CGGAGAG&GG CTTTAGATAA TCCATATATT

-~ 1751 AGCGGCGACA CTTAACTGTG ACGCGTTAAA TAAAGGCATA C~ lACC
ri 1801 TCCGCTGCAC TTAGGAAC'TA TAGTTTATGA AGGGGATAAT ATTTAGATGG
'~. 1851 CTTAGl~l-lC AAGCAAGCAA GGATCCCTTA TGGGTTGCGC GATATGCCAT
:~, 1901 GTGAAGTTTT TGATGAACGT CACCACAAAC GTC~l-l-lAGG ATTCACACCT

2151 CGAACTGAGC GTAATAATTA CCAAGAAAAG CAl~l~-lCA TTCATTTAGA
2201 ATTTGATAAA GC~CTAAATG CTGGTCAAGG CGTATTTAAG CAAACCCACA

2401 GAAACGGTAT ~l-l-l~l~lAA TAATACACGC GCGACACTAA CTGTGACGCG

2501 GAATAACCGA GATACGGTTC AGl~l-l~l-lC CAACCAGTTG CGATGGCCCA

r - 73 ~
.

~ 2 1 64274 2701 Gl~ l~GTA CCACCCTTGA TATTAACCGG AGTCAATTAT AAAAACGAGT
2751 TACGTGGAGC GCAATTTATA Al~l~ATGT CAGATACTGT AAAACTCTAT
2801 ATTACCGTGG GCAGCAATTA G'GTGACAGGG CCACGGGGCA AGCGAAACCA
2851 GACG&GTACC AATTACACCG ATTTGCCGCC CCCGGGAGAG AAATTTCAGT

2951 GAAAGTATAT TCAGCGCGTT l-l-l~l-l~CTC TAACGGATTA CATACGAATT
' 3001 CAAAATGTTT TAACGGTCGG TTA

~ ' Figure 7. The promoter region of P. haerno~ytica tbpB (PHTBPB).
The putative Fur con~ncll~ sequence is indir~tff3 by asterisks. The Fur consens~ls sequences of N. gonorrhoeae tbpB (NGTBPB) and N. meningitid~s tbpB (NMTBPB) are also indicated.

i--, ~-~ OT- S~- .
_ _ _ ~&Y~N ~Y-Y'''~''Y~D~SYY'''~''Y~LY''~ '''Y_D

~-~ OT- SE- r~
D_Y_~ 'YY~_LY~-'''~''~al~''~ ~ lS~ ~, ~-~ OT- S~-' W;YY~SY_;CD~Y_~S~Ys~ v .~

of homology and may have a similar structure.
The Kyte~ olittle plot of P. haemo~ytica Tbp2 reveals several large hydrophobic regions in the centre of the protein and two smaller hydrophilic regions at each end (Figure 9a). This is significantly different than the hydropathy plot of N. gononhoeae Tbp2 (Figure 9b). This suggests that the two proteins may have different structures.
Surface e~cposed regions of Tbpl and Tbp2 were determined using the Emini surface probability method (Emini et al., 1985) and shown in Figures 10 and 11. The peaks on the graph correspond to the regions with the highest probability of being exposed. Surface e~posed regions of each protein may be involved in ligand binding and may be antigenic. The Emini plot of P. haemo~ytica Tbpl suggests that the hydrophilic regions near amino acids 330, 410, 460, 560, 610 and 820 of the protein may be exposed at the cell surface (Figure lOa). The Emini plot of N. gonorrhoeae Tbpl (Figure lOb) shows a few common exposed regions with P. haemohytica Tbpl (regions at 330, 580, 810).
The Emini plot of P. haemo~ynca Tbp2 suggests that the hydrophilic regions at amino acids 140, 160 and 620 have the greatest probability of being e~posed (Figure 1 la). The Emini plot of N. gonorrhoeae Tbp2 shows that only the e~cposed regions at 160, 330 are similar to the surface regions of P. ha~mo~ca Tbp2 (Figure 1 lb).
The sccond~ ~ structure of both 'rbpl and Tbp2 were predicted using the method of Chou-Fasman (1978) and shown Figures 12 and 13. This method analyzes each protein for regions of sheet or heli~c forme.s and determines the extent of each structure.
Each of the possible secondary structures is presented on a sepa~te line of the graph.

~, If more than one structure is possible at a particular location, the computer will present both possibilities. The Chou-Fasman plot of P. haemo~ynca Tbpl predicts that the protein is primarily a B-sheet and B-turn structure (Figure 12a). The Chou-Fasman plot of N.
gonorrhoeae Tbpl predicts a similar structure (Figure 12b). These predictions are consistent with other topology predictions of other iron-regulated outer membrane proteins, which are also composed of amphipathic B-sheets (Moeck et al., 1994). It is also interesting to note that the location of the B-sheets co,-~spond to the location of the hydrophobic domains in the two Tbpl Kyte-Doolittle graphs (Figure 10).
The Chou-Fasman plot of P. haemo~ynca Tbp2 in~icqt~ that it also consists primarily B-sheets and B-turns (Figure 13a). The predicted pattern of B-sheets is different than N. gonorrhoeac Tbp2, which also has B-sheet regions (Figure 13b). These results add to the evidence that the Tbp2 proteins have different structures.

IV. Distribution of tbpA in P. ho~moly~ca and related species Southern hybridization analysis was carried out to determine whether or not all si~cteen serotypes of P. )u~emobtica carried the gene for the Tbpl protein. Chromosomal DNA from each of the serotypes was digested with r~l. ic~ion e~donucle~ce and probed with the S' end of the tbpA gene from P. haemobtica Al. Similar hybridization e~periments were pe.rol.l,ed on ~igcsted chrom~somql DNA from A. pleuropr~umon ac CM5 and shope 4074 and A. suis 3714.
High-stringency Southern hybridi~lion (50% formamide) with theP. haemobt~ca Al tbpA probe demonstrated the presence of tbpA homologous sequences in ..11 si~cteen igure 8. The Kyte-Doolittle hydtop~ y plot of the P. haemo~yt~ca, N. gonorrhoeae Ibpl proteins.
The X-a~is is the amino acid seque-nce and the degree of hydrophobicity/hydrophilicity is located on the Y-a~is.

s - - 2 1 64274 ~olyel~ pl ~f~P~ c L hydrop~$11c b ) N. go~o~ D ~
hyd,ro~ lc .
-~, ic igure 9. The Kyte-Doolit~e hydropathy plot of P. haemo~yhca and N. gonorrhoeac Tbp2 proteins.
The X-a~is is the arnino acid sequence and the degree of hyd~oyhobicity/hydrophilicity is loeated on ~e Y-asis.

:

J
-,.;
~) t. h~olytic- Tbp~
2~y~1rop~obic 1~ t~

,r ~ L

~ydrophilic , . b) ~. gonorrho~ bp~
: ~y~o~hc;~lc ., .

~'Jl i hydrop~il$c -Figure 10. The Emini sur~ace probability plot of P. haemo~ica and N. gonorrhoeae ~:
Tbpl proteins.
The arnino acid sequence is on the X-a~is and the surface probability values are located on the Y-asis.

F~ 21 64274 ~ P h - ~nol yt l c~

~ --r-~ldu- ~r . S~orrho~
t~
tJ~-t~
~o~ .

r-~du- uu~r igure 11. The Emini surface probability plot of ~e P. haemo~ica and N.
gonorrhoeae Tbp2 proteins.
The amino acid sequence is on ~e X-a~cis and the surface probability values are lo~ted on the Y-asis.

) t. ~ct~ C~ t~

~ ' .
re~ldu- nu~b-r b ~ N. go~o~ ~ D ~ ~ T~t2 .. .

~
~ ''' .
: ,, ~t.
_ . ". . , ~

re-ldu- nu~r ~ 1 Figure 12. The Chou-Fasman plot of P. haemo~nca and N. gonorrhoeae lbpl proteins.
The amino acid sequence is on the X-a~cis. Each line on the Y-asis r~pl~sen~ a possible se~ondary structure.

~ 2~ 64274 ~) p. J~ nolytic- Tbpl ';~
' ;

H~
__ _ r--ldu- ' ~r b ) N goDorrho-~c ~ .

r~ldu- ' ~r igure 13. The Chou-Fasman plot of P. haemo~y~ica and N. gonorrhoeae Tbp2 proteins.
The amino acid sequence is on the X-a~is. Each line on the Y-asis re~)resen~ a possible secondary structure.

) P. h-~olytic- T~2 .
.
.

r--ldu- ~r b) N. goslorrbo~ 2 r~ldu~

serotypes of P. haemo~y~ica (Figures 14,15). In addition, there is a considerable difference in the size of fragments which hybridized with the probe between the A and T biotypes. It is important to note that in Figure 14, there was a problem with the quality of the serotype 7 DNA, which did not give a reactive band with Southern hybridization. The reaction of this serotype should be identical to that of serotype 1.
A similar problem is seen in Figure 15, where serotypes 12 and 16 DNA was not properly digested. The reaction of these serotype should be identical to that of serotype 1.
Low-stringency Southern hybridization (25% formamide) with the tbpA probe in~icate~ that A. suis 3714, A. pleuropneurnoniae CM5 and Shope 4074 genomic DNA
which hybridized with the P. haemo~ytica tbpA probe (Figure 16). The two strains of ,t. pleuropneumoniae both belong to serotype 1 and hybridized in the sarne fashion. A
preliminary restriction map of the tbpA, tbpB regions in P. haerno~ica A1, A. suis and . pkuropr~umoniae is shown in Figure 17.

V. Homology Studies The predicted amino acid sequence of P. haerno~ynca lbpl was compared with the predicted sequences for the Neisseria spp. and ~. pleuropneumoniae transferrin binding proteins as well as for several E. coli TonB~c4er~kn~ r~tor proteins. All of the comparisons were performed according to the Higgins and Sharp algorithm (Higgins and Sharp, 1988).
The predicted amino acid sequence of P. haemo~ynca lbpl was found to have a Figure 14. Southern hybridi7~ion of P. haemofytica genomic DNA digested with ClaI and probed with the tbpA gene. Lanes 1-16 represent P. )taerno~ytica serotypes 1 to 16. I~ne M represer.ts Iambda DNA digested with Hind Ill and hybridized with lambda DNA radiolabelled separately as size ,arl~e.~.

, 21 64274 ~1 .

-0 r 0 o~ ' '3 1 kb ~
9 .4 kb ~ ~ ~

' . 1 )cb --igure 15. Southern hybridization of P. haemo~tica genomic DNA digested with HindlII and BamHI and probed with the tbpA gene. Lanes 1-16 represent P. haerno~tica serotypes 1-16. The molecular sizes are as in~i~ted on the left.

~ 216~274 3 o 6il~
4.4 kb ~
2 3 ~

Figure 16. Southern hybridization of A. suis 3714, A. pkuropneumoniae CM5 and shope 4074 genomic DNA digested with various r~lliCliOn endonucleases and probed with P. hacmolynca tbpA. Lane M represents lambda DNA
digested with Hind III and hybridized with lambda DNA radiolabelled separately as size markers.

A - A. suis 3714 B - A. pleuropneumoniae CM5 1)CI4I/AvaI 2)ClaI/PstI 3)ClaIlHindlII
4)ClaI/BarnHI S)ClaI/Xbal 6)ClaI/EcoRl 7)ClaI/EcoRV

C - P. haerru~ica A1 1)ClaIMvaI 2)ClaIlPstI 3)ClaI/HindIII
4)ClaI/BarnHI 5)ClaI/XbaI 6)ClaI
7)ClaI/PvuII 8)ClaI/HincII

2 ~ 64274 .~

f a b c ~igure 17. Restriction maps of the tbpA, tbpB regions in P. ~2aemo~ytica Al, A.
plcuropncumon~ac CM5, Shope 4074 and A. suis 3714.
Line 1 r~rese.~b the tbpA probe used in Figure 14.
Line 2 represents the tbpA probe used in Figures 15 and 16.
Abbreviations:
C - Clal E - EcoRl H - HindlII P - PstI

- 98- :

~ 2164274 o W ~.

g o ~
-m O
. ;, o --~ . ~
-~:r ~ ~h degree of homology with both thc N. gonorrhoeae and N. meningitidis Tbpl proteins (Cornelissen ct al., 1992; Legrain et al., 1993) (Figure 18). The homology, including identic~l and conserved amino acids, was found to be 41 X. This result agrees with the protein topology studies which suggested that the P. haerno~ytica and Neisseria spp. Tbpl proteins share a similar structure. A homology comparison between P.
haemo~ytica Tbpl and ~. pleuropneumoniae serotype 7 and serotype 1 TfbA proteins (Gerlach et al., 1992a; Gerlach et al., 1992b) reveals only a low degree (22%) of homology (Figure 19). The degree of genetic relatedness among the Paste~rella, Neisseria, and Aaiw'oacillus transferrin binding proteins is shown in the form of a dendrograrn in Figure 20. It is intc~estillg to note that P. haernohynca Tbpl is more closely related to NeisseAa Tbpl than to ~c~inobncill~s TfbA, which is also a member of the family Pasteurellaceae.
P. haerno~tica Tbpl also has loc~ Pd regions of homology with E. coli TonB
dependent outer membrane receptors (Figure 21). Homology with these proteins implies that P. haernotyhca Tbpl is also a TonB dependent receptor protein. The first homologous domain includes the TonB bo~, which has been implicated in the direct interaction between TonB and the receptor protein (Bell et al., 199~). The significanoe of the other homologous domains is not known, however, it is possible that they are also involved in TonB interaction.

VL T7 e~ ss;c.. of Tbpl 1-7 e~pression was pelrO.I.led in order to express the protein encoded by tbpA

2~ 64274 Figure 18. Alignment of the amino acid of Tbpl of P. haemo~ynca Al (PHTBP) and the Tbpl of N. gonor~hoeae (NGTBP1) and N. meningi~dis (NMl). The numbers to the right indic~ amino acid positions. Asterisks indic~te positions of complete identity in ~lignmpnt~ dots indicate similar amino a~id residues. Gaps were introduced to m~simi7~ sequence ~ nmçl~t and are indic~tPd by dashes (-).

~-PHT~P MIMXYHHPRYSTVALrYLPALSHSY~AAT8NXXIB~YYD!~VLDBVIVSB SO
NGT8Pl MCQQ-HLFRLNILcLsLMTALp-AyA~NvQAGQAQ8xQ----LDrtQv~A
NMl MOQQ-HLE~RLNrLCLStM~ALP-WAl~Nv9ABQAQBl~Q----~IQv~
. -- ... ... ~--. ...... . .-.... -..
PHT~p SHYAFIRDQm~LCKW~ ~lSl~QrL~rRDL'rRYDPGIS~ s~kG 100 NGTBPl KKQ~TRRDNBNTGLC~LVXTADSLSKBQVLDIRDLTRYDPGIAVV~G 94 NMl KKQlCl~RDNB~LGltLVXSSDl%SKBQVI,NIRDLTRYgPGIAV~KG 94 . -.--.-.. .. -- -- -- ----.------ --------NG~BPl ASSGYSIRGMDKNRVSLTVDGLAQIQSYTAQAALGGTRTAGSSGAINBI8 14~
NMl ASSGYSIRGMDKNRVSL~ W ~SQIQSYSAQAALGGSRTAGSSGAIN8I8 144 P} m P YBNIRSI8LSKGASSABYGSG ~ GGArGPRTXDAQD~IIu ~ ~GLDSRT 194 NGIBPl YBNVXAV~ISKGSNSVBQGSGALAGSVAPQTKTADDVIGBGRQWGIQSXT 194 NMl YBNVKAVBIS~GSNSSBYGNGALAGSVAPQrKTAADIIG3GKQWGIQSKS 194 --....-.---,.- .~- .-...-.--.t.-.- - - t-PHTLP SYAS~NSH~LQ-IAAAGBAGGPBALVIATY~G~ IHSBA~nrUNIR 243 NGTBPl AYSGKNRGLTQSIALAGRIGGAEALLIRTGRXAGBIRAH3AAGRGVQSPN 244 NMl AYSGKDHALTQSLALAGRSGGABALLIYIXRRGRBI~AH~DAGKGVQSPN 24q -- - ---.- - ~.. -.. ..-.. ....
PHTBP RITGPBNRYDPTQIPERMPPGGS---PPIv~v ~-Y~DCTP~ARV ~ 290 NGTD~Pl R~APVDD-------~------GSXYA~rlv~ UR-KCKANP~D 279 NMl RLVt-nRn~----------GGS~K~v~ ~-X~ t.Y~n 282 PHTBP NYPV~-~rlS~ B~ABQIP~K~SQr.S~QIC~G~ r~0NPLD~A~ V- 3~0 NGTBPl W --------GBDKRQT---------VSSRD~-G~hKPLADPLSYBSRSW 312 NMl AS--------VKDB~AT---------VS~QD~GSNRLL~NPLBf~SQS~ 315 ..-.. .~... ~- .-. ..--.- -.-PXTBP Ph~G~ SS-HYLGAI~v~AQK-T$SV$CKRQ~$Q~rILTYHIGTM 38 NGTBPl LPRPGPRPBNXRHYIGGIL~K~Q~r~KDMrVPAPLT~AVFn~ ~rQA~ 362 NMl LPRPGWH$DN-R~YVGAV~BRTQ~ K~.~v~A~Sk~vP-----G 359 PHTBP. P~KGIIrKWISVQAA_DPLWVAHMPCB~___________DRDU~U~PPtr~ ~26 NGTBP1 StDGNr~U~YAGNHKYGGLPSSGENN~PVGA-~G~V~IUBTKT~SRYGL ~12 NM1 SLRGLG__KYSGDNKA_RLPVQG~GSTLQGI~G~GV~DBDAHSKNRYGV 407 PHTBP Tnt-y-~kh~DsINsc~ r-ocro~ s~cQl~I8Lys~oT~T~DT~ucsDyp ~75 NG~Pl 8YVYT~KD5WAD----YARL~ -S~vRQGIGr~.~ OSA-D ~51 NM1 ~V~NADKDTWAD----YARL------S~URQG~DT~nN~T~X~ S8-D ~6 .-- ' .... ~ .......... . .. . .-- .... '--PHTBP W DKNOGPTT-nrSWS~K~K~.t~-~KVrHL8PVLALNb5QGVPLQrH 525 NGSBP1 GSDXYCRPSAb~PPS~ URV1.~SY~T4~A~S~V.AX----I~H ~97 NM1 GSDXN CKP~h~tS~SDR~1~KKSR~-T~AVP~XAnDTA~----IKH ~92 PHTBP ~T Nn~T~PBSINS~ ~GL~.~AQYTLGRLYQL-__PRRDPKSIWTVSLCN 572 NGTBP1 NLSVNLGYDRFGS~-~QDYYY~SAN_RAYSLKTPPQM~AA1SP~K~A 546 NM1 NLSINLGYDRPASQLSHSDYYLQNAV_QAYDLITPA----A-PPFY~SA;U 537 .~...-~.... ' . - ' ' . . - ~ . .. .

NGT8Pl NPYWVSIGRGN W TRQICLPGN~l~lU~lPRSINGKSYYAAV--K~DNVRL 594 NMl NPYRVSIGKTTVNTSPICRFGNNIYl~Cl~RNIGGNGYYAAV--QDNVRL 585 .... .. . . . .. .. . ..
PH1~3P HRTWTPTSLGBLPSI~AMAYY~NHHPNQVpWGRGAVRXLT------LLSS 659 NGTBPl GR-wADvGAG----LRy--DyRsTHsDDGsvsl~ lLswNAGIvLRpA 637 NMl GR-WADVGAG----IRy--DyRsTHs~DKsvsTGTxRNLswNAGvvLKpp 628 . . . . . .
PHTBP PWM-LKPAASG--RHVTLSVISG-ATDRpLYppLILTGVNYRN~S---Yv 702 NGT8Pl DwLDLT-yKlsl~KLpspA~MyGwRsGDRlKAvxIDpBRspN~BAGIvpR 687 NMl TWMDLTYRAsTGPRLPsPAEMYGwRAGBsLKTLDLRPBRsPNREAGIVP~ 678 t t .--. ... . .. . . . .. ....--- -PH1~3P SAIyNvDvRycxrLy-------yRGQQLGDRATGQARpDGy---QLxRFA 742 NGTBPl GDFGNLBAswpNNAyRDLIvRGyBAQI~ Q~GNp-AyLNAQsARIT 736 NMl GDPGNLEASYFN~AYRDLIAr~Y~lKlylr~QTSASGDP-GYRNAQNARIA 727 ... ..... .. .. .... .. .
PHTB~ APG-~ --------------RNPSYHSKKFRPAR---~NTKNAESIPS 770 NGT8Pl GINILGRIDWNGVWDKLPBGWYSTBAYNRVRVRDIKXRADRTDIQSHLFD 786 NMl GINILGRIDWHGVWGGLPDGLYSTLAYNRI~VRCADIRADRTP~TSYLPD 777 .... .. . . . .. .
PHTBP A-----PPVGSNGLHTNSRSCPNr-PT-U~PIPYPPNPLRNVPRPNBYHCCC 815 NGT8Pl AIQPSRYVVGSGYDQPBGRWGVNGMLT------YSRA~BITBLLGSR--- 827 NMl AVQPSRYVLGLGYDHPDGIWGINTMPT------YSRARSVDBLLGSQ--- 818 --.... ... .. ...

; NGT8Pl -ALLNGNSRNTXATAR~TRphyI~vVSG~v~Hr~RAGvy~TT.T.uU~y 876 NMl -ALLNGNANu?~ou5RRTRp~y~v~sGr~NI~ RAGvyNLLNyRy 867 .... . . . . .............. . . . .
PHT8P --RBVVYLTCCACA~Nl~-~ W P----CVGCCSNILABMKP 898 NGT8P1 VTWENVRQTAAGAVNQHKNVGVYNRYAA~ rSLEMRP 917 NM1 V~WENVRQTAGGAVNQHXNVGVYNRYAA~K~Y~PSLEMKP 908 t . . . ' -Figure 19. Alignment between P. haemolynca A1 Tbpl (PHTBP1) and the A. ~.
pleuropneumoniae serotype 1 and 7 TfbA proteins (APL, APL7~.
Asterisks in lic~te positions of complete identity in ~ nmPnt dots indicate --similar amino acid residues. Gaps were introduced to ma~imize sequence nme-1t and arc jr~ir~ted by dashes (-). -:-21 642~4 PHTBPl MI~ ~nn~ ~LSVLPALSHSYGAASEN~r~ENNDLAVt~ SO
APL ~HP~LNPY---AL~PTSLPLVACSrJr.rGSFD~ ----L~DVRPNQ 36 APL7 ~HP~LNPY~ LAFSSLPLVACSW ~GSFD-----------L~DVR~NX 3 P~I~Pl SHYAH8R9Nlm'~;LGm~ usMSI~- -NQrL~;- - Iil.'DL~YDP~;rSW~ 9C
APL ~A~A~ S~QD~ LD~LMBPALGYVTOrLRRN~APlrBra SC
APL~ rrGNs~-8--yxDv8TA~ RQ~r~t~BpALGyvv~v------pvsspB ~
.. . .... .. -P~TaPl QGRGAssGyArR~v~hKvsLLvDGLp~AHsyHTts~nuyGr-~T~R-r--y l~S
APL B~RNBR---- wBLsBDxI~LyQBsvBrrp8--rnBr~ sn~vIns 130 APL7 NK~--------VDISD----------I8VIlNGNLDDVpY~ANSS~-YNY 109 .... . . .. .. ..
PHTBPl RNIRSI8LS~SS~R~GSGW~3GAIG~rAJA0-Drr~BG--------- 185 APL HDsrDrnr~DnL~yvRs~v~Lbstn~IRRNcsGr~vraQ~IuG~v~a 180 APL1 PDIa--r~DSS~4YVRS~vlu~`--S~Sr--------------C~v~a~ l~S
... . . .
PRT~Pl ----QH~GLDSrTSYAS~Sn~O~A~9~GGPBALV~ATXRN------ 225 APL Vrps~BLpaG~vIs~a~Jrvs~lh~gRBIDGnv~s~v~hvsATsI~B 230 APLt NSPA~BLPV~QLLTYSGSWDPSSNANlNNB------BGRPNYlNDDYYTa 189 .. . . .. . . . . .
PHTBP~ --GaKl~I-NS-9~UlT ~-IRR$TGPENRYDPTOIP ~PoGSPPIVED 272 APL ~V~K~aV~ K va~vANSSBPA~DPDN~lLTGSLYRNG--YINRN 2t8 APLt PIG~--RVGLVSGDA~PA~a~SQ ~v~T~TG~L------~~~~D 228 .. .. .. . . .. .... .. .
PRTBPl TCPILDCTPRaR~LNRDN~CrPr~a--LSAQ 320 APL AAQ~AKYS~BADLAGNRPRa------r~GnrIFr-DSNYLeOG 321 APL7 ~B~TI---YIVNADIAGNRFTr'~T~C~rr~ S~ c~SQ~e!BGO 275 .. ... .. -- . ....... .. . .
PXT~Pl ~r~A~R~APNPLL~S~Sv~kr~rn~hS5~r~r~rt~T~QRTISVIC 370 APL PYGPA-A88MAG~8PnKN~SLPA~PAa~S8h~ Irn'T~ TQPN 371 APL7 PyGpA-AsBMAG~FyAND~sLpavpsa~x~G~R~ rn~sr~ s 32S
. .. . ...--.-- . . .... . . ... ... .
PH~Pl A-RQLTI--Q-rILsyHl~nnsrq~3lIpR~Lsvc~DpL~vA~pcp~DER ~lS
APL A~BLNNPGr~SVL~DDGQ~IDLAGvn~AhS~-~DNG~n~VAVACC~U~ ~21 APL7 ISBLN ~ SVL~ r~ S~nAh--~L~A--nu~v~cr~u~P 375 .. . . ..--. . .. . . . . .. ..-- .
PNTBPl UUlr~DD~T~ fhrr~PsNNR~LDSINSCVP~J--erOr~ S~QDIBLY--~ 63 APL YM~----PGQ~P~ V~u~SLPLQG8RSATD~MPAGG~ v~ 67 APL7 YM~----P~QL.~A~5~,r~BMNSLP~4r~TAT~ ~d~ lCT~D ~ 21 .. .. .. . ... .
PHTBPl ---SD~ 5~rvv~NCGPl~ NNYQB~ATCHSPCI 510 aPL aLVS~GTNW~A3U~ 5~-k-~ S~VNG~ AG~rv~-~ 517 A~PL7 AQ~s~BNN~rATA3DDK~A~ ~v--~cur~n-o~ u~br~ 71 . . .... . .. ... ... . . . .. .. . .
PHTBPl ~r~ ~,v~Y~ U~tJ::PBSl~~-TIIGIILPNIP~ à~KC 560 ~ ~PL DATIN-GNGPIGSA~SDSG~n'GSSO~VPSDl~vbOG~CP--T 56~
APL7 DA1CID-GNGPTG~- ~v~SGL~ ~S~SSC~ ~NDVA'v~r~Gr--T 515 .. .-- .... . ...... . . . . . ..... .
PHTBPl RGRLDUP~l~KkDPRSIBTVSLCNNTRATLLLLRYNXGIRt-~-D 60~
APL AGRLr~GQ~ VGAv------------Fc~rDQTR---~ 593 APL7 A~Rt~GQPH~S_NGSVG~V------------P~AXQQ~ 5~7 .... .... ... . ... .

Figure 20. Dendrogram illustrating the genetic re!~tedn~ss among P. haerno~ynca lbpl (PHTBP), N. gonorrhoeae ~bpl (NGTBP1), N. rneningitidis Tbpl (NMl) and the TfloA proteins from A. plel~ropneumoniae serotype 1 and 7 (APLl, APL7).

.

~ 2164274 I

-nn~P
.

.. --1~6TIIPl , N111 `

. ~L

~PL7 ~0 30 40 50 60 7l 8l 90 1~0 Percent S~lar~ ty Figure 21. Peptide alignment between P. hacmo~tica Al Tbpl and TonB-depen,dent outer membrane receptors of E. coli. Asterisks show arnino acids with complete identity in alignrnent, dots indicate similar amino acid residues.
Gaps were introduced t~ ma~imize sequence alignrnent and are in-ii~t~d by dashes (-).
PHTBP1- P. haemo~tica Ibpl CIRP - E. coli colicin I re~ptor FEPAP - E. coli ferric enterobactin receptor FHUAP - E. coli coprogen and rhodc!cl-ric acid r~ce?tor FECAP - E. coli ferric citrate receptor 2 ~ 64274 1 TonB box PHTBPl DEVIVTE 22 . FBCAP FTLSVDA 30 ~ . . ~.

t, 2 PHTBPl YAIRGVD---KNRVSLLVDG 121 t ~
.~
-.~
. 3 PHTBPl IBLSKGASSABYGSGAHGGAIGFRTRD 177 CIRP IB W RGPMSSLYGSDALGGVv~IITRK 159 FBPAP IBVLRGPARARYGNGAAGGVVNIITK~ 180 ;~, FHUAP ABIMRGPVSVLYGRSSPGGLLNMVSKR 188 F8CAP IDVVRGGGAVRYGPQSVGGvv~PVTRA 251 ~ ~t . ~ . . . -PHTBPl FRQTHKLNLGLGF 533 FEPAP ABTSINK~IGLBF 447 FBCAP PSKGKQY~v~v~ 560 FBCAP PB~ARTWBLGTRY 5 7 8 . . ~

21 ~27~

(Figure 22). An attempt to express tbpA by maxi~ell analysis of E. coli under iron-depleted and iron-repleted conditions was not successful (data not shown). The 17 e~pression did not produce any reactive band at 100 kDa. A 30 kDa positive control is shown in lane 1. There was no difference between the plasmid carrying tbpA and the pBluescript vector alone (Figure 22, lanes 2 and 3).

VII. Westem Immunoblot Analysis Inner and outer membrane fractions from P. haemo~ytica A1 and E. coli HB101 cells grown under iron-limiting and iron-sufficient conditions, were prepared and analyzed by Western immunoblotting. llle purpose of these e~periments was to deterrnine whether or not the iron-regulated proteins would react antigenically with antiserum prepared against the soluble antigens of ,D. hllemolynca. The inner and outer membrane fractions were immunoblotted with rabbit ~anti-autologous~ antiserum which was raise~ against the soluble antigens of P. haemo~y~ica A1 cultured in RPMI 1640 that had been supplemented with the rabbit's own serum (to avoid inclusion of antibodies to serum proteins). The antiserum was preabsorbed with E. coli HB101 cells in order to minimi7e reactivity with E. coU antigerls. Immunoreactive bands corresponding to the transferrin binding proteins were not observed in the outer membrane fraction from P.
h~emo~ytica Al cells grown under iron-limitinP conditions (Figure 23, lane 3).
The inner and outer membrane fractions were also immunoblotted with the serum from a calf vaccinated with Blesponse as a first ~ntibody (Figure 24). The an~i~u~ was preabsorbed with E. coli HB101 cells to limit the number of E. coli immunoreactive 2 ~ 64274 bands. Bands of 71, 77 and 100 kDa were observed in the outer membranes of P.
haem~b~tica cells which were grown under iron-limiting conditions (Figure 24, lane 3).
These protein bands correspond to the size of the P. haemo~ytica the transferrin binding proteins. If these antigenic bands are the transferrin-binding proteins then this result suggests that these peptides are antigenic and also immunogenic in cattle.

2~ 64274 igllre 22. 17 analysis of the P. hacrno~ytica Tbpl protein. The molecular weight markers in IcDa are as indic~ted on the left.
Lane 1 - positive control recombinant plasmid I~ne 2 - the recombirsnt plasmid cont~inin~ tbpA
Lane 3 - the vector plasmid pBluescript (SK) ~ 21 64274 ~' ,.~

~- 1 2 3 2 0 0 IcD -- _ 6 8 lcDa .~. ~a 43 kDa 2 9 IcDa ~ ~_ ~. ~' ~ - 113-~' ~1 64274 Figure 23. Western immunoblot of inner and outer membranes from P. )~emo~ca Al and E. coli HBlOl. The first antibody was a rabbit antiserum raised to the soluble antigens of P. haemo~tica Al and the second antibody was goat anti-rabbit alkaline phosphatase conjugate. Lane M represents the mole~ weight markers in kDa. Lanes 1-4 represent outer membrane fractions and lanes 5-8 are inner membrane fractions. 6 ~g of protein was added to each lane.

Lanes 1 and S - E. coli proteins from cells grown in LT
Lanes 2 and 6 - proteins from cells grown in BHIB
Lanes 3 and 7 - proteins from cells grown in BHIB plus plus 100 ~M EDDA
Lanes 4 and 8 - proteins from cells grown in BHIB plus plus 100 ~M EDDA with 1 mM FeS~4 added 200 ItD~ ~

97 lcD~ -~3 IcDa --~9ICD
.~

Figure 24. Western immunoblot of inner and outer membranes from P. haemo~tica Al and E. coli HBlOl using sera raised in calves to soluble antigens by vaccination with Presponse~. The se~ond antibody was goat anti-bovine alkaline phosphatase conjugate. Lane M represents the molecular weight markers in kDa Lanes 14 are outer membrane fractions and lanes 5-8 are inner membrane fractions. 6 ~g of protein was added to each lane.

Lanes l and 5 - E. cok proteins from cells grown in LT
Lanes 2 and 6 - proteins from cells grown in BHIB
Lanes 3 and 7 - proteins from cells grown in BHIB plus 100 ~M EDDA
Lanes 4 and 8 - proteins from cells grown in BHIB plus 100 ~M EDDA
with l mM FeSO, added 2~ 64274 2 0 0 ~cDa 9 7 lcDa ~
6 8 )~Da ~j~

__ _ 2 9 ~cDa ~ __ _ ~164274 I. Preliminary Sequence analysis Upon preliminary sequence analysis of the cloned DNA, the two tbp genes were found in tandem with tbpB directly upstream of tbpA. This genetic organization is consistent with iron uptake systems in other bacteria such as Neisseria spp. where the genes where are arranged in an operon (Anderson at al., 1994). It is likely that the genes involved in P. haemolytica A1 iron uptake are also arranged in an operon. The tbpA gene has only a ribosomal binding sequence whereas the tbpB gene is preceded by a ribosomal binding site and has a Fur consensus sequence in its promoter region.
The presence of a putative Fur consens.ls sequenoe implies that the two genes could be coordinately regulated by iron concentrations and that a Fur homolog exists in P. hac~wlynca A1. Fur homologs have been cloned and se~uenced in pathogenic Neissena spp. (Berish a al., 1993; Thomas and Sparling, 1994). If a P. haerno~ytica A1 Fur homolog e~ists, it may be involved in the regulation of other antigens such as the Ieukoto~in. Strathdee and Lo (1989) repo- Led that under iron-limiting conditions, there was a de~.ease in the amount of leukoto~in produced. This is the opposite of the situation in the diphtheria to~in (Boyd ct al., 1990) where toxin production incteases when the cells are grown under iron-limiting conditions. It is possible that Fur acts as a positive regulator in P. haemo~y~ica leukoto~cin production. This would be consistent with the earlier observation by Gentry et al. (1986) of inc~eascd to~in production in iron cont~inin~ media. N. meningitidis also produces iron-regulated proteins which are related ~ 21 6427~
.
to the RTX family of exoproteins (Thompson ct al., 1993).
The first 28 predicted amino acids in the tbpA sequence form a putative signal sequence. A signal sequence is essential for inserting the precursor protein into the membrane during the process of translocation across the membrane. The signal sequence also acts as a recognition site for the proteolytic cleavage of the precursor protein into 'A its mature form (von Heijne, 1983; Benson and Silhavy, 1983). The presence of a signal sequence confirms that rbpl is located beyond the cytoplasmic membrane, but it does not contain any sorting information. The predicted amino acid sequence of lbpl has a terminal phenylalanine residue at the carboxyl-terminal of the protein. Phenyl~l~nin~ is a hydrophobic aromatic amino acid which facilitates the partitioning of the hydrophobic environment in the membran~. The presence of a terminal phenyl~l~nin<~- residue has been shown to be important for outer membrane localization (Struvé a al., 1991) and suggests that P. haemo~ca Ibpl is located in the outer membrane.
Sequence analysis of tbpB revealed a putative cleavage sequence for lipoproteins.
Lipoproteins have a charac~,islic cleavage sequence of Leu-X-Y-Cys, where X and Y
are small neutral amino acids (Wu, 1987). This suggests that Ibp2 is processed and lipid modified. I~ is interesting to note that Tbp2 lacks a terrninal phenyl~l~nine residue which is involved in outer l,.el"br~nc localization. Analogous transferrin binding lipoproteins have been found in H. infl~enzae, N. gonorrohoeae and N. rneningiridfs (Legrain et al., 1993; Anderson et al., 1994) Griffiths et al. (1993) has demonstrated common antigenic domains among the Ibp2 proteins in N. gonorrohoeae, N. meningitidis and N.
influenzae type b.

-Thc isoelectric point (pl) of a protein is defined as the pH at which the peptide has a net charge of zero. The pI calculation assumes that there are no three-dimensional structures which i"le~f~.e with the ionization states. Therefore, calculated pl values are only approximate values and may differ from experimental results. The pl of P.
haerno~ytica Tbpl and lbp2 has been calculated to be 9.16 and 9.71, respectively. It has been suggested that cationic polypeptides enhance in vivo membrane interactions.
It is possible that the basic nature of Tbpl e~h~nces interaction with the host transferrin proteins. The Neisserial Tbpl (Cornelissen et al., 1992) and ~bp (Berish et al., 1990) proteins are also basic proteins. In Legionella pneumophilia, basic surface proteins act to inhibit phagolysosomal fusion (Cianociotto ct al., 1989).

'f II. Preliminary Predicted protein topology The hydropathy plots of lbpl and Tbp2 were ger~.ated using the method of Kyte and Doolittle (1982). In this method, a sliding window of 20 amino acid residues is run along the protein. For each arnino acid, the water vapour free energy of transfer is calculated and then adjusted for the preference of the arnino acid for internal versus e~posed enviromnents. These values are plotted on a graph, with hydrophobic amino acids as positive values and hydrophilic values as negative. The Kyte-Doolittle method has been found to be reasonably accurate in preAicti~ hydrophobic membrane-spanning regions in proteins (Fasman and Gilbert, 1990; Jahnig, 1990). It is important to note that the Kyte-Doolittle method predicts hydrophobic sequences but it cannot diffe,el.liate between ~-helices and B-strands (Fasman and Gilbert, 1990).

In the hydropathy plot of P. haemo~tica Tbpl, the first peak is located from the amino acids 1 to 30. This represents the hydrophobic core which is common to all signal sequences (Hayashi and Wu, 1990). The other hydrophobic regions may be transmembrane domains. Hydrophilic domains may be regions of the protein which are e~p~sed to either the cell surface or to the periplasm. The location of the transmembrane regions in the P. haemo~ca Tbpl protein is similar to many transmembrane regions predicted in N. gonorrhoeac Ibpl. This suggests that the Tbpl proteins may have a similar structure and that they share a certain degree of homology at the amino acid level. The P. haemo~`ca Tbp2 protein possess a hydrophobic leader sequence as well as several large hydrophobic regions in the centre of the protein and is significantly different than the hydropathy plot predicted for N. gonorrhoeac Tbp2. This implies that both proteins are structurally different.
Surface e~posed regions of Tbpl and Tbp2 were predicted using the Emini surface probability method. This method utilizes the surface probability values calculated by Janin et al. (1978). A window of si~ amino acid residues is run along the protein.
For each window, the surface probability values for each amino acid are multiplied together and then multiplied by a constant. The value for each window is then plotted on a graph with the peaks co,les~)onding to regions of the protein which have the highest probability of being e,~ The Emini surfa~e probability m.othod gives results which are comparable to the method of Hopp and Woods (Emini et al., 1985). Regions of Tbpl and lbp2 which are e~posed to the cell surface may be involved in ligand interaction and may be antigenic.

The Chou-Fasman method is commonly used in predicting the secondary structure of a protein. This method is based on the tendency each amino acid has for being in an -heli~, a B-sheet or a B-turn. These tendencies are found from a set of 29 proteins with known secondary structures (Chou and Fasman, 1978). The accuracy of the prediction will depend on the similarity between the protein to be studied and the original proteins of Chou and Fasman. This method has been found to be appro~imately 60% correct for all residues (Fasman and Gilbert, 1990).
The Chou-Fasman method predicts that P. haemo~ynca Tbpl consists of many ,B-sheets and ,B-turns. It is possible that these B-sheets cross the outer membrane repeatedly and that the intervening sequences constitute surface or periplasm e~posed loops. The Chou-Fasman method also predicts that N. gonorrhoeae Tbpl also consists of B-sheets.
This structure has already been proposed for E. coli outer membrane proteins such as FepA (Moeck ct al., 1994). P. haemo~ynca and N. gonorrhoeac Tbpl proteins share a common structure which implies that they may also have a similar meclt~nicm of removing iron from the host trarufel.in molecule. The Chou-Fasman plot of P.
haemo~ca Tbp2 also predicts a predominantly B-sheet and B-turn structure which is significantly different than the prediction for N. gonorrhoeac Tbp2.

m. Distribution of tbpA in P. hfremQly~ca and Related Species Southern hybridi;~tion of the genomic DNA of the si~cteen P. haemobnca serotypes with the ~bpA probe demorLs~,a~d that a highly homologous gene is present within the A biotype. The results also suggest that the genetic organi7~tion of the tbpA

21 ~42~
gene is significantly different in the T biotypes. This supports the observations by Murray et al.(1992) who demonstrated that iron-regulated proteins from the A and T
biotypes were antigenically dictinct. Previous work on P. haemo~tica antigenic determinants demonstrated that the sialoglycoprotease, the serotype specific antigen, three lipoproteins and a LPS biosynthetic gene were either missing or had a different genetic organi7~tion in the T biotype (Burrows, PhD thesis, 1993). The A and T biotype do share phenotypic and biochemical traits (Holt, 1977), but they are only distantly related based on DNA:DNA hybridization (Bingham et al., 1990). Sneath and Stevens (1990) proposed that the biotype T serotypes be renamed as the species P. trehalosi.
Diversity in the genetic org~ni7~tiQn of trancferrin binding proteins has also been demonstrated in A. pkuropncumoniac TfbA (Gonzalez ct al., 1990; Gerlach et al., lg92b) and N. rneningitidis Tbp2 (Legrain ct al., 1993; Rokbi, 1993). In A.
pleuropm emoniae, serotype 1 and serotype 7 TfbA proteins share only 55 % homology at the amino acid level (Gerlach et al., 1g92b). N. meningindis Tbp2 proteins are divided into two classes based on their molecular weight, sequence similarity and antigenic heterogeneity (Robki et al., 1993). The diversity in transfe, l hl binding proteins within a species may facilitate the binding of different epitopes of the transferrin molecule. Alternatively, variation may allow the different serotypes to avoid the host immune response against heterologous strains (Gerlach et al., 1992b).
Southern hybridization e~periments demonsl,atc~ that chromosomal DNA from A. pleuropneumoniac strains CMS and Shope 4074 hybridized with the tbpA probe only under low-stringency conditions. This suggests that P. haemob~ica and A.

plcuropneurn~niae transferrin binding proteins share only a low de~ree of homology.
This result was confimed by homology studies on the amino acid sequence from both P.
haerno~ynca Tbpl and ~. pler~ropneumoniac TfbA proteins.
The A. suis genomic. DNA also hybridiæd ~ith the tbpA probe, which suggests that it may have an analogous transferrin binding protein. The results also suggest that . suis transferrin binding proteins may be more closely related to the Tbpl in P.
haemo~ytica than to A. pleuropneurnoniae TfbA. The iron uptake system of A. suis has not yet been characterized and further e~cperiments must be carried out to prove that a transferrin binding protein homolog exists.

IV, Homology StudUes All of the protein sequences were aligned by PCGene (Clustal), which compares sequences according to the method of Higgins and Sharp (1988). The first step in this n~etho~ is to calculate all pairwise sequences similarities. A dendrogram is then generated from the similarity matri~ generated in the first step. l~e final calculation involves a multiple alignment of the sequences in a pairwise manner, following the order of clustering in the dendrogram created in the second step. The Higgins and Sharp method has been shown to be a fast and sensitive method for multiple sequence alignrnents by computer. One drawback to this method is that only a single optimal alignment is generated, which in some cases may not be the most ~biologically significant~ one (Higgins and Sharp, 1989). The dendrog~rn in Figure 20 was generated by a Higgins and Sharp alignment of the PasteureUa, Ac~inobaciUus, and Neisseria transferrin binding proteins.

a) Ncisseru~ spp.
The predicted amino acid sequence of P. haemo~ynca tbpA has regions of homology with the predicted amino acid sequence of tbp~. in N. gonorrhoeae. This suggests that the transferrin binding proteins are structurally similar and agrees with the observations m~de in the protein topology studies. Ogunnariwo and Schryvers (1990) reported that P. haemo~ca A1 Tbpl was similar to N. gonorrhoeae Tbpl proteins in size and properties. Both species produce 100 kDa receptor proteins which cannot bind transferrin after SDS-PAGE, which suggests that the conformation of the native protein is important in ~ansferrin binding. However, the two proteins differ in their binding specificities: N. gonorrhoeae Tbpl bound only human transfe..in whereas P.
haemo~ytica A1 Tbpl bound only bovine trar~f~llin. This suggests that dirrerences between the two tbpA sequences may be regions which encode for specificity of iron source.
The degree of homology between P. haerno~y~ica Tbpl and N. gonorrhoeae Tbpl was highest at tihe amino-terminal and was lower at the carbo~cyl-terminal. This implies that thc call~o,.yl-terminal of the protein may be involved in ligand binding and the amino-terminal is involved in the meell~nicm of iron uptake. Previous work by Alcantara ct al. (1993) has demor,allatw that the region of the transferrin r~ptor r~yonsll,le for ligand binding in N. meningitidis and H. influen~ae, is loC;3li7~d on the C-lobe of the protein. More recently, Yu and Schryvers (1994) has demonstrated that P. haemo~tica 21 64274 ~

Tbpl and Tbp2 transferrin binding is located on the C-lobe of the protein.

b) A. pkuropnuemon~
The predicted arnino acid sequence of P. haemo~ytica A1 Tbpl has a low degree of homology with the sequence of A. pleuropneumoniae TfbA. This result is confirmed by the Southern hybridization e~periments, which demonstrated that chromosomal DNA
from ~. pleuropneumoniac strains CM5 and Shope 4074 hybridized with the tbpA probe only under low-stringency conditions. This is interesting because both bacteria belong to the family Pasteurellaceae and would therefore be e~pected to have a similar transferrin binding protein. Previous work has suggested that the two proteins are functionally similar but structurally different. The TfbA protein in A. pleuropneumoniae has been shown to be a lipoprotein (Gonzalez a al., 1990), whereas the 100 kDa P.
haerno~y~ica Al Tbpl is not (Ogunnariwo and Schryvers, 1990). A. pleuropneumoniae is able to distinguish between iron-saturated and iron-depleted transre,l in (Gerlach a al., 1g92a), whereas N. meningitidis cannot (Tsai a al., 1988). It is h~r~ing to note that A. pleuropneumoniae TfbA has homology with the N. gonorrhoeae Tbp2, which is also a lipoprotein. This suggests that the TfbA protein is analogous to Tbp2 and that Tbpl of A. pleuropneurnoniac has not yet been identified.

c) TonB dependent-recept~- proteios The P. haemo~hca Tbpl sequence also has amino acids which are common to a group of E. coli TonB dependent r~plor proteins. This finding suggests that P.

hacmotytica Al it belongs to this family and that a TonB homolog exists in Pasteurella species. The first homologous domain or TonB bo~c~ has b~en implicated in direct interaction between the receptor protein and TonB (Bell ct al., 1990, Brewer et al., 1990). The significance of the other homologous regions is not kno~vn, but they may be required for TonB interaction or may be ne~esc;lry for outer membrane localization. P.
haemo~ytica Tbpl, like many other TonB-dependent proteins, is a tr~nc~nembrane protein which is iron-regulated and involved in iron utilization (Mietzner and Morse, 1994). It is possible that P. haemo.~tica Tbpl functions as a gated channel as has been proposed for E. coli FepA (Rutz et al., 1992). Both N. gonorrhoeae (Cornelissen et al., 1992), and H. ir~enzal (Jarosik et al., 1994) also belong to the family of TonB~ependent r~ptor proteins.

V. Proposed Model for P. h~emol~a lron Uptake The e~istence of many analogous proteins in Neisseria, Pastel~rella and Haemophilus suggests that a common m~h~nicm may be utilized for iron acquisition.
A hypothetical model of iron acquisition has been proposed for Neisseria (Chen et al., 1993) which may be used as a model for P. haemo~ytica A1. As shown in Figure 25, iron deprivation activates trans~;liylion of the iron-regulated proteins by a Fur-like regulatory system. Host transferrin binds to the bacterial cell surface via a specific iron r~ptOr comple~c composed of two or more proteins. The iron is removed from the transferrin and tran~l ~d across the outer membrane of the bacterium with the energy -Figure 25. Hypothetic~l model of iron uptake in Neissena spp.
(Chen ct al., 1993) hTf - human transfe.lin Fbp - ferric binding protein OM - bacterial outer membrane CM - bacterial cytoplasmic membrane ~N 2~64274 ,~ ~
*~.

~ . . e o ~ J~q provided by TonB. In the periplasm, the iron is transiently comple~ed to a periplasmic component, Fbp, which transports it to a cytoplasmic membrane pell--ease. The iron is transported across the cytoplasmic membrane by a periplasmic binding protein transport system. In the cytoplasm the iron is reduced to Fe2+ and assimilated by the cell.
One feature which may be unique to P. haemobnca A1 iron uptake is the presence of a third iron-regulated outer membrane protein (71 kDa) which may form part of the receptor comple~ (Ogunnariwo and Schryvers, 1990). In addition, P. haemo~ynca does not have a receptor protein which is capable of binding transferrin after SDS-PAGE
and electrobloning, while N. go~wrrhoeae does (Schryvers and Morris, 1988). This suggests that the binding mechani~rn of P. haemolynca r~eptor comple~ may be slightly different than the r~ptor comple~ in N. gonorrhoeae.
Proteins which are similar to N. gonorrhoeae Fbp have been identified in the family Pasteurellaceae. In H. infl~enz~c, a 40 kDa periplasmic protein was identified and its N-terminal sequence was found to be 81% homologous to N. gonorrhoeae Fbp (Harkness et al., 1992). In P. haemolytzca A2, a 35 kDa periplasmic iron-regulated protein has been described but no function has been found (Lainson et al., 1990). In addition, a 37 IcDa iron regulated protein has been isolated by affinity procedure from P. haernolytica Al (Ogunnariwo and Schryvers, 1990). Based on size and location similarities, it is possible that both of these proteins are analogous to M gonorrhoeae Fbp.

VI. 17 Proteill Exprcssion T7 RNA polymerase-dependent production of a Tbpl gene product in E.coli JM109 (DE3) was unsuccessful. One possible e~planation may be that the ribosomal binding site of tbpA was inefficient. In future studies the gene could be cloned into a vector that carries a functional ribosome-binding site. Alternatively the Tbpl protein may be unstable and requires the presence of other proteins or factors in order to be c~I~tly produced. Components of heterodimeric proteins are of~en unstable when they are synthesized singly. Future e~cpression studies would likely utilize a cons~uct containing the entire tbpA, tbpB region.

VII. Westem ~m~ r~blot Analysis Western immunoblots were performed on the inner and outer membrane fractions from P. haerno~ynca A1 cells which were grown under iron-sufficient or iron-limiting conditions. The iron-limiting conditions were SilT ul~t~d by adding the iron chelator EDDA which is a common synthetic iron chelator used to limit the availability of iron in culture media. EDDA was chosen for these studies because of its specificity for iron and its lack of to~ic side effects to bacteria (Neilands 1981).
P. hacmo.~ytica A1 membrane fractions im",llnoslAin~ with rabbit anlisc~ to soluble antigens did not react with the iron-regulated proteins. Neither the 100 kDa nor the 77 kDa iron-regulated proteins were observed in this ;...~ noblot possibly because dle original P. haemo~y~ica A1 culture (used in the hyperimmuni7~tiQn of the rabbit) was not grown under iron-resll icled conditions. The medium used contained 7 % rabbit serum ~ 1 ~4274 to avoid the inclusion of antibodies to serum proteins. In contrast, peptides which may be the iron-regulated proteins reacted with antisera from calves vaccinated with the Presponse. Presponse is produced from P. haemo~tica Al oells which are grown to late log phase in serum-free RPMI medium 1640 (Shewen and Wilkie, 1987; Shewn et al., 1988). It is possible that the low iron conoentration of this medium induoed production of the transferrin binding proteins. It is also possible that the calf responded to transferrin binding proteins produced by P. hacmolynca which are commensal organisms in the nasopharynx. The presence of antibodies to transferrin binding proteins suggests that these proteins are immunogenic.

2 1 642~4 CHArl ~;K S - SUMMARY AND CONCLUSIONS

The P. haemo~rtfca A1 transferrin binding proteins, T~bpl and Tbp2, were found to be encoded by two genes (tbpA, tbpB) which are closely associated in tandem.
Sequence analysis suggests that Tbpl is a highly basic tran~membrane protein located on the outer membrane of P. haemo~tica. The Tbp2 protein is also a highly basic transmembrane protein. The predicted secondary structures for both proteins suggest that they consist of B-sheets and B-turns. There is also evidenoe that the transferrin binding proteins are coordinately regulated by iron concentrations in a Fur-like manner.
The tbpA gene was found in all sixteen serotypes of P. hacmo~ytica but the genetic org~ni~tiQn of the gene was different in the A and T biotypes. This result confurms previous work which suggests that the two biotypes be reclassified as separate species.
The predicted amino acid sequence of Tbp 1 protein has significant homology with the Tbpl found in pathogenic Neisseria spp. A low but significant homology was revealed between Tbpl and several E. coli TonB~ependent r~eptor proteins. This suggests that the energy required to transport iron across the outer membrane is supplied by a TonB homolog in P. haemo~ica A1. Comparison between P. haemo~t~ca Tbpl and ~. pleuropnellmoniae TfbA found only a low degree of homology, indicating that the two proteins were funclionally similar but structurally different.
The ~;~nce of many analogous proteins involved in iron uptake in Pasteurella, ~eisseria, and Naemophilus suggests that all three utili~e a similar merh~nicm Based 2 1 6~274 on the hypothetical model of Neisseria iron uptake, a mechanism for iron uptake in P.
haemaiytica was proposed.
Antibodies to iron-regulated outer membrane proteins were found in antiserum from calves vaccinated with Presponse. This suggests that the transferrin binding proteins are immunogenic.

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APPENDIX A. MEDIA RECIPES

Brain ~Ieart Infusion Broth.
Difco BHI powder 37.0 g dH2O to IL.

Davis minimql medium.
K2HPO4 7.0 g KH2PO, 3.0 g (NH4)SO4 1.0 g Na citrate 0.5 g de~trose 2.0 g dH~O to lL.
after autoclaving, add 1% thiamine 0.5 ml MgSO4-7H20 Luna Thymi~le broth (LT).
Difco Bac~tryptone 12.0 g Difco Bact~yeast e~ctract 5.0 g NaCl 5.0 g de~trose 1.0 g thiamine 0.05 g dH2O to lL.
for plates, add agar 12.0 g for soft agar, add agar 6.0 g antibiotic supplements (add after autoclaving) .
ampicillin 100.0 mglL
chlorampenicol 25.0 mg/L

_ . _ _ _ 2 ' 64274 SDS-PAGE Sample Buf~er (2X strength).
O.S M Tris-HCI (pH) 6.8 2.5 ml 10% SDS 4.0 ml glycerol 2.0 ml Bromophenol blue 0.005 b-mercaptoethanol 1.0 ml dH2O 0.5 ml 21 ~4274 Bacterial strains. The bacterial strains used in this study are listed in Table 1. P. haemolytica strains hl73, hl74, hl75 and hl76 were field isolates from ruminants with pneumonic pasteurellosis and were provided by Dr.
5 Frank Milward, Rhone Merieux, Lyon, France. P. haemolytica strains h44-h46 were bovine clinical type Al isolates from bovine pneumonia obtained from S. Lundberg ,Veterinary Laboratory, Regional Agricultural Building, Airdrie, Alberta. h44 has been described previously (26). P. haemolytica strains h93-h97 were bovine clinical type Al isolates from bovine pneumonia obtained from 10 by Dr. A. Potter of the Veterinary and Infectious Diseases Organization (VIDO), Saskatoon. Strains h98-hlO7 are ATCC P. haemolytica strains (5) also obtained from Dr. A. Potter. Actinobacillus (Haemophilus) equuli strain h50 was obtained from Dr. Jane Pritchard, Veterinary Laboratory, Regional Agricultural Building, Airdrie, Alberta.

Table 1. List of strains included in this study.
Species Strain SerotypeHost Species Source P. haemolytica h44 Al cattleS. Lunberg, Air~
P. haemolytica h45 Al cattleS. Lunberg, Air~
P. haemolytica h46 Al cattleS. Lunberg, Air~
P. haemolytica h93 (ph21) Al cattleA. Potter, VID
P. haemolytica h94 (ph24) Al cattleA. Potter, VID
P. haemolytica h95 (ph27) Al cattleA. Potter, VID
P. haemolytica h96 (ph45) Al cattleA. Potter, VID
P haemolytica h97 (ph46) Al cattleA. Potter, VID
P. haemolytica hl96 Al cattleR. Lo, U. of Gu P. haemolyticah98 (ATCC33366) A2 sheepA. Potter, VID
P. haemolytica*h99 (ATCC33367) T3 sheepA. Potter, VID
P. haemolytica*hlOO (ATCC33368) T4 sheepA. Potter, VID
P. haemolyticahlOl (ATCC33370) A6 sheepA. Potter, VID
P. haemolyticahlO2 (ATCC33371) A7 sheepA. Potter, VID
P. haemolyticahlO3 (ATCC33372) A8 sheepA. Potter, VID
P. haemolyticahlO4 (ATCC33373) A9 sheepA. Potter, VID
P. haemolyticahlO5 (ATCC33369) A5 sheepA. Potter, VID
P. haemolytica*hlO6 (ATCC33374) T10 sheepA. Potter, VID
P. haemolyhcahlO7 (ATCC33375) Al l goatA. Potter, VID
P. haemolyticahl73 (77020-15184) Untypable goatF. Milward, Rhone r P. haemolyticahl74 (90020-16266) A7 goatF. Milward, Rhone r P. haemolyticahl75 (84020-15786) A7 sheepF. Milward, Rhone ~
P. haemolyticahl76 (84020-15792) A9 sheepF. Milward, Rhone r A. equuli h50 horseJ. Pritchard, Air T-type strains are now considered as a new species, P. trehalosi (34).

2~ ~4274 Growth conditions. All bacterial strains were stored frozen at -70C in 30% glycerol. Isolates from the frozen stocks were streaked onto chocolate agar plates and incubated at 37C in a 5% CO2 incubator. Iron-restricted growth was achieved by growing the bacteria in Brain Heart Infusion5 broth (BH1, Difco Laboratories) or O'Reilly-Niven broth (25) supplemented with 2 llg/ml thiamine monophosphate and 3 ,ug/ml nicotinamide adenine dinucleotide (NAD) and containing the iron chelator ethylenediaminedihydroxyphenylacetic acid (EDDHA, Sigma) at a final concentration of 100 ~M. Growth experiments for use of different transferrins 10 as iron source was performed as previously described (26) Preparation of transferrins and derivatives. Bovine transferrin was obtained from Sigma. The preparation of equine (horse), ovine (sheep) and caprine (goat) transferrins (2), the iron loading of transferrins to30% or 100% saturation (22) and conjugation of horse-radish peroxidase (HRP) 15 to trannsferrin (37) was essentially as described previously. In the preparation of conjugates of bovine, ovine, caprine and equine transferrins (HRP-bTf, HRP-oTf, HRP-cTf and HRP-eTf), the mixture of HRP and transferrin were subjected to gel filtration after chemical conjugation. The fractions demonstrating maximal activity were pooled, dialyzed and aliquots frozen and 20 stored at -70C.
Solid-phase binding assays. The solid phase binding assay was essentially derived from methods described previously (32). Aliquots of intact cell suspensions or crude total membrane preparations were spotted onto nitrocellulose/cellulose acetate membranes (HA paper, Millipore Corporation, 25 Bedford, MA) and after drying the HA paper was blocked with buffer containing 0.5% skim milk (blocking solution). For the transferrin binding assay, the paper was exposed to blocking solution containing 450 ng/ml of the HRP-conjugated transferrin, washed and developed with HRP substrate mixture essentially as previously described (32). For assessment of binding of 30 anti-receptor antibody by intact cells a similar procedure was utilized except that the first binding solution contained a 1/1,000 dilution of the anti-TbpA

and anti-TbpB antisera and, after washing, the membrane was exposed to a second binding solution containing a 1/3,000 dilution of a HRP-conjugated goat anti-rabbit antibody preparation.
Affinity isolation of transferrin binding proteins (TbpA and 5 TbpB). Bovine, ovine, caprine and equine transferrins were individually coupled to CNBr-activated Sepharose 4B according to the manufacturers instructions using solutions containing 3.5 mg/ml of iron-saturated transferrin. Activated groups were blocked by addition of ethanolamine.
Noncoupled transferrin was removed by washing with 10 to 20 column volumes of a 50 mM TrisHCl, 1 M NaCl, pH 8.0 buffer containing 6.0 M
guanidine hydrochloride and after further washing the bound transferrin was reloaded with iron using a solution containing 5 ~g/ml FeCl3 in 0.1 M
sodium citrate/0.1 M NaHCO3 pH 8.6 buffer.
Iron-deficient total membrane (200 mg protein) from P.
haemolytica or A. equuli prepared as previously described (32) was diluted to 2 mg/ml in 50 mM Tris pH 8.0 containing 1.0 M NaCl. The diluted membrane was solubilized by addition of EDTA and sarkosyl to a final concentration of 10 mM and 0.75%, respectively followed by incubation of the mixture at room temperature for 15-30 min with gentle rocking. The solution was centrifuged at 10,000 rpm for 10 min to remove insoluble debris. The supernatant containing the solubilized membrane was applied to a 1.5 x 10 cm transferrin-affinity column and then washed extensively (at least 10 bed volumes) with 50 mM Tris pH 8.0 containing 1.0 M NaCl, 10 mM EDTA, 0.75% Sarksosyl to remove non-specifically bound protein. In experiments using low salt washing conditions the washing buffer contained 100 mM NaCl in lieu of lM
NaCl. In some instances, additional washing with 2-3 bed volumes of washing buffer containing 0.2 M guanidine hydrochloride was necessary to remove contaminating proteins.
Coelution of both transferrin binding proteins (TbpA and TbpB) was achieved by application of 2-3 bed volumes of 2.0 M guanidine hydrochloride in 50 mM Tris pH 8.0, containing 1.0 M NaC1, 1 mM EDTA, 2 1 ~4274 0.01% sarkosyl. The eluant was collected for immediate dialysis against 50 mM
Tris pH 8Ø Further treatment with higher concentrations of guanidine hydrochloride usually did not result in any further yield of receptor protein.
Individual isolation of TbpA and TbpB was attained by sequential elution with 2 bed-volumes of each buffer containing 0.2, 0.5, 0.75, 1.0, 1.5, 2.0 and 3.0 guanidine hydrochloride, respectively. The eluates were dialyzed against 3 changes of 3 litres 50 mM Tris pH 8.0 over an 18-hour period and concentrated by ultrafiltration. After SDS-PAGE analysis the fractions from the 0.5 and 0.75 M guanidine HCl elution buffers were pooled for a preparation of TbpB and fractions from the 1.5 and 2 M guanidine HCl elution buffers were pooled for a preparation of TbpA.
Expression of recombinant receptor protein. An E. coli strain carrying the appropriate recombinant plasmid (DH5aF'/pCRIIPHtbpB for TbpB, DH5aF'/pCRIIPHtbpA for TbpA) was used to inoculate six 50 ml L-broth starter cultures containing 0.2% maltose and 150 ~lg/ml ampicillin. After growth at 37OC for several hours the cultures were used to inoculate 1.5 liter of the same medium to a starting A600 of 0.05. Once the A600 reached 0.4, glucose was added to 4 mg/ml and grown until A600 reached 0.7-0.8. At that time MgSO4 was added to 10 mM and 150-155 ml of a 1010 pfu/ml suspension of CE6 phage. The cell culture was incubated for an additional 2-3 hrs at 37OC after exposure to the phage and then was harvested, resuspended in ice cold 50 mM
Tris-HCl pH 8.0, 1 M NaCl to a final volume of 300 ml (50 ml/1.5 liter of culture).
Isolation of recombinant receptor protein from intact cells.
After expression, cells are centrifuged and resuspended into 1/30 volume in 50 mM Tris-HCl pH 8.0, 1 M NaCl buffer. EDTA is added to 4 mM and sarkosyl to 5% and the mixture incubated with agitation for 0.5-2 hrs at room temperature until solubilization is complete.The mixture is centrifuged for 15 min at 8,000 rpm (40C) and the supernatant carefully decanted. The supernatant was diluted 3/5 with 50 mM Tris pH 8.0, 1 M NaCl buffer and applied to a ml bTf-Sepharose column previously equilibrated with 50 mM Tris-HCl pH 8.0, 1 M

NaCl. The column was washed with 12 column volumes of 50 mM Tris-HCl, pH 8.0,1 M NaCl,10 mM EDTA, 0.75% sarkosyl followed by 4 column volumes containing only 0.05% sarkosyl. The receptor proteins were eluted from the affinity column by applying the elution buffer (buffer containing 3 M
5 guanidine hydrochloride) in a reverse direction. Early fractions were collected and dialyzed against 3 changes of 50 mM Tris-HCl, pH 8.0 buffer prior to storage at 40C (for short term) or at -70 oC.
N-terminal amino acid sequence analysis. Samples of affinity-purified TbA and TbpB were subjected to SDS-PAGE, electroblotted onto PVDF
(Immobilon-P, Millipore IPVH 00010) membrane, briefly stained with Coomassie Blue, and strips containing the individual protein bands were cut from the membrane. N-terminal amino acid sequence analysis was performed by Sandy Kieland at the University of Victoria.
Preparation of anti-TbpA and anti-TbpB monospecific rabbit 15 sera. Approximately 500 .m of purified TbpA and TbpB from P. haemolytica strain h44 obtained from the appropriate fractions in the affinity procedure after dialysis and concentration was mixed with Freund's complete adjuvant and injected intramuscularly into two white female New Zealand rabbits, respectively. The rabbits were boosted twice at 3-week intervals with the same 20 amount of antigens plus Freund's incomplete adjuvant. Two weeks after the final boost, blood was collected to determine the serum titre to the respective antigens using the dot assay in a dot-blot apparatus. The rabbits were either further boosted if titre was unsatisfactory or terminally bled, if the titre wassatisfactory. The specificity of the sera against TbpA and TbpB was tested by 25 SDS-PAGE and immunoblotting using goat anti-rabbit IgG conjugated to HRP
as secondary antibody in western blot analysis.
Analytical methods. Protein samples were analyzed by SDS-PAGE followed by silver staining as previously described (32). For Western blot analysis, about 1-2 .m of purified receptor proteins or 40 .m of 30 outermembrane protein from iron-poor cells were separated on 10%
polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose (Millipore, Bedford, MA) overnight at 15V in 20 mM Tris, pH

7.5, 150 mM glycine, 20% methanol and 0.1% SDS. The filters were blocked with 0.5% skim milk in 20 mM Tris pH 7.5, 500 mM NaCl (TBS) for 30 minutes at room temperature. A 1/300 dilution of the appropriate antibody in the blocking solution was applied to the paper for 1 hour at room temperature followed by two, 10-minute washes each with TBS. A 1/3000 dilution of secondary antibody (goat anti-rabbit IgG-horse-radish peroxidase conjugate from BioRad) was allowed to bind for 1 hour at room temperature. The conjugate was removed by three, 10-minute washes in TBS and developed using the HRP-substrate mixture.
PCR amplification.The primers for PCR were synthesized in an Applied Biosystems Model 390E Synthesizer and purified according to manufacturer's instructions. PCR was carried out in thin-walled 500 l GeneAmptudes in a Perkin-Elmer Cetus 480 Thermal Cycler using the PCR
core reagents and Ampli-Tag as recommended. The PCR conditions consistof 950C for 2 min., followed by 30 cycles of denaturation,annealing and extension at 95OC (1 min.), 52OC (1 min.) and 72OC (2min.) respectively. A negative control which did not contain template DNA was included in each PCR run.
Nucleotide sequence analysis. Two separate strategies for sequencing the tbp region were adopted. The preliminary approach reported in Example 1 herein primarily involved subcloning fragments from recombinant plasmids into the M13 vectors and then sequencing subsequently isolated single stranded DNA prepared by the dideoxy chain termination method using vector primers. In a limited number of cases, oligonucleotide primers were synthesized on the basis of the sequence results from the cloned inserts and used to complete the sequence of the cloned insert. In this preliminary analysis the nucleotide sequences were compiled and analyzed by the Pustell programs (IBI).
The alternate approach primarily involved sequence determination of a succession of cloned inserts obtained by PCR amplification from chromosomal DNA. Oligonucleotide primers were synthesized on the basis of the preceeding preliminary sequence analysis. The PCR amplified products were cloned into the PCRII cloning vector. Double stranded DNA

sequencing was performed purified recombinant plasmids by the oligonucleotide primer-directed procedure using synthetic oligonucleotides, fluorescent dye-labelled dideoxynucleotide triphosphate terminators, and cycle sequencing with Taq polymerase. Sequence reaction products were analysed 5 on a Applied Biosystems(ABI) model 373A automated fluorescent sequencer.The results from successive sequencing runs were compared and the composite sequence was determined by comparison of the chromatograms using the SeqEd program. This sequence was subsequently compared to the sequence obtained by single strand sequencing using the Mac-DNASIS
10 program. In addition the sequence was analyzed by comparing the predicted protein sequence in all three reading frames with aligned sequences for Tbps from several different species. Any areas of uncertainty identified by this analysis were subjected to repeated runs of sequence analysis.

Results:
15 1. Comparison of receptor specificity:
Prior studies had demonstrated differences in specificities towards different ruminant transferrins (i.e. cattle, sheep and goat) by transferrin receptors from various pathogenic bacterial species of ruminants (38). This probably reflects differences in the regions of the receptor proteins20 involved in ligand binding and thus suggests that these regions could not serve as the basis of a broad-spectrum transferrin receptor-based vaccine for ruminant pathogens. However, it does not preclude the possibility that a group of related ruminant pathogens, such as the various Pasteurella species, may have common ligand binding domains that could provide the basis for 25 generation of a cross-protective response. Thus it was important to determine whether the transferrin receptors from a collection of representative Pasteurella isolates possessed the same specificity for ruminant pathogens.
As a preliminary analysis of receptor specificity, a collection of representative isolates were assessed for their ability to utilize various 30 ruminant transferrins as a source of iron for growth (Table 2). A simple plate assay described in the methods section was utilized. The growth of all the representative ruminant isolates of Pasteurella haemolytica and P. trehalosi was stimulated by Fe-saturated transferrins from ruminant (bovine, caprine and ovine) but not from non-ruminant (equine) hosts. The stimulation of the 5 growth of the equine pathogen, Actinobacillus equuli (strain h50), by equine transferrin indicated that the inability of the P. haemolytica strains to use equine transferrin as iron source was not due to deficiencies in the preparation.

Table 2: Growth on different transferrins.
Species Strain Serotype Host Source of Tf for growth bTf oTf cTf eTf P. haemolytica h44 Al cattle + + +
P. haemolytica hl73 Untypable goat + + +
P. haemolytica hl74 A7 goat + + +
P. haemolytica hl75 A7 sheep + + +
P. haemolytica hl76 A9 sheep + + +
P. haemolytica hlO6 TlO sheep + + +
A. equuli h50 horse - - - +

As a further assessment of the receptor specificity, binding of transferrin by intact cells or isolated membranes was assessed by a simple binding assay utilizing horse-radish peroxidase (HRP) conjugates of transferrin. Conjugates were prepared from bovine, ovine and caprine transferrin and then tested for their ability to bind to total membranes isolated 15 from iron-deficient cells of several representative strains of P. haemolytica and P. trehalosi. The results illustrate that all the selected strains were capable of binding the three ruminant transferrins (bovine, caprine and ovine) but not equine transferrin (Figure 26), which is consistent with the results of the growth studies (Table 2). To confirm that the observed binding by all three 20 ruminant transferrins was due to the same receptor in the selected species, competitive binding assays were performed in which the ability of unlabelled ruminant transferrins were tested for their ability to block binding of the labelled tranferrins. In these experiments reciprocal inhibition by the various ruminant transferrins was equally effective, indicating that they bound to the same receptor with similar affinities (data not shown).
The results of the growth and binding studies suggested that bovine, ovine and caprine transferrins were capable of interacting with the 5 receptor components involved in iron acquisition in P. haemolytica. We, therefore, utilized the affinity procedures described in the methods section to identify the proteins interacting with the ruminant transferrins by employing bovine, caprine or ovine transferrin-sepharose resins. As illustrated in Fig. 27, a predominant receptor protein of approximately 100,000 molecular weight 10 was isolated with membrane preparations from the bovine isolate (h44), the caprine isolate (hl73) or the ovine isolate (hl75) when either bovine (lanes A
and B), ovine (lane C) or caprine (lane D) transferrin affinity columns were used. This protein is analogous to receptor proteins of similar size that are found in other bacterial pathogens (18,27,30,31), which have conventionally 15 been termed transferrin binding protein 1 (Tbpl). Recently an alternate name,TbpA has been recommended (21)to be consistent with existing conventions of nomeclature.
A second protein of approximately 60,000 molecular weight was also evident in the samples isolated by affinity chromatography with the 20 ruminant transferrins (lanes B, C and D) using membranes from the bovine isolate (h44). This protein is comparable to the lower molecular weight receptor protein, transferrin binding protein 2 (Tbp2), isolated from other pathogenic bacterial species (18,27,30,31). For reasons alluded to above, the alternate name, TbpB, has been recommended (21). A protein of this 25 molecular weight is also detectable in most samples obtained with the caprine(hl73) and ovine (hl75) isolates but the presence and yield of this component was sensitive to the conditions of isolation. The characteristically low yield of TbpB (Tbp2) relative to TbpA (Tbpl) observed in these species is not a general property of the bacterial receptor proteins and may even reflect common 30 properties of TbpB from related species.
Neither of the proteins were isolated when equine transferrin-Sepharose was used in the affinity isolation procedure (lane E) indicating that their isolation was specifically due to the presence of ruminanttransferrin. When less stringent washing conditions were used during the affinity isolation procedure, additional proteins of approximately 38,000 and 70,000 molecular weight were retained by the affinity column (lane A) when membranes from the bovine (h44), caprine (hl73) or ovine (hl75) isolate were used. An additional protein of approximately 77,000 molecular weight was also evident in the sample obtained with membranes from the bovine isolate.

2. Comparison of the immunological properties of the receptor proteins.
The observation that bovine, caprine and ovine transferrins compete for the same receptors suggested that there is conservation at least in the binding domain of the receptors. In order to determine whether there was also a similarity with respect to presence of common immunological epitopes, we decided to prepare antisera against purified receptor proteins from one strain and evaluate their crossreactivity with receptor proteins from other isolates. Thus we obtained affinity purified preparations of TbpA and TbpB
from strain h44 (see methods section) and used these proteins for generation of monospecific antisera in rabbits. These antisera were then tested against receptor proteins isolated from representative strains of different serotypes including isolates obtained from cattle, sheep and goats. The results in Fig. 28A
demonstrate that the anti-TbpB antisera reacted strongly with a protein of approximately 60,000 molecular weight (TbpB) that was affinity isolated with bTf-Sepharose from all of the representative strains. Similarly, the anti-TbpA
antisera crossreacted with TbpA isolated from all seven representative strains (Figure 28B). Extension of this analysis to the additional serotypes of ruminantisolates (Table 1) continued to show considerable cross-reactivity with both receptor proteins (data not shown). These data suggest that both receptor proteins are conserved amongst the different serotypes of P. haemolytica causing pneumonic pasteurellosis in cattle, sheep and goats.
Although the immunological cross-reactivity illustrated in Figures 28A and 28B indicates that there are conserved epitopes in receptor proteins from different species, there is no indication whether any of these epitopes are exposed at the bacterial surface, where they could serve as effective targets for the host immune effector mechanisms. In order to address this issue, a solid-phase binding assay was used to assess the binding of antireceptor antibodies by intact cells. This assay demonstrated that there was 5 strong binding by cells grown under iron-deficient, but not iron-sufficient conditions, when a selection of bovine type A1 isolates were tested (data not shown). When a selection of sheep isolates of varying serotypes were tested, there was a variable degree of reactivity (Figure 29). Other serotypes of type AP.haemolytica strains (h98 and hlO5, Figure 29) showed considerable reactivity 10 against the anti-TbpA and anti-TbpB antisera. In contrast, the T-type strains (P.
trehalosi, h99, hlOO and hlO6) showed only very weak reactivity against both of the anti-receptor antisera. However, the fact that there was also weak binding by labelled bTf indicates that there was limited production of receptor proteins under the iron-deficient growth conditions used in this experiment.
15 Thus the lack of reactivity of the anti-receptor antisera cannot be attributed to a lack of surface-exposed, cross-reactive epitopes in the receptor proteins from these species.

3. Cloning of the transferrin receptor genes from a type A1 strain.
Antireceptor antisera and N-terminal amino acid sequences 20 were obtained in order to facilitate cloning of the P. haemolytica transferrin receptor genes. Using a modified affinity protocol we were able to obtain purified preparations of the individual receptor proteins; Tbpl (TbpA) and Tbp2 (TbpB) from a type A1 strain of P. haemolytica. Immunization of rabbits with the purifed receptor protein preparations provided monospecific antisera 25 that was suitable for detection of recombinant receptor protein. Amino acid sequence analysis of an electroblotted preparation of purified mature Tbpl (TbpA) yielded a readable sequence of 20 amino acids;
TENKKIEENNDLAVLDEVIV. A similar analysis with the purified Tbp2 (TbpB) failed to provide any sequence information suggesting the N-terminus 30 of this protein is blocked.

The sequence of the first eight amino acids of the purified Tbpl was used to design an oligonucleotide primer based on a P. haemolytica preferred codon usage table (023, Table 3). To facilitate polymerase chain reaction (PCR) amplification of a portion of the tbpA gene from an existing P.
5 haemolytica genebank derived from hl96 chromosomal DNA, two other primers based on the junction sequences next to the BamHI site on pBR322 were also prepared (RL2 and RL3, Table 3). Appropriate restriction endonuclease sites were included to facilitate cloning of the PCR product. PCR
amplification reactions were performed with the tbpA primer (023) in 10 combination with either of the vector primers (RL2 or RL3) using a aliquot ofthe P. haemolytica plasmid genebank as a template. An 800 bp PCR product was obtained when the tbpA primer (023) was used in combination with vector primer RL2 but not vector prime RL3. The PCR product was purified, digested with EcoR1 and HindIII, cloned into M13 mpl8 and subsequently into 15 pBluescript, resulting in plasmid pBphGT1. The predicted protein sequence from the cloned PCR product immediately downstream of tbpA primer region had an identical sequence to that obtained from sequencing the purified TbpA, confirming that the PCR product was from the authentic tbpA gene. This fragment corresponded to approximately the first third of the tbpA gene and 20 encoded stretches of amino acids that are highly conserved in all known TbpAs.

2 ~ 64274 Table 3: Oligonucleotide primers.

Primer # Description (gene/region - location) Direction* Sequence 023 tbpA - S'end,1st 8 N-terminal aa's 5'-3' GGM~II~CI~I~
088 tbpA - S'end, 3'-5'* CACTACTTTCCCCAAGCCAG
RL2 pBR322 - upstream of BamHI site 3'-5'* GGAATTCCCTCCTGTGGATC
198 tbpA - 3' end, 3'-5'* GCIGCII(G/C)IGCICGIAA(T/C)T(T/A)(T/C) 190 tbpB - S' end, leader peptide region 5'-3' CAAAGCTTGCITG(TC)TCIGGIGG
352 upstream of tbpB - S' end 5'-3' A~l ~CCGCTTGTAT
192 tbpB - conserved aa sequence near 3' end 5'-3' GTI(T/A)(A/G/C)IGGIGGrTT(C/l)TA(T/C)GG
401 tbpB - S' end 5'-3' TAAATTAAAGGAGACATTATGTTTAAACT
350 tbpB - 3' end, flanking Ncol site 3'-5'* CGACGCCCATGGTTAl l l l l~lATTTGACGTI
199 tbpB - 3' end, flanking Hindlll site 3'-5' * GCGCAAGCTTTTAl l l l l~lATTTGACG
349 tbpA - S' end, BamHI/Bglll sites upstream of rbs 5'-3' GGATTCAGATCTTAAAGGAGACCCTATCTAA
255 tbpA - S' end, Ndel site at start codon 5'-3' CCCTATCATATGATAATGAAATATCATC
256 tbpA - 3' end, Hindlll site after stop 3'-5'* TA~ I G~ACTTCATTTCAAAT
Direction relative to orientation of coding strand for the relevant gene.

The 800 bp product was subsequently used as a hybridization probe for Southern analysis of restriction endonuclease-5 digested chromosomal DNA and for screening a plasmid genebank. The Southern analysis enabled us to prepare a restriction map of the chromosomal DNA in the tbp region and compare it to cloned inserts obtained from the plasmid and phage genebanks. Initially two strongly hybridizing colonies were identified after transformation of E. coli with 10 the plasmid genebank DNA. Plasmid p(clone 9) contained a 9 kb insert which included most of the tbpA gene with adjacent downstream regions but was fused with DNA from another chromosomal locus. The second plasmid, p(clone 10), only contained a small insert that was primarily situated around the 5' end of the tbpA gene.
The artificial junction in plasmid p(clone 9) was remniscent of similar artifacts observed while attempting to clone the meningococcal tbpB gene and the ensuing difficulties that were encountered (23) prompted us to consider alternative strategies for cloning 21b4214 the P. haemolytica tbpB region. One strategy was based on the observation that in other species the tbpB gene was located upstream of the tbpA gene (19,20,23) and that there were short stretches of amino acid identity in the predicted sequences of the respective TbpBs. To obtain the remainder of 5 the tbpA gene, the intergenic region and a portion of the 3' end of the tbpB
gene we used a conserved amino acid sequence near the carboxy terminus of TbpBs for designing a degenerate oligonucleotide primer (primer 192, Table 3). This primer was used in combination with a primer based on the sequence from the 5' end of the (primer 088, Table 3) to amplify a 700 bp 10 fragment from chromosomal DNA which was subsequently cloned into the pCRII vector (producing pCRPHT1/2). The sequence from this insert enabled us to design an oligonucleotide primer based on the authentic sequence of the 3' end of the tbpB gene (primer 199, Table 3) which was used in combination with degenerate oligonucleotide primer based on a 15 conserved amino acid sequence present in the leader peptide region of known TbpB's (oligo 190, Table3) for PCR amplification. The resulting 2.4 kb PCR product obtained when chromosomal DNA was used as a template, which was larger than anticipated, contained the authentic 3' end of the tbpB gene. However, when this PCR fragment was cloned in the 20 pCRII vector (resulting in plasmid pCRIIPHtbpB) and used in expression experiments utilizing the T7 promoter an intact recombinant Tbp2 was produced (see below), indicating that the ribosomal binding site and start of the tbpB gene was contained within the insert.
A second strategy utilized anchored PCR in which PstI
25 digested pBluescript plasmid was ligated to PstI digested chromosomal DNA and used as template for a PCR reaction utilizing a primer from the 3' end of the tbpB gene (oligo 199, Table 3) and the M13 reverse primer from the vector. The resulting 3.5 kb product containing the entire tbpB
gene and a considerable amount of adjacent upstream regions was 30 subcloned into the PCRII vector.
After obtaining cloned segments of the tbp genes, they were subjected to DNA sequence analysis by using several different sequencing strategies concurrently as described in the methods section.
Sequence analysis of the tbpA gene region from type A1 P. haemolytica strain hl96 revealed and open reading frame (ORF) of 2,790 bp (Figure 30) encoding a protein with a predicted molecular mass of 106,921 Da (Figure 5 31). The putative signal peptidase cleavage site at residue 28 was confirmed by comparison with the known N-terminal amino acid sequence of the mature protein (underlined aa in Figure 31). The predicted protein sequence of TbpA was compared to the sequences of TbpA from N.
meningitidis (23), N. gonorrhoeae (8), H. influenzae (20) and 10 Actinobacillus pleuropneumoniae (19) as well as LbpA from N.
meningitidis (28). It is evident that there is considerable identity between the various TbpAs and LbpAs and that most of this identity occurs in clusters of amino acids that are found throughout the length of the linear amino acid sequence. These clusters of amino acids correspond to the short 15 transmembrane ~-sheets in the topology model predicted by Tommassen (28).
Analysis of the sequence in the tbpB gene region gene from type A1 P. haemolytica strain hl96 revealed an ORF of 1752 bp (Figure 32) encoding a protein with a predicted molecular mass of 63,419 20 kDa (Figure 33). This predicted protein sequence of TbpB was compared to the published sequences of TbpB from N. meningitidis (23), N.
gonorrhoeae (1), H. influenzae (20) and Actinobacillus pleuropneumoniae (14). This predicted amino acid sequence included a 18 amino acid leader peptide, a signal peptidase II recognition sequence with a cysteine as the 25 predicted N-terminal amino acid of the mature protein. The presence of an N-terminal cysteine, which has been shown to be lipidated in other species (14,24), may explain our inability to obtain an N-termimal amino acid sequence for this protein and may serve as the primary means of anchoring the protein to the outer membrane. The presence of several 30 short stretches of amino acids that are identical in all TbpB proteins analyzed to date (i.e. GGFYG), appropriately located throughout the linear amino acid sequence, confirms the identity of this protein.

4. Comparison of the transferrin receptor genes.
As a complement to the immunological studies we decided to evaluate the variability of the tbp genes from the various strains of P. haemolytica and P. trehalosi. Using the sequence information 5 obtained for the tbpA gene from the serotype A1 P. haemolytica strain hl96, we prepared specific primers for the 5' and 3' ends of the gene (primer 255 and 256, Table 3). These primers were capable of amplifying the intact tbpA genes from all strains of P. haemolytica and P. trehalosi tested which included representatives of all serotypes. The intact genes 10 were then subjected to digestion by the Sau3A1 restriction endonuclease and the resulting fragments were analyzed by electrophoresis on polyacrylamide gels. There are 13 predicted Sau3A1 sites in the tbpA gene from strain hl96A resulting in 14 predicted fragments. A specific pattern consisting of 9 of these fragments was readily resolved on the 15 polyacrylamide gels and this pattern was observed in all type A1 strains. In fact this pattern was observed in representative strains from the other serotypes of type A (P. haemolytica) and type T (P. trehalosi) with the exception of strain hlO7 (type A11). This indicates that there must be a considerable degree of identity in the tbpA genes and correspondingly in 20 the TbpA proteins from the various strains. Many of the Sau3A1 sites were in the regions encoding segments of the TbpA protein, proposed to be surface loops, which show the greatest variation in amino acid sequence among proteins from different species (21,28).
In order to evaluate the variation in the tbpB genes, we 25 first attempted to amplify the intact genes from the various strains using specific primers for the 5' and 3' ends of the gene based on the sequence from strain hl96 (primer 401 and 350, Table 1). We were able to amplify the intact genes from all the strains except strain h99, a serotype 3 P.
trehalosi strain. The intact genes were then subjected to digestion by the 30 Sau3A restriction endonuclease and the resulting fragments were analyzed by electrophoresis on polyacrylamide gels. A specific pattern Sau3A1 restriction, consistent with the known DNA sequence of tbpB from strain hl96, was observed in all type A1 strains. In fact an identical pattern was observed in representative strains from the other serotypes of type A (P.
haemolytica) and type T (P. trehalosi). This indicates that there must be a 5 considerable degree of identity in the tbpB genes and correspondingly in the TbpB proteins from the various strains.

5. Expression of transferrin receptor genes, isolation and characterization of the recombinant receptor proteins.
The intact tbpB and tbpA genes were PCR amplified from 10 chromosomal DNA and subcloned into expression vectors for production of recombinant proteins. For our initial attempts at expression of the tbpB
gene, the subcloned tbpB gene was obtained by PCR amplification with an upstream sequencing primer (352, Table 3) and a primer containing the authentic 3' end flanked by an NcoI site (350, Table 3). When the PCR
15 amplified fragment was subcloned into the pCRII vector, all five of the resulting clones were in the same orientation; downstream of the T7 promoter and in the opposite direction of the lac promoter. Since the lac promoter would not be tightly regulated in a high copy number vector, this result suggests that expression of TbpB in E. coli may be selected 20 against. Expression of TbpB from the T7 promoter was accomplished by infection with CE6 lamba phage, which encodes the T7 RNA polymerase.
Several hours after infection a protein of the anticipated molecular weight for TbpB was evident and this protein reacted with anti-TbpB antisera after electroblotting. The recombinant TbpB could be purified to homogeneity 25 directly from intact cells by using a modified affinity isolation procedure described in the methods section. The affinity-purified protein retained bTf binding activity after elution from the column.
In our initial attempts at expressing the tbpA gene, PCR
amplification was performed with a set of primers (oligos 349 and 256, 30 Table 3) that maintained the predicted ribosomal binding site. When the PCR product was subcloned into the pCRII vector, expression from clones in the appropriate orientation was accomplished by infection with CE6lamba phage. Under these conditions, very little recombinant TbpA was detected with anti-TbpA antisera after SDS-PAGE and electroblotting.
Restriction digestion analysis of several of the clones and sequencing of 5 one of the clones confirmed that the restriction sites and putative ribosomal binding site had been retained. On the assumption that the limited expression might be related to the ribosomal binding site, we decided PCR amplify the tbpA gene with an alternate primer (255, Table 3) that introduced an NdeI site at the putatitive start codon. When this PCR
10 product was subcloned into the pT7-7 expression vector (which supplies a properly position ribosomal binding site) and expression was induced by CE6 phage infection much higher levels of rTbpA were produced . The recombinant TbpA could be purified to homogeneity directly from intact cells by using a modified affinity isolation procedure and the affinity-15 purified protein retained bTf binding activity after elution from thecolumn .Figure 26. Binding of labelled transferrins by iron-deficient bacterial membranes. Aliquots of total membranes (4 ~g protein) prepared from iron-deficient cells from the indicated bacterial strains were spotted onto 20 strips of nitrocellulose/cellulose acetate paper and, after blocking, the papers were exposed to mixtures containing 450 ng/ml of the indicated HRP-conjugated transferrin. The filters were subsequently washed and developed with HRP substrate mixture as described in the Methods section. hl73, hl74, hl75, hl76 and h44 are representative strains of P.
25 haemolytica whose serotype and source are listed in Table 1. h50 - A.
equuli. HRP-bTf,-oTf,-cTf and -eTf - HRP conjugates of bovine, ovine, caprine and equine transferrins.

Figure 27. Isolation of receptor proteins with transferrin affinity columns.
Affinity isolation experiments were performed with iron-deficient total 30 membranes prepared from P. haemolytica strain h44 (top panel), hl73 (middle panel) and hl75 (bottom panel). Experiments were performed 21 ~42~4 with bovine transferrin-Sepharose (lanes A and B), ovine transferrin-Sepharose (lane C), caprine transferrin-Sepharose (lane D) or equine transferrin-Sepharose (lane E) using standard washing conditions (lanes B-E) or low salt washing conditions (lane A) as outlined in the methods section. The samples eluted with buffer containing 2M guanidine HCl were dialyzed, concentrated and aliquots analyzed by SDS-PAGE and silver staining as described in the methods section.

Figure 28. Immunological analysis of receptor proteins from different serotypes of P. haemolytica from bovine, sheep and goats. Aliquots of purified receptor proteins from representative serotypes of P. haemolytica were subjected to SDS-PAGE, electroblotted and then probed with specific anti-TbpB serum (Panel A) or with anti-TbpA serum (Panel B) as described in the methods section. The following P. haemolytica strains of the indicated serotype were included in the analysis; Lane 1- strain h44 (A1), Lane 2 - hl73 (untypable), Lane 3 - hl75 (A7), Lane 4 - hl76 (A9), Lane 5 -hlOO (T4), Lane 6 - hlO6 (T10), and Lane 7 - hlO7 (A11). The numbers on the left represent the molecular weights (X 1000) of standard proteins.

Figure 29. Binding of labelled transferrin and anti-receptor antibody by intact cells. The indicated bacterial strains were grown under iron-limiting conditions, harvested by centrifugation and resuspend to a A600 of 1-2 in 50 mM TrisHCl, 150 mM NaCl, pH 7.5 buffer. A 5 ,ul aliquot of the suspensions were applied to HA membrane, the membrane was dried, blocked and then exposed to blocking solution containg labelled transferrin (HRP-bTf) or antireceptor antibody (anti-TbpA, anti-TbpB). The latter membranes were washed and subsequently exposed to labelled second antibody prior to development with substrate.

Figure 30. DNA sequence of the tbpA gene from Pasteurella haemolytica strain hl96.

2164~74 Figure 31. Predicted amino acid sequence of the TbpA protein from Pasteurella haemolytica strain hl96. Underlined amino acids correspond to the N-terminal amino acids of the mature protein determined by protein seqeunce analysis.

5 Figure 32. DNA sequence of the tbpB gene from Pasteurella haemolytica strain hl96.

Figure 33. Predicted amino acid sequence of the TbpB protein from Pasteurella haemolytica strain hl96. Underlined amino acids correspond to the predicted N-terminal amino acids of the mature protein determined 10 by protein seqeunce analysis.

2~274 HRP- HRP- HRP- HRP-bTf oTf cTf eTf h173- ~
h174- ~
h175- ~
h176- O
h44- o h50-Figure 26 Binding of labelled transferrins by iron-deficient bacterial membranes. Aliquots of total membranes (4 ,ug protein) prepared from iron-deficient cells from the indicated bacterial strains were spotted onto strips ofnitrocellulose/cellulose acetate paper and, after blocking, the papers were exposed to mixtures containing 450 ng/ml of the indicated HRP-conjugated transferrin. The filters were subsequently washed and developed with HRP
substrate mixture as described in the Methods section. hl73, hl74, hl75, hl76 and h44 are representative strains of P. haemolytica whose serotype and source are listed in Table 1. h50 - A. equuli. HRP-bTf,-oTf,-cTf and -eTf - HRP
conjugates of bovine, ovine, caprine and equine transferrins.

A B C D E
1 1 6 ~

49~ ~ ~ i) h44 36-~

1 16 ~

84 i ~ ii) h173 36~

1 1 6--¦

58--~a iii) h175 36 ~

Figure 27. Isolation of receptor proteins with transferrin affinity columns.
Affinity isolation experiments were performed with iron-deficient total membranes prepared from P. haemolytica strain h44 (top panel), hl73 (middle panel) and hl75 (bottom panel). Experiments were performed with bovine transferrin-Sepharose (lanes A and B), ovine transferrin-Sepharose (lane C), caprine transferrin-Sepharose (lane D) or equine transferrin-Sepharose (lane E) using standard washing conditions (lanes B-E) or low salt washing conditions (lane A) as outlined in the methods section. The samples eluted with buffer containing 2M guanidine HCl were dialyzed, concentrated and aliquots analyzed by SDS-PAGE and silver staining as described in the methods section.

A B
2 3 4 5 6 7 1 ' 2' 3' 4' 5' 6' 7' Figure 28. Immunological analysis of receptor proteins from different serotypes of P. haemolytica from bovine, sheep and goats. Aliquots of purified receptor proteins from representative serotypes of P. haemolytica were subjected to SDS-PAGE, electroblotted and then probed with specific anti-TbpB serum (Panel A) or with anti-TbpA serum (Panel B) as described in the methods section. The following P. haemolyfica strains of the indicated serotype were included in the analysis; Lane 1- strain h44 (A1), Lane 2- hl73 (untypable), T ane 3 - hl75 (A7~, Lane 4- hl76 (A9), Lane 5 - hlOO (T4), Lane 6 -hlO6 (T10), and l ane 7 - hlO7 (A11). The numbers on the left represent the molecular weights (X 1000) of standard proteins.

2 1 o~74 ~8 1 hlO5 Figure 29. Binding of labelled transferrin and anti-receptor antibody by intact cells. The indicated bacterial strains were grown under iron-limiting conditions, harvested by centrifugation and resuspend to a A600 of 1-2 in 50 mM TrisHCl, 150 mM NaCl, pH 7.5 buffer. A 5 111 aliquot of the suspensions were applied to HA membrane, the membrane was dried, blocked and then exposed to blocking solution containg labelled transferrin (HRP-bTfl or antireceptor antibody (anti-TbpA, anti-TbpB). The latter membranes were washed and subsequently exposed to labelled second antibody prior to development with substrate.

2 1 6427~

ATGATAATGAAATATCATCATTTTCGCTATTCACCTGTTGCCTTAACAGTGTTATTTGCTC
TTTCTCATTCATACGGTGCTGCGACTGAAAATAAAAAAATCGAAGAAAATAACGATCTAGC
TGTTCTGGATGAAGTTATTGTGACAGAGAGCCATTATGCTCACGAACGTCAAAACGAAGTA
ACTGGCTTGGGGAAAGTAGTGAAAAATTATCACGAAATGAGTAAAAATCAAATTCTTGGTA
TTCGTGATTTAACTCGCTATGACCCTGGTATTTCGGTGGTGGAACAAGGTCGCGGTGCAAG
TAGTGGCTATGCCATTCGAGGTGTAGATAAAAACCGTGTCAGCTTACTTGTTGATGGGCTA
CCACAAGCGCACAGTTATCATACGCTAGGTTCAGATGCTAATGGTGGTGCAATTAATGAGA
TTGAGTATGAAAACATTCGTTCAATTGAGTTAAGCAAAGGAGCAAGTTCTGCGGAATATGG
CTCTGGTGCGCATGGTGGTGCTATTGGTTTTCGTACTAAAGATGCGCAGGATATTATTAAA
GAGGGGCAGCATTGGGGCTTAGATAGTAAGACCTCTTATGCCAGCAAAAATAGCCATTTTT
TACAGTCTATCGCAGCGGCTGGTGAGGCGGGTGGTTTTGAAGCACTTGTTATTGCAACTCA
CCGACACGGTAAAGAGACCAAAATTCATTCCGAGGCAAATAAATTAAAACATAATATTCGG
CGTATAACCGGCTTTGAAAATCGCTACGACTTTACCCAAATTCCGCACAGAATGCTCCTGG
AGGATCTCCTTTTAATTGTGGAAGATACTTGCCCAACATTAGATTGTACTCCTCGTGCAAG
GGTTAAGTTGAACCGCGATAATTTCCCAGTGAGAACATTTCCGGAATATACGCCTGAAGAG
CGCAAACAGCTTGAGCAGATTCCTTATCGCACTGAGCAGCTCTCAGCCCAAGAATATACCG
GTAAAGATCGCATTGCACCAAACCCTTTAGATTACAAGAGTAATTCTC~'l"l~l"l"l'ATGAAGTT
TGGCTATCACTTCAACTCGTCTCATTATCTTGGCGCAATCTTAGAAGATACAAAAACACGC
TACGATATCCGTGATATGCAAACGCCAGCTTACTATACAAAAGACGATATTAACTTATCAC
TTAGGAACTATGTTTATGAAGGGGATAATATTTTAGATGGCTTAGTGTTCAAGCCAAGGAT
CCCTTATGGGTTGCGCTATAGCCATGTGAAG'l"l"ll"l"l'GATGAACGTCACCACAAACGTCGT
TTAGGATTCACCTATAAATATAAACCAGAGAATAATCGCTGGTTGGATAGCATTAAACTCA
GTGCGGATAAACAAGATATTGAACTATATAGCCGGCTACATCGCTTGCATTGTAGCGATTA
TCCTGTGGTAGATAAAAATTGCCGCCCGACTTTGGATAAATCTTGGTCTATGTATCGAACT
GAGCGTAATAATTACCAAGAAAAGCATCGTGTCATTCATTTAGAATTTGATAAAGCGCTAA
ATGCTGGTCAAGGCGTATTTAACCAAACCCACAAACTGAATTTAGGGTTGGGCTTTGATCG
ATTTAATTCGCTTATGGATCATGGGGATATGACTGCCCAATATACCAAAGGCGGTTATACC
AGCTACCGCGGTAGAGGGCGTTTAGATAATCCATATATTTATCGCCGCGATCCACGCAGTA
TTGAAACGGTATCTTTGTGTAATAATACACGCGGCGACATCTTAAACTGTGAACCGCGTAA
AATTAAAGGCGATAGCCATTTTGTTAGCTTCCGCGATCTAGTGATAAGCGAGTATGTGGAT
TTGGGATTAGGGGTGCGTTTTGATCAACATCGATTTAAATCTGATGATCCGTGGACACTTA
GCCGAACTTATCGAAATTGGTCTTGGAATGGTGGGATTACGCTTAAACCAACAGAGTTTGT
ATCGCTTTCTTATCGCATTTCAAACGGTTTTAGAGTGCCTGCATTCTATGAACTTTATGGT
AAACGTGATCATATTGGGCTTAAAGATAACGAATATGTGCAACGCGCGCAACGTAGCCACC
AGTTAGAGCCAGAAAAATCGACTAATCATGAGATTGGAGTTAGCTTTAAAGGTCAATTTGG
TTACCTTGATGTGAGCTATTTCCGTAATAACTATAAAAATATGATTGCGACAGCATGTAAA
AGAATAATACAAAAATCACACTGTTTCTATAACTACCATAATATTCAAGATGTAGCACTAA
ACGGGATAAATTTAGTCGCTAAATTTGACTTACACGGTATTTTATCTATGCTGCCAGATGG
TTTTTATTCATCAGTTGCTTATAACCGTGTAAAAGTAAAAGAGCGGAAACTAACCGACTCA
AGACTCGATAGCGTAAACGATCCTATTCTAGATGCGATTCAGCCAGCACGCTATGTGCTTG
GATTCGGCTACGATCACCCAGAAGAAAAATGGGGAATTGGCATTACTACCACCTATTCTAA
AGCCAAAAACGCCGATGAGGTGGCAGGCACACGTCATCACGGNATACATCGCGTTGATTTA
GGTGGCAAACTGACCGGTTCTTGGTACACCCATGATATTACCGGTTACATCAATTATAAAA
ACTACACCTTACGTGGAGGAATTTATAATGTGACTAATCGTAAATATTCCACTTGGGAATC
AGTGCGCCAATCCGGTGTGAATGCAGTAAACCAAGACCGGGGTAGCAATTACACTCGATTT
GGCGCTCCGGGGAGAAATTTCAGTTTAGCATTTGAAATGAAGTTTTAG

Figure 30. DNA sequence of the tbpA gene from Pasteurella haemolytica strain hl96.

~ t ~42~4 lMIMKYHHFRYSPVALTVLFALSHSYGAA

931~

Figure 31. Predicted amino acid sequence of the TbpA protein from Pasteurella haemolytica strain hl96. Underlined amino acids correspond to the N-terminal amino acids of the mature protein determined by protein seqeunce analysis.

- l~g-2t 64274 .

A~ lAAACTTAAAAGTA~l-l~ AcTGcTTAATGcGGcGcTAcTI ~ :l1~11~1 CAAATG&TGGAAGClTl~ i'll~AATCTGCCAAAGTlY~AATCTCAAACGCAAACTACCCC
CAAAAAGCCAAGTTTACAAGATGATAATAGTAACGCAA~ACGTACAGTAAGCGCTTCTGAA
ACTGAAGCTTTATTGCAGCCGGG&TTTGG'lllll~AGCCAAAATTCCGCGTCGTAATCTCC
TTCC ~ lGT ~ CCTATT&GT&ATAT~AAAC.Ar~TTAClY~GAGATCT
GCCAAAAATTCCGI~r~AG~r~lAA-GCGTGCGGTAGTAGTGCT&AT&GATTTAGC
CATACTCATGATAGAAATCATAAC;l1~lATAcAAGAGATTTTAA~ Gll~C( ~ T
ATGTTGTGcATTcTGGTccAAAAccTGAAATAAAGcCTAAAGAAATTTTGAGAACAGGTGc ACATGGGTAl~lllaC5~511~rCrAT~GAGCCGCCCAAAGCAATACCTACCCAAAAACTA
ACT5~n.~XOo,U~ o~A~TACCTATGCGGCTAAGGGGAGAGATAGTAATATTT
TTCTAATTCCCGCAGGCATCAATAGT&GCGCCATACCGGAAAATAGTCACGATATTAATGT
TGATGATTCTGAAAAACCAATGGGGCATACAGGAGAATTTACGGCTGATTTTGCTAATAAA
ACTTTAACTGGAACATTGGTTCGTAATGG&TATGTTAGTCGTAGCAAAGAGCaaaAAATTA
CAACAATTTACGATATTGATGCGAAAATTAAAGGTAATCGCl'll~l~l~GTAAAGCAAACCC
AAAAAAACCGATGATCCTTATTTTTGGGAAAAGCTCCACGACACTTGAAGGTGGAl-l-lll-l G&TG&GGAGGcTcAAGAAcTTGccG&TAAATTcTTAGcTGAT&ATAAGTcGGTA~
'l'l'l'll~CTGGCACACGAGATGCTAAAAAAGATGATAGTGAATCTGCCTTTGATGCTTTCCC
AATTAAAcTTAAAGATTTAAATAAATcl~AGATGGATA~ITTcGGGAAT&cGAcAcATTTG
ATTATTAACAATAAGCAGATTCCACTTATTGCGGAAGCCACAAAAAGCTTTGCCGAGATGA
AATlTGATGATTTGGT~AcccGTAcTATTGATGGAAAAAcGTATcGAG111CAGTCTGCqG
TAATAATTTAGATTATGTCAAATrT~GGATTTATAGCGAGGGAAATAATAGTGATACT&CT
CTCCAAGAATATTTAGTA&GAGAACGTACAGCTCTGGCAGATTTGCCAACAGGGACAGTAA
AATATCGAGGTAcTrGGGACGGG&TAATGTAcAGTAAATCTGGCTCGGCAGGGGTTGAATC
GCCAAGTAACAGCGAAAGTGGTACTCGTTCACTATTCGATGTAGA'l-l~ AATAAAAAA
A'IYAATGGCAAGCTGATTGCTAATGATGGl~ll~AAGAACGCCCAATGCTGACACTGGAAG
GCAATCTGAAAGGGAATG~l-l~ll~GAGGCACAGCCAAAACGGGCAATTCTG~ l'AATCT
TGATCCCAAAAGTACGAAT~GTGGCACGGTAGGGCATATAAATACTCAATTTGAAGGGGGC
TTTTATGGCCCTAAGGCGACGGAATTAGGTGGTATTGTACAAAATACAGAAACGGATAAAG
ATAGAGTCAGTATTACATTCGGCGGAAAACGTCAAATAGAAAAATAA

Figure 32. DNA sequence of the tbpB gene from Pasteurella haemolytica strain hl96.

241 P A N R T L T G T L V R N G Y V S R S R E Q K r T T I Y D I

:~01 E G G F F G G E A Q E L A G R F L A D D K S V F V V F A G T

391 L V T R T I D G R T Y R V S V C C N N L D Y V K F G I Y S E:

Sll L R G N G F G G T A R T G N S G F N L D P R S T N G G T V G

571 R V S I T F G G R R Q I E K ~

Figure 33. Predicted amino acid sequence of the TbpB protein from Pasteurella haemolytica strain hl96. Underlined amino acids correspond to the predicted N-terminal amino acids of the mature protein determined by protein seqeunce analysis.

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25 Interaction of ruminant transferrin receptors in bovine isolates of 2 ! 64274 Pasteurella haemolytica and Haemophilus somnus. Infect.Immun.
60:2992-2994.
Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those 5 skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim all modifications coming within the scope of the following claims.
All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent 10 as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims (16)

1. A purified and isolated nucleic acid molecule comprising a sequence encoding TbpA of P. haemolytica.

2. A purified and isolated nucleic acid molecule as claimed in claim 1, having the amino acid sequence as shown in Figure 31.

3. A purified and isolated nucleic acid molecule as claimed in claim 1, comprising the nucleotide sequence as shown in Figure 30.

4. A purified and isolated nucleic acid molecule comprising a sequence encoding TbpB of P. haemolytica.

5. A purified and isolated nucleic acid molecule as claimed in claim 4, having the amino acid sequence as shown in Figure 33.

6. A purified and isolated nucleic acid molecule as claimed in claim 4, comprising the nucleotide sequence as shown in Figure 32.

7. A naturally occurring nucleic acid molecule which is characterized by the ability to hybridize to the purified and isolated nucleic acid molecule as claimed in claim 1 or 4 under stringent hybridization conditions.

8. An oligonucleotide comprising at least 15 contiguous bases of a nucleic acid molecule as claimed in claim 1 or 4 which is characterized by the ability to hybridize to the nucleic acid molecule under stringent hybridization conditions.

9. A recombinant expression vector adapted for transformation of a host cell comprising a nucleic acid molecule as claimed in claim 1 or 4 and one or more transcription and translation elements operatively linked to the nucleic acid molecule.

10. A host cell containing a recombinant expression vector as claimed in claim 9.

11. A purified and isolated TbpA of P. haemolytica.

12. A purified and isolated TbpA as claimed in claim 11, which has the amino acid sequence as shown Figure 31.

13. A purified and isolated TbpB of P. haemolytica.

14. A purified and isolated TbpB as claimed in claim 13, which has the amino acid sequence as shown Figure 33.

15. Antibodies having specificity against an epitope of TbpA
or TbpB of P. haemolytica.

16. A vaccine for augmenting the immune response of a ruminant to P. haemolytica comprising at least one of TbpA and TbpB of P.
haemolytica and a pharmaceutically acceptable carrier.