EP0738325A1 - Adhesines bacteriennes specifiques de recepteurs et leur utilisation - Google Patents

Adhesines bacteriennes specifiques de recepteurs et leur utilisation

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Publication number
EP0738325A1
EP0738325A1 EP95906910A EP95906910A EP0738325A1 EP 0738325 A1 EP0738325 A1 EP 0738325A1 EP 95906910 A EP95906910 A EP 95906910A EP 95906910 A EP95906910 A EP 95906910A EP 0738325 A1 EP0738325 A1 EP 0738325A1
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EP
European Patent Office
Prior art keywords
adhesin
fimh
val
gly
thr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP95906910A
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German (de)
English (en)
Inventor
Evgeni Veniaminovic Sokurenko
David Long Hasty
Per Klemm
Lars Pallesen
Soren Molin
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GX BioSystems AS
University of Tennessee Research Foundation
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GX BioSystems AS
University of Tennessee Research Foundation
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Publication of EP0738325A1 publication Critical patent/EP0738325A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)

Definitions

  • the present invention pertains to naturally occurring bacte ⁇ rial adhesins and derivatives and variants hereof, having the ability to bind to pre-determined, specifically selected receptors, and to the use of such adhesins in the targeting of active compounds and microbial cells to locations compris ⁇ ing such selected receptors.
  • adhesion of bacteria to host surfaces is common ⁇ ly regarded as an essential step enabling bacteria to become established as members of the normal flora of host organisms or to cause an infection (refs. 7, 18) .
  • Bacterial lectins are the most common and most thoroughly studied type of adhesins among both gram negative and gram positive bacteria (ref. 40) .
  • Evolutionary pressures have selected lectins for adhes ⁇ ive functions probably due to the abundance of glycoconjugat- es on animate and inanimate surfaces.
  • CFA Colonization Factor Antigen
  • Type 1 fimbriae are heteropolymers of four different subunits (refs. 28, 44). For each fimbria, about 1000 copies of a 17-kDa primary structural subunit designated Fi A (or PilA) , are polymerized into a right-handed helix surrounding a hollow axial core (ref. 11) . Three ancillary subunits, FimF, FimG and FimH, are also polymerized into the fimbrial structure, but comprise only 1-3% of the fimbrial mass (refs. 20, 24, 27, 32) .
  • the 28 kDa FimH subunit has been shown by several direct and indirect tests to be the actual fimbrial lectin (refs. 2, 4, 20, 21, 27, 29, 32, 36, 55), although its function may be affected by other subunits (ref. 55) .
  • the FimA subunit is highly variable, but the FimH subunit is highly conserved antigenically and genetically among enterobacteria (ref. 1) .
  • Interactions between type 1 fimbriae and D-mannose-containing receptors have been shown in a number of studies to play a key role in the infectious process (refs. 2, 4, 9, 19, 25, 26, 31, 33, 44, 50) .
  • the combining site of the type l adhesin is in the form of an extended pocket corresponding to the size of a trisaccharide and fitting best the structure ⁇ -D- Manp- (1-3) -0-D-Manp- (1-4) -D-GlcNac (ref. 16).
  • a hydrophobic region within or close to the combining site was also pre ⁇ dicted in these studies.
  • a similar pattern of specificity was found independently in indirect adhesion-inhibition studies, as well as in direct adhesion studies using "neoglycolipids" as receptors (refs.
  • CSH-50 exhibits mannose-sensitive peptide-binding activity (ref. 51) .
  • CSH-50 E. coll bound to yeast mannan (Mn) , a highly mannosylated glycoprotein, and to human plasma fibronectin (Fn) when immobilized on assay wells. Adhesion to Mn, but not to Fn, was essentially elimin ⁇ ated by periodate treatment. Furthermore, CSH-50 E. coli adhered in a mannose-sensitive fashion to non-glycosylated peptide fragments of Fn and to a synthetic peptide copying the first 30 residues of the Fn molecule, FnSpl.
  • Fimbriae purified from these organisms also bound to Fn and FnSpl.
  • a well-characterized recombinant strain of E. coli PC31 expres ⁇ sing type 1 fimbriae, HB101(pPKL4) , adhered to Mn, but did not adhere to the other substrata.
  • Fimbriae purified from HB101(pPKL4) did not adhere to Fn or FnSpl.
  • E. coli type 1 fimbriae appeared to be functionally heterogeneous.
  • E. coli isolates obtained from human urine also expressed peptide-binding activity similar to that of CSH-50, indicating that this new phenotype was not restricted to a laboratory strain.
  • Other isolates expressed an adhesive activity similar to that of HB101(pPKL4) .
  • a third class of type 1 fimbriae-mediated adhesive phenotype was also observed among these isolates.
  • the FimH subunit is the D-mannose-sensitive adhesin of type 1 fimbriae, common i.a. to the Enterobacteriaceae . It is pres- ently widely accepted that host receptors are strictly limited to glycoproteins containing terminal mannosyl resi ⁇ dues (refs. 16, 37, 41, 42, 43, 47) . Hereinbelow functional and genetic evidence is provided demonstrating that this generalization is not correct. Allelic variants of E. coli fimH genes encoding proteins differing by as little as a single amino acid substitution confer distinct adhesive phenotypes and accordingly, the fimH gene is not a single gene but rather a family of fimH genes.
  • active receptors for FimH proteins were found to include glycoprotein domains where mannosyl residues are not terminal and even protein domains devoid of saccharide.
  • This unexpected adhesive diversity within the fimH family broadens the scope of potential receptors for bacterial adhesion and may lead to a fundamental change in the under- standing of the role(s) type 1 fimbriae and other bacterial adhesins may play in bacterial ecology or pathogenesis.
  • the present findings also opens up a completely new field of technology, since it provides the means to design bacteria expressing adhesins that bind to pre-determined, specific receptors in a wide range of animate and inanimate locations.
  • This new technology is referred to herein as Designer Adhesin Technology.
  • the present invention relates in one aspect to a recombinant or mutant bacterial adhesin variant derived from a naturally occurring adhesin, said adhesin variant having altered binding properties relative to the naturally occur ⁇ ring adhesin from which it is derived.
  • the invention provides a FimH adhesin having an amino acid sequence which differs from the E. coli PC31 FimH adhesin by at least one amino acid and a recombi ⁇ nant replicon comprising a DNA sequence selected from the group consisting of a sequence coding for a recombinant bacterial adhesin variant as defined above and a sequence coding for a FimH adhesin as also defined above.
  • a fusion protein comprising an adhesin selected from the group consisting of a recombinant bacterial adhesin variant as defined above and a FimH adhesin as also defined above, and a heterologous polypeptide.
  • the invention also pertains to a recombinant bacterial cell which expresses an adhesin selected from the group consisting of a recombinant bacterial adhesin variant as defined above and a FimH adhesin as defined above, and to a composition comprising a population of such cells.
  • a method of isolating a bacterial cell expressing an adhesin having modified binding properties relative to a natively expressed adhesin comprising identifying in the bacterial cell DNA sequence(s) coding for the binding domain(s) of said natively expressed adhesin and substituting at least one codon herein, whereby a modified adhesin molecule is expressed that is different in at least one amino acid from the adhesin expressed natively, and selecting a bacterial cell expressing the modified adhesin having an altered adhesion phenotype relative the natively expressed bacterial adhesin.
  • the invention relates to a method of preparing a recombinant bacterial cell that binds to a specific receptor moiety, comprising introducing into a bacterium that does not produce an adhesin binding to said receptor moiety, a DNA sequence coding for an adhesin binding to the receptor moiety, and selecting a bacterial cell ex ⁇ pressing the DNA sequence.
  • a method of targeting a bacterial adhesin to a specific location comprising (i) identifying in said location an adhesin-interacting receptor moiety which is recognizable by bacterial adhesins, said moiety preferably being one which is occurring abundantly, (ii) isolating a bacterial cell that grows in said location and expresses an adhesin recognizing and interacting with said receptor moie ⁇ ty, and administering to the location the bacterial cell or the adhesin under conditions where the adhesin and the receptor moiety are brought into interacting contact whereby the adhesin is associated with the receptor moiety.
  • bacterial adhesins denotes proteins which recognize and bind to a large variety of target mole- cules such as polysaccharides, glycolipids, glycoproteins, polypeptides and proteins. More than a hundred different adhesins have been described so far originating from a large variety of gram-negative and gram-positive bacteria. Adhesins can be present on the bacterial surface as components of organelles such as fimbriae, also called pili or fibrillae, these three terms being used interchangeably herein, or as non-fimbrial or afimbrial adhesins (ref. 64) . Examples of fimbrial or pili adhesins include the following surface structures in E.
  • coli P pili, type 1 fimbriae, S pili, K88 pili, K99 pili, CS3 pili, F17 pili and CS31 A; in Klebsiella pneumoniae: type 3 pili; in Bordetella pertussis: type 2 pili; in Yersinia enterocolitica: Myf fibrillae; in Yersinia pestis: pH6 antigen and FI envelope antigen.
  • non-fimbrial cell surface structures which have adhesin function or which may comprise proteins having such a function include capsules, lipopolysaccharide layers, outer membrane proteins, NFA (non-fimbrial adhesin) -1, NFA-2, NFA- 3, NFA-4, AFA (afimbrial adhesins)-!, AFA-II and AFA-III.
  • Fimbriae designates long thread-like bacterial surface organelles. Fimbriae are hete- ropolymers each consisting of about 1000 structural compo ⁇ nents, mostly of a single protein species. However, in many cases a few percent minor components are also present.
  • Adhes ⁇ ins can either be identical to the major structural protein as in Escherichia coli K88 and CFA1 fimbriae and type 4 fimbriae of Pseudomonas, Vibrio and Neisseria, or they may be present as minor components as in E. coli type 1 and P fimbriae [for reviews see Krogfelt 1991 (ref. 62) ; Kaufman and Taylor, 1994 (ref. 60): Kuehn et al., 1994 (ref. 63); Klemm and Krogfelt, 1994 (ref. 61)]. In the latter case, i.e.
  • adhesins when present as minor compounds, the adhesins are closely related in amino acid sequence to the major fimbrial compo- nent.
  • bacterial adhesin will also include adhesins isolated from non-bacterial sources includ ⁇ ing viruses, and which are expressed in a bacterium.
  • FimH adhesin of type 1 fimbriae will be described structurally and functionally as a representative example of a bacterial adhesin.
  • FimH is located at the tip of the type 1 fimbriae and also intercalated at intervals in the fimbrial organelle. Most forms of the FimH adhesin target to (bind to) oligosaccharide structures containing terminally located ⁇ -D-mannoside resi- dues [Krogfelt et al., 1990 (ref. 29)]. Based on studies with various D-mannose derivatives the receptor binding site of the FimH adhesin is assumed to be shaped like an elongated pocket large enough to accommodate a trisaccharide motif [Sharon, 1987 (ref. 65)].
  • the fimH gene encodes the precursor FimH protein of 300 amino acids [Klemm and Christiansen, 1987 (ref. 27)]. This precur ⁇ sor is processed into a mature form of 279 amino acids.
  • the amino acid sequence of the E. coli PC31 FimH protein is shown in Table 1 below wherein cysteine residues are indicated by asterixes, the signal peptide is outlined in bold letters, and two regions contributing to the binding site are under ⁇ lined (SEQ ID NO:l) . (It should be noted that residue 176 is a proline residue and not as previously indicated when the PC31 FimH protein was first published, an arginine residue) :
  • FimH contains 4 cysteine residues assumed to direct folding of the molecule into distinct functional domains.
  • FimA and the minor components FimF and FimG only have two cysteine residues.
  • the localization of the cysteine residues in FimH points to a tandem arrangement of two ances ⁇ tral genes.
  • similar amino acids can be found in similar positions in the two halves of the FimH protein.
  • the "midway" point is located roughly around residue 150 in the mature protein.
  • the two halves or domains of FimH have evolved differently with the N-terminal section becoming the domain harbouring the receptor binding site, whereas the C- terminal sector became the domain of the molecule required for integration into the fimbrial organelle structure, i.e. having the features of a structural component.
  • the recombinant bacterial adhesin as defined above is one which is derived from an adhesin having certain binding properties, but which recombi- nant bacterial adhesin has altered binding properties rela ⁇ tive to the naturally occurring adhesin (the parent adhesin) from which it is derived.
  • this feature encompasses situations where the adhesin variant recognizes and binds to receptor moieties not being recognized by the parent adhesin irrespective of whether the adhesin variant has lost its normal ability to recognize and bind to a cer ⁇ tain receptor moiety or certain receptor moieties, or not.
  • binding indicates that the adhesin has a degree of affinity to the receptor moiety which enables it, when brought into contact herewith, to interact in a binding manner with this moiety whereby an adhesin-receptor moiety association occurs.
  • the strength of this binding depends on the type of binding force which causes the inter ⁇ action between the receptor moiety and the adhesin.
  • binding forces include covalent binding and binding by non-covalent binding forces including hydrogen bonds, hydrophobic interactions, van der Waal forces and ionic interactions.
  • receptor moiety as used herein encompasses any moiety to which an adhesin may interact by the above binding forces.
  • the adhesin variant is a FimH mannose-sensitive adhesin normally binding to a receptor selected from a domain where mannosyl residues are not ter ⁇ minal and a domain devoid of saccharide and having an amino acid sequence which differs from the E. coli PC31 FimH adhes- in by at least one amino acid residue substitution, including an amino acid sequence differing by at least 2 amino acids, preferably by at least 3 amino acids, more preferably by at least 4 amino acids, most preferably by at least 5 amino acids.
  • the amino acid sequence may even differ by more than 5 amino acids such as at least 6, preferably by at least 7, more preferably by at least 8, even more preferably by at least 9 and in particular by at least 10 amino acid residues, such as by at least 12 amino acids including by at least 15.
  • the above FimH adhesin variant is preferably at least 90% homologous to the PC31 FimH adhesin, such as at least 92% homologous, more preferably at least 93% homologous, even more preferably at least 94% homologous, most preferably at least 95% homologous, and in particular at least 96% homologous, e.g. at least 97% homologous.
  • the adhesin is at least 98% homologous, including at least 99% homologous such as at least 99.5% homologous.
  • the above FimH adhesin variant can be a chimeric adhesin comprising amino acid sequences from different FimH adhesins having identical or different binding specificities.
  • the present invention is generally aimed at providing the means to design bacterial adhesins having specific binding properties whereby bacteria expressing the adhesin variants or the adhesin variants in isolated or purified form can be designed to bind to a speci ⁇ fic desired target receptor moiety.
  • the adhesin variant may in accordance with the invention be an adhesin variant as defined above which binds to an animate receptor moiety.
  • Such receptors include receptors located on inner surfaces of humans and animals, such as e.g. the mucosal membranes of the gastrointestinal tract including the teeth and the oral cavity, and the mucosal membranes of the respir- atory and the genitourinary systems. Included are also adhesin variants that bind to outer surfaces, including the skin, of humans and animals.
  • the adhesin variant is designed so as to acquire the ability to bind to a plant receptor moiety.
  • This aspect is of particular interest in relation to deliber ⁇ ate release to out-door or in-door environments where plants are cultivated, of useful recombinant bacteria having a desirable effect on the growth and yield of the plants.
  • useful bacteria are e.g. bacteria expressing a pesti- cidally active substance, i.e. a biopesticide including as examples pesticidal toxins produced naturally by Bacillus spp such as the Bacillus thuringiensis (Bt) toxin.
  • Bacillus spp Bacillus thuringiensis
  • Bacillus thuringiensis (Bt) toxin another example is bacteria which protect plants against low temperature damages or bacteria which express gene products protecting plants against detrimental effects of herbicides.
  • these useful bacteria will, when administered to the plant growing envi ⁇ ronment, be selectively associated with the target plant species or a specific target area of the plant. It may thus be desirable to have these useful bacteria administered to the leaves of the plants or to have the root system colonized herewith.
  • the present invention encompasses adhesin vari ⁇ ants as defined herein which bind selectively or specifically to a phylloplane receptor moiety or which bind to receptors on plant roots.
  • adhesin variants can be provided which are targeted to the stem or the flowers of the plants.
  • bacterial adhesins include adhesins having an inherent capability to bind or interact with inani ⁇ mate surfaces carrying receptor moieties with which the adhesin can interact to become bound to the surfaces. It is known that certain bacterial adhesins can bind to inanimate surfaces including as examples glass, hydroxyapatite (a tooth enamel model compound) or polymer structures including plas ⁇ tics and polysilicates.
  • the present invention has made it possible to design bacteria which bind selectively to any inanimate surface which carries a receptor moiety for which an adhesin variant binding thereto may be constructed. Accor ⁇ dingly, the present invention also provides an adhesin vari ⁇ ant as defined herein which binds to an inanimate receptor moiety.
  • adhesin variants are particularly interesting in connection with the concept of bioremediation, i.e. a tech ⁇ nology designed to enhance degradation of chemical pollutants in the environment. It is clearly a significant improvement of this technology to have at hand bacteria which comprise genes coding for pollutant-degrading gene products and which also express adhesins targeting the bacteria selectively to the environment where the pollutants are present, e.g soil, aquatic environments and drinking water supply systems. Furthermore, adhesin variants capable of binding to tooth enamel are useful in the protection of teeth against caries.
  • an adhesin variant which is part of a fusion protein comprising the adhesin variant and a non- adhesin, heterologous polypeptide.
  • FimH as an example, it has been found that fusions between a bacterial adhesin and other proteins can be made whereby the resulting fusion proteins are inserted into the cell surface organelle of which the adhesin is a structural part.
  • the non-adhesin region of a fusion protein comprising an adhesin variant as defined herein include a heterologous polypeptide which is selected from an epitope, an enzyme, a toxic gene product and an antibody.
  • the heterologous polypeptide is an ' epitope or an epitope-carrying domain forming an integrated part of the fusion protein, and thus presented on the surface of the host cell expressing the fusion protein, the epitope- carrying polypeptide can be presented in a conformational form similar to its natural conformation.
  • the above fusion proteins can be overproduced by the bacteria compris- ing hybrid genes coding for fusion proteins, resulting in excretion of the fusion proteins to the growth medium in large quantities. Accordingly, the excreted fusion proteins are then readily isolated and purified, e.g. by means of affinity chromatography.
  • adhesin variant-fusion pro ⁇ teins is useful for a range of industrially important pur ⁇ poses such as:
  • fusion proteins as carriers of non-covalently linked chemical moieties whereby the adhesin part of the protein is used to target the chemical moiety to specific locations and the non-adhesin part carries and then releases the moiety when the fusion protein has reached its target.
  • chemical entities which may be linked to the fusion protein include imaging agents and pharmacologically active components. Examples of applications for this use include imaging of atherosclerotic plaques or tumor tissues, and delivery of chemical agents at specific locations in or on microbial, human, animal or plant cells including specific tissues or tissue components;
  • E. coli FimH adhesin is not a single gene but rather a family of fimH genes, and accordingly it has now been established that allelic variants of E. coli fimH genes exist that encode adhesin proteins which, relative to the known E. coli PC31 fimH gene product differ by as little as a single amino acid substitution and confer distinct binding or adhesive phenotypes.
  • the present invention relates in a further aspect to a FimH adhesin having an amino acid sequence which differs from the E. coli PC31 FimH adhesin as defined above by substitution of at least one amino acid.
  • an adhesin encompasses naturally occurring adhesins as well as adhesins which are encoded by recombinant or mutant fimH genes.
  • the term "fimH gene” denotes a gene coding for a gene product which can be integrated into a type 1 fimbria and which confers to the fimbria the ability to recognize and bind to a receptor.
  • the FimH adhesin as defined above may be an adhesin having its inherent binding properties or an adhesin variant which in relation to an adhesin encoded by a naturally occurring gene from which the gene coding for the adhesin variant is derived, has altered binding properties. Furthermore, the FimH adhesin may be either mannose-sensitive or mannose- insensitive. The term "mannose-sensitive" is used herein to designate that the binding of an adhesin is inhibited in the presence of mannose residues.
  • the FimH adhesin may be a FimH adhesin normally binding to a receptor moiety selected from a domain where mannosyl resi- dues are not terminal and a domain devoid of saccharide such as e.g. a glycolipid, a glycoprotein, a protein, a polypeptide and a peptide, including a hormone.
  • a FimH adhesin according to the present invention may bind include as examples animal proteins such as a casein including /c-casein, a gelatine, a globin, an albumen and a collagen, and vegetable proteins including soy protein.
  • the FimH adhesin according to the invention include an adhes ⁇ in having an amino acid sequence which differs from the E. coli PC31 FimH adhesin by at least 2 amino acid residues, such as an amino acid sequence differing by at least 3 amino acids, preferably by at least 4 amino acids, more preferably by at least 5 amino acids, most preferably by at least 6 amino acids.
  • the amino acid sequence may even differ by more than 6 amino acids such as at least 7, preferably by at least 8, more preferably by at least 9, even more preferably by at least 10 and in particu ⁇ lar by at least 11 amino acid residues, such as by at least 12 amino acids including by at least 15.
  • the above FimH adhesin is preferably at least 90% homologous to the PC31 FimH adhesin, such as at least 92% homologous, more preferably at least 93% homologous, even more preferably at least 94% homologous, most preferably at least 95% homologous, and in particular at least 96% homologous, e.g. at least 97% homologous.
  • the adhesin is at least 98% homologous, including at least 99% homologous or at least 99.5% homologous.
  • the FimH adhesin as defined above is one which, when tested for binding to yeast mannan (Mn) , human plasma fibronectin (Fn) , periodate treated Fn and the synthetic peptide FnSpl comprising the first 30 amino acids of Fn, only binds to Mn.
  • Mn yeast mannan
  • Fn human plasma fibronectin
  • FnSpl synthetic peptide FnSpl comprising the first 30 amino acids of Fn
  • FimH adhesin is an adhesin which, when tested for binding to the above compounds, binds to Mn and Fn (MF class FimH adhesin) or an adhesin which among these compounds bind to all of these (MFP class FimH adhesin) .
  • bacteria expressing FimH adhesins of the above MFP class bind in a mannose-sensitive (MS) manner to polyoxyethylene sorbitan monolaurate (Tween 20) and a little less well to polyoxyethylene sorbitan monooleate (Tween 80) .
  • bacteria expressing MFP class FimH adhesins make a much tougher pellicle than bacteria expres ⁇ sing other types of adhesins.
  • the term "pellicle” indicates a layer or film of bacteria that forms at the air/liquid interface of a liquid growth medium. This noticeable phenomenon might be of particular interest where there is a reason to concentrate microorganisms at the surface of an aquatic environment, such as e.g.
  • bacteria expressing a MFP class adhesins bind to hydroxyapatite to a higher degree than do bacteria expressing a M class adhesin.
  • Hydroxyapatite, especially saliva-treated hydroxyapatite is i.a. used as a model for tooth enamel, and accordingly, this finding indi ⁇ cates that bacteria expressing MFP class adhesins are par ⁇ ticularly useful in bacterial compositions intended for colonization of teeth.
  • MFP class adhesins bind to a large range of synthetic peptides and accordingly seem to have a broad specificity in terms of amino acid motifs.
  • the FimH adhesin is an adhesin which, when tested for binding to the five Fn-fragments obtained by thermolysin treatment as it is described in reference No. 51, only binds to the 40-kDa gelatin-binding fragment or which binds to all of these Fn- fragments, or to none of these.
  • M L low adhesive
  • M L mannose-sensitive adhesin
  • adhesins of this low adhesive M L class adhere poorly to MDCK, buccal cells and erythrocytes as compared with M class adhes ⁇ ins.
  • Example of a M class adhesin is one expressed by the recombinant E.
  • M class adhesins adhesion is found that is not sensitive to inhibition by D- mannose.
  • M R adhes ⁇ mannose-insensitive (or mannose-resistant) M class adhesin is designated in the following as an M R adhes ⁇ in.
  • M R adhes ⁇ mannose-insensitive (or mannose-resistant) M class adhesin.
  • a FimH adhesin as defined above can be a chimeric adhesin comprising amino acid sequences from different FimH adhesins.
  • Such chimeras are constructed e.g. by providing multiple restriction fragments of a fimH gene, followed by exchanging under ligation condi ⁇ tions these fragments with corresponding fragments of an other fimH gene and cloning the ligation product as it is described in Example 1 below.
  • recombinant plasmids containing such chimeric fimH genes can be transformed into a host cell and transformants tested for adhesive phenotype, allowing determination of the regions of each gene capable of conferring functional activity (Fig. 5) .
  • the invention encompasses in one embodiment a FimH adhesin comprising an amino acid sequence which differs from the E. coli PC31 FimH adhesin by at least one amino acid occurring between residues 27 and 119 of the mature FimH sequence, including a FimH adhesin comprising an amino acid sequence which differs from the E. coli PC31 FimH adhesin by at least one amino acid occurring between residues 33 and 78 of the mature FimH sequence.
  • the selected potential receptors for a FimH adhesin as defined above include those animate and inanimate receptors mentioned above for a recombinant bacterial adhesin variant and the potential uses of the FimH adhesins are also the same as those uses described above for this recombinant bacterial adhesin variant.
  • the invention relates in a further aspect to a recombinant replicon comprising a DNA sequence coding for a recombinant bacterial adhesin variant as defined herein or a DNA sequence coding for a FimH adhesin as also defined herein.
  • a replicon is suitably selected from a chromo- some or a plasmid.
  • the DNA sequence includes a sequence which is inserted by conventional recombination techniques such as insertion by means of restriction enzymes and subsequent ligation, or the DNA sequence is provided by subjecting a replicon comprising a naturally occurring sequence coding for an adhesin to a mutagenization procedure including site- directed mutagenesis, insertion of a transposable element, mutagenization by radiation or chemical mutagenization, followed by selection of cells comprising a mutated sequence conferring altered binding properties relative to a cell comprising the wild-type sequence.
  • the recombinant replicon is one having a broad host range including bacterial species na ⁇ turally occurring in soil, in aquatic environments, on inner and outer surfaces of humans and animals, and which is com- patible with replicons occurring in potential host strains.
  • the recombinant replicon as defined above is one wherein the DNA sequence codes for a FimH adhes ⁇ in having an amino acid sequence which differs from the E. coli PC31 FimH adhesin by at least one amino acid, including an adhesin having an amino acid sequence which differs from the E. coli PC31 FimH adhesin by at least 2 amino acid resi ⁇ dues, such as an amino acid sequence differing by at least 3 amino acids, preferably by at least 4 amino acids, more preferably by at least 5 amino acids, most preferably by at least 6 amino acids.
  • the amino acid sequence may even differ by more than 6 amino acids such at least 7, preferably by at least 8, more preferably by at least 9, even more preferably by at least 10 and in particu- lar by at least 11 amino acid residues, such as by at least 12 amino acids including by at least 15.
  • the above recombinant replicon preferably com ⁇ prises a DNA sequence coding for a FimH adhesin which is at least 90% homologous to the PC31 fimH gene, such as at least 92% homologous, more preferably at least 93% homologous, even more preferably at least 94% homologous, most preferably at least 95% homologous, and in particular at least 96% homologous, e.g. at least 97% homologous.
  • the adhesin is at least 98% homologous, including at least 99% homologous such as at least 99.5% homologous.
  • the above replicon comprises a DNA sequence which is a chimeric fimH gene as it has been defined above, comprising DNA from different fimH genes.
  • the replicon can also be one which comprises a further DNA sequence e.g. derived from a microorganism selected from a bacterium, a virus, a protozoan, a fungus and a yeast.
  • This further DNA sequence is e.g. one coding for a heterologous polypeptide, including an epitope, an antibody, a toxic gene product, an enzyme, a pesticidally active gene product and a pollutant- degrading gene product.
  • the replicon as defined herein com ⁇ prises a DNA sequence which is isolated from an -EnteroJbacte- riaceae species, including a DNA sequence which is isolated from E. coli , a Klebsiella sp., an Enterobacter sp., a Yersinia sp. or a Salmonella sp.
  • sequence in addition to being a DNA sequence as defined above, the sequence can be a synthetic sequence constructed by conven- tional techniques of DNA synthesis.
  • the present invention encompasses a fusion protein comprising a recombinant bacte ⁇ rial adhesin variant or a FimH adhesin as defined above, and a heterologous polypeptide.
  • a polypeptide is in useful embodiments an immunologically active gene product i.e. an epitope (antigenic determinant) from a pathogenic organism, which polypeptide, when administered to the body of a human or an animal is capable of stimulating the formation of antibodies therein.
  • a cell in which such an epitope is expressed is advantageously utilized in the preparation of live vaccines.
  • Such vaccines have several advantages over known live vaccines:
  • the epitope forms a structural part of an adhesin which is embedded in a surface organelle of the vaccine cells.
  • the hybrid DNA sequence coding for the epitope further comprises the means for transporting the epitope, when expressed, to the outer surface of the cell, i.e. translocating it through the cell membrane.
  • This is immunologically highly advantageous, since the epitope will be brought more closely in contact with immunologically competent cells of the body to which the fusion protein- expressing vaccine cells are administered.
  • the adhesin part of the epitope-carrying fusion protein can be selected so as to have specific binding prop- erties whereby the vaccine cell may be targeted to a particu ⁇ lar location in the body where an immunological response to the epitope is desirable.
  • the adhesion of the epitope-carry ⁇ ing cell to a particular location or region of the body will in this manner ensure that the cell is retained in the human or animal body in that particular location for a period of time which is sufficient to obtain the desired immune response.
  • a useful cell for expres- sion of the above fusion protein is one selected from a bacterial species which inherently contains an adhesin-carry ⁇ ing surface organelle.
  • bacterial species which inherently contains an adhesin-carry ⁇ ing surface organelle.
  • Such species include as examples gram- negative species of Enter obacteriaceae such as E. coli , Klebsiella spp, Salmonella spp, Yersinia spp, Vibrionaceae, Hemophilus spp, Bordetella spp and Pseudomonadaceae , and gram-positive species such as Neisseria spp and Streptococcus spp.
  • the epitope part of a fusion protein according to the inven ⁇ tion can be an epitope derived from any pathogenic organism or agent against which it is desirable to develop vaccines.
  • pathogenic organisms include viruses, bacteria and eucaryotic organisms such as fungi, yeast or protozoa.
  • cells expressing an epitope-carrying fusion protein as defined herein may be used as a live vaccine
  • the fusion protein according to the invention comprises as the non-adhesin polypeptide part a toxic gene product e.g. having a selective toxic effect on particular cells in the body such as e.g. cancer cells.
  • a toxic gene product e.g. having a selective toxic effect on particular cells in the body such as e.g. cancer cells.
  • the adhesin part By selecting the adhesin part as one having a specific binding affinity to receptors in such cells it is possible to have cells expressing the toxic gene product bound selectively to such target cells whereby these cells may be killed or damaged by the toxic gene product. It is also possible to use isolated or purified cell organelles containing a fusion protein comprising the cell toxic (cytotoxic) gene product for the purpose of targeting the toxic product.
  • the fusion protein comprises an antibody.
  • an embodiment is, inter alia, particularly interesting with respect to the provision of fusion proteins which may be used in affinity purification of biological compounds having binding affinity to the antibody part of the fusion protein. It is contemplated that cells expressing as part of a surface organelle, such a fusion protein may be utilized directly as a means of concentrating a biological compound, or the isolated surface organelles comprising the antibody-carrying fusion protein may be used for this purpose.
  • the fusion proteins as defined herein are useful as carriers of non-covalently bound compounds such as pharma ⁇ cologically active, diagnostically active and imaging com- pounds with the purpose of providing cells or cell organelles carrying the active compounds, which thereby become targetab- le to particular regions or locations of a body to which these cells or cell organelles are administered.
  • the inven ⁇ tion encompasses any combination of a fusion protein as defined herein and an active compound which can be covalently bound to a fusion protein.
  • the present invention encompasses in one aspect a recombinant bacterial cell which expresses a recom ⁇ binant bacterial adhesin variant or a FimH adhesin as defined above.
  • the bacterial cell is one which comprises the above-defined recombinant replicon.
  • it may e.g. be selected from a soil bacterium, an aquatic bacterium, a bacterium which is normally associated with plants, a bacterium which is member of the human or animal indigenous bacterial flora, or a bacterium which is adapted to colonize certain ecological niches such as e.g. sewage purification plants or certain inanimate surfaces.
  • the major significant advantages which have been achieved by the present invention is the possibility to provide recombi ⁇ nant bacterial cells which are not only ecologically well- adapted to grow in a particular ecological environment, but which are also provided with means for colonizing more perma ⁇ nently in their ecologically natural environment.
  • These means for improved ability to colonize an environment are the adhesins expressed by the bacteria which have been con ⁇ structed and/or selected so as to enable the recombinant bacterial cell to adhere to or bind to specific receptors in the environment, i.e. the bacterial cells are targeted to that environment.
  • the bacteria according to the present invention will have an ecologically competitive advantage relative to organisms in the particular environment which do not have surface structures comprising adhesins binding to receptors present in the environment, at least not to the same extent as the bacterial cells according to the invention.
  • the cell will have a phenotype which is desirable in the environment to which it is tar- geted.
  • a cell according to the invention which is originally isolated from the human or animal indige ⁇ nous bacterial flora may typically be one which expresses an epitope including an epitope which is part of a fusion pro ⁇ tein expressed by the bacterial cell.
  • a bacterial cell which is isolated from a plant and which expresses a pesticidally active compound such as a Bacillus thuringiensis toxin.
  • Further examples include a plant root-associated nitrogen-fixating bacterium isolated from soil which in accordance with the invention is provided with adhesins improving the capability of the bacterium to become permanently colonized to the roots of a specific plant or specific plants, or a bacterium which is ecologically associated with an aquatic or terrestrial environment con ⁇ taining pollutants to be degraded or removed.
  • the recombinant bacterial cell can be derived from any gram-negative or gram-positive bacterium for which a need exists to obtain improved colonization in a particular inanimate or animate environment.
  • bacteria include as examples Enterobacteriaceae spp, Hemophilus spp, Neisseria spp, Bordetella spp, Streptococcus spp, Pseudomonadaceae spp, Vibrionaceae spp, Baccilaceae spp.
  • the present recombinant bacterial cell is provided as one which, when it is administered to a particular location or environment, will not persist in that environment.
  • a recombinant bacterial cell may further comprise a gene coding for a gene product which, when expressed has a killing or cell function-limiting effect in said cell, the expression of said gene coding for the cell killing or cell function-limiting gene product being regulated in such a manner that the bacterial cell when targeted to receptor in a specific location will be killed or limited in its function in a pre-determined manner.
  • the gene coding for the cell killing or cell function-limiting gene product is suitably regulated by a factor selected from the group consisting of a stochastic event, the presence/absence of a chemical compound in the location, and a physical factor.
  • the invention relates to a method of isolating or constructing a recombinant bacterial cell ex ⁇ pressing an adhesin having modified binding properties rela- tive to a natively expressed adhesin such as a natively expressed FimH adhesin.
  • this method comprises identifying in the bacterial cell DNA sequence(s) coding for the binding domain(s) of said natively expressed adhesin and substituting at least one codon herein whereby a modified adhesin molecule is expressed that is different in at least one amino acid from the adhesin expressed natively, and selecting a bacterial cell expressing the modified adhes ⁇ in having an altered adhesion phenotype relative to the natively expressed bacterial adhesin.
  • the binding domain can e.g. be identified by constructing chimeric adhesin-encoding genes and screening for cells which by having a region in the adhesin gene replaced by a corresponding heterologous region of a different DNA sequence, acquires a new binding phenotype.
  • recombinant cells having desirable binding phenotypes may be obtained by substituting one or more codons in the binding domain(s) to obtain expression of recombinant adhesins and selecting cells having the desirable phenotypes.
  • the substitution of codons may be achieved by methods know per se such as site-directed mutagenesis using synthetic oligonucleotides and PCR technology or transposable elements or by conventional radiation or chemical mutagenization.
  • the above method includes steps whereby a non-adhesin compound is associated with the adhesin, e.g. a step where a gene coding for the recombinant adhesin is part of a hybrid gene comprising a gene coding for a non-adhesin polypeptide which thereby is expressed with the recombinant adhesin as part of a fusion protein comprising the adhesin.
  • recombinant adhesins resulting from the above method may in specific embodiments comprise a non- covalently bound compound which is associated with the adhes ⁇ in when expressed.
  • the invention also encompasses recombi- nant bacterial cells having selected binding properties whereby cells with desirable phenotypes can colonize environ ⁇ ments where the presence of bacteria having a particular phenotype is advantageous. Accordingly, there is in a further aspect of the invention provided a method of preparing a recombinant bacterial cell that binds to a specific receptor moiety, comprising introducing into a bacterium that does not produce an adhesin binding to said receptor moiety, a DNA sequence coding for an adhesin binding to the receptor moie ⁇ ty, and selecting a bacterial cell expressing the DNA sequence.
  • the primary objective of this method is to provide the means of constructing a bacterial strain having the capacity to colonize an environment, based on a parent strain which has an inherent, useful phenotype in this particular environment but which does not express an adhesin binding to receptor moieties in the environment.
  • the method includes as a first step the isolation of an environmentally adapted bacterium not binding to appropriate receptor moieties and in subsequent steps, the identification of heterologous genes encoding adhesins which bind to receptor moieties occurring in said environment, preferably moieties occurring abundant ⁇ ly, isolating this gene and introducing it into the above parent strain.
  • the adhesin gene may e.g. be a gene coding for a naturally occurring FimH adhesin or a recombinant FimH adhesin as defined above.
  • the adhesin-encoding gene is introduced by transforming a parent bacterial cell with a recombinant replicon as defined herein.
  • the method is designed so as to obtain a cell wherein a non-adhesin compound is associated with the adhes ⁇ in, e.g. by introducing the gene coding for an adhesin as a hybrid gene coding for a non-adhesin polypeptide whereby non- adhesin compound is expressed with the adhesin as part of a fusion protein comprising the adhesin, or by binding non- covalently a compound to the adhesin when expressed.
  • an adhesin carrying bacterial cell having an altered pattern of adhesion can be provided by using a selection procedure comprising contacting an appro ⁇ priately sized population of wild-type adhesin-carrying bacterial cells with a potential receptor moiety to which the wild-type cells do not adhere, e.g. in a manner as it is disclosed in Example 6 below whereby spontaneously or random ⁇ ly mutated cells having acquired the ability to adhere to the receptor moiety in question, become progressively enriched. From such an enriched culture, cells with the new adhesion ability can readily be isolated and further characterized.
  • one primary objec ⁇ tive of the present invention is to provide the means of targeting a compound to a specific location.
  • the invention relates in an important aspect to a method of targeting an adhesin to such a location.
  • the method comprises the identification in the location of a receptor moiety, said moiety preferably being one which occurs abundantly in the particular location, which moiety can recognize and interact with an adhesin, and the isolation of a bacterial cell which is capable of growing in the location and expressing an adhesin which recognizes and interacts with the identified receptor moiety, and administering the cell or the adhesin in an isolated form to that location.
  • the identification of a suitable receptor moiety in a par ⁇ ticular location can be carried out in several manners.
  • One example is a screening procedure where cells expressing known adhesins or known isolated adhesins are administered to the location e.g. being isolated cells or tissues of microbial, animal or plant origin or an inanimate surface as defined herein, and screening for binding/adhesion of the tested adhesins e.g. according to adhesion assays as disclosed herein. If binding of one or more adhesins occurs, it is an indication that receptor moieties for that or those tested adhesin(s), is/are present in the location.
  • adhesins which are known to bind to one or more of the identified major moieties are selected and their binding to this/these structure(s) is tested e.g. according to the assays as used herein.
  • Chemical moieties which are considered potential adhesin-interacting receptor moieties include as examples glycolipids, glycoproteins, proteins, polypeptides, saccharide moieties and peptides.
  • a library of modified adhesin molecules may e.g. be selected for specificity towards a given receptor by running clones of these adhesins through a column or matrix containing the receptor moiety in question or cells or tissues isolated from the location without knowing what the receptor moiety is. The clone(s) expressing the adhesins with affinity to receptor moie ⁇ ty/moieties will adhere/bind to the column or matrix, and can subsequently be isolated therefrom.
  • one embodiment of the above method is one wherein the isolated bacterial cell expresses an adhesin having modified receptor moiety-binding properties relative to an adhesin natively expressed by the cell, the isolation of the cell comprising identifying in a parent bacterial cell, DNA sequence(s) coding for the binding domain(s) of said natively expressed adhesin and substituting at least one codon herein, whereby a modified adhesin mole ⁇ cule is expressed that is different in at least one amino acid from the adhesin expressed natively, and selecting a bacterial cell expressing the modified adhesin having an altered adhesion phenotype relative to the natively expressed bacterial adhesin or a method wherein the bacterial cell expressing an adhesin that recognizes and binds to the receptor moiety is a recombinant bacterial cell derived from a parent bacterial cell that does not produce an adhesin binding to said receptor, by inserting into the parent cell a DNA sequence coding for an adh
  • One primary objective of the present invention is the target- ing of useful non-adhesin compounds to a particular location. Accordingly, the invention encompasses in an interesting embodiment a method as defined above wherein a non-adhesin compound is associated with the adhesin, whereby said com ⁇ pound is targeted with the adhesin to the location comprising the receptor moieties recognizable by the adhesin.
  • the compound can be associated with the adhesin by a covalent binding or by any of the above mentioned non-covalent types of molecule interaction forces.
  • the compound to be co-targeted to the selected location with the adhesin can be an enzyme, an antibody, an epitope or a toxin which is part of a fusion protein comprising the adhesin.
  • a compound which is associated with the adhesin by a non-covalent bind ⁇ ing is typically a pharmacologically active, diagnostically active or imaging compound.
  • Locations to which it is desirable to have an adhesin tar ⁇ geted by the present method include a human or animal sur ⁇ face, a plant surface and an inanimate surface as defined above.
  • the bacteri ⁇ al cell being administered to the location expresses a recom ⁇ binant bacterial adhesin variant derived from a naturally occurring parent adhesin, said adhesin variant having altered binding properties relative to the naturally occurring adhes ⁇ in from which it is derived, the altered binding properties including binding to at least one receptor moiety to which the parent adhesin does not bind.
  • Such an adhesin variant ' is advantageously derived from a naturally occurring adhesin isolated from a cell structure selected from the group con ⁇ sisting of a capsule, a lipopolysaccharide layer, on outer membrane protein, a flagellum, a pilus, a fimbria, a non- fimbrial adhesin (NFA) or an afimbrial adhesin (AFA) .
  • a naturally occurring adhesin isolated from a cell structure selected from the group con ⁇ sisting of a capsule, a lipopolysaccharide layer, on outer membrane protein, a flagellum, a pilus, a fimbria, a non- fimbrial adhesin (NFA) or an afimbrial adhesin (AFA) .
  • the above adhesin variant as used in the present method is a protein having an amino acid sequence differing in at least one amino acid residue from its parent protein adhesin such as a FimH adhes ⁇ in having an amino acid sequence which differs from the E. coli PC31 FimH adhesin as defined herein in at least one amino acid.
  • Such a FimH adhesin includes an adhesin which binds to a receptor selected from the group consisting of a domain where mannosyl residues are not terminal and a domain devoid of saccharide and an adhesin variant which is at least 90% homologous to the PC31 FimH adhesin as defined herein, such as at least 92% homologous, more preferably at least 93% homologous, even more preferably at least 94% homologous, most preferably at least 95% homologous, and in particular at least 96% homologous, e.g. at least 97% homologous.
  • the adhesin is at least 98% homologous, including at least 99% homologous or at least 99.5% homologous.
  • the above FimH adhesin can be a chimeric adhesin as defined above, comprising amino acid sequences from different FimH adhesins and constructed according to the methods below.
  • an adhesin can be adminis ⁇ tered to a location in the form of an adhesin-expressing bacterial cell.
  • Such a cell is one capable of growing in that particular location.
  • the bacterial cell is suitably derived from a bacterial species which is normally occurring in the location including human or animal body surfaces, plant surfaces such as plant root surfaces and inanimate surfaces.
  • an animal body surface includes the insect gut, whereto it is desirable to adminis- ter a bacterial cell expressing an insecticidally active toxin.
  • a suitable bacterial cell is preferably isolated from a strain which has colonized the rhizosphere of that plant to a large degree, i.e. the strain is a major member of the natural plant root flora.
  • Such an isolate is then provided with a gene coding for an adhesin which will recognize and interact with an abundantly occurring moiety on the roots of said plant.
  • a suitable adhesin which is expressed naturally in a bacterium which is not adapted to grow in a plant rhizosphere, becomes expressible in a normal inhabitant of the rhizosphere environment (loca ⁇ tion) .
  • the adhesin is a FimH adhesin as defined above, having an amino acid sequence which differs from the E. coli PC31 FimH adhesin as defined herein in at least one amino acid.
  • the adhesin-carrying bacterial cell being targeted is a cell which further comprises a gene coding for a gene product which, when it is expressed, has a killing or cell function-limiting effect in said cell, the expression of said gene coding for the cell killing or cell function-limiting gene product being regulated in such a manner that the bacterial cell, when targeted, will be killed or limited in its function in a pre-determined manner.
  • the expression of such a "suicide” or cell function-limiting gene may suitably be regulated by a factor selected from the group consisting of a stochastic event, the presence/absence of a chemical compound in the location and a physical factor.
  • such "suicide" or cell function-limiting genes providing the means of biological containment may be men ⁇ tioned those disclosed in WO 87/5932 and WO 93/20211
  • the present Designer Adhesin Technology provides very useful means of obtaining colonization with desirable bacteria in a particular environment with the purpose of obtaining beneficial changes of the microbial flora in the environment.
  • certain bacterial species in the gastrointestinal (GI) tract of humans and animals have beneficial effects on the health condition of the host organism e.g. by suppressing pathogenic organisms or by contributing to the digesting of certain diet components.
  • the present technology makes it possible to select particu ⁇ larly useful bacteria from the GI-tract and have them designed in accordance with the present invention, to have improved colonization abilities.
  • Similar examples include desirable bacterial colonizations of biological sewage puri ⁇ fication systems, plants where invasion of pathogenic organ ⁇ isms may be controlled by colonizing the plants with harmless bacteria, and teeth where caries may be controlled by coloni ⁇ zing the dental enamel with bacteria suppressing those caus ⁇ ing the caries attacks.
  • the invention provides the means of isolating a compound from a solution or suspension containing the compound.
  • the method comprising contacting the solution or the suspension with a fusion protein as defined herein wherein the heterologous polypeptide has an affinity to the compound to be isolated.
  • composition comprising a population of a bacterial cell as defined herein.
  • the invention is further illustrated in the below Examples and the Figures, wherein
  • Fig. 1 is a schematic model for the construction of recom ⁇ binant plasmids pGBl-24 (containing fimH from Cl #10) and pGB2-24 (containing fimH from PC31) used for transforming E. coli AAEC191A(pPKLll4) with cloned fimH genes.
  • Plasmid pGB2- 24 was used as the vector for all other cloned fimH genes described herein;
  • Fig. 2 is a restriction map of fimH genes. Five unique res- triction sites are present in the PC31 fimH gene. Numbers in parentheses following enzymes are the base pair positions of the cut sites. Some of these sites are found in the other fimH genes, as marked. Chimeric genes were produced by ex ⁇ changing each available restriction fragment from the other five fimH genes with corresponding fragments in the PC31 gene and then recombinant strains expressing resulting chimeric fimH subunits were tested for adhesion. Fragments indicated by boxes are those which conferred MF or MFP adhesive phenotypes on the chimeric genes;
  • Fig. 3 illustrates adhesion of representative "wild-type" (A) and recombinant (B) M-class, MF-class and MFP-class strains to Mn (1), Fn (2), periodate-treated Fn (3) and to FnSpl (4). Strain designations given for the "wild-type" strains are given in AS.
  • Strain designations KB31, KB12, KB4, KB7, KB50 and KB10 are for recombinant strains of AAEC191A(pPKL114) , which is fimH ⁇ , after transformation with plasmids that contain fi J from strains HB101(pPKL4) , Cl #12, Cl #4, Cl #7, CSH-50 and Cl #10, respectively.
  • Fig. 4 illustrates the adhesion of representative M-class, MF-class and MFP-class strains (CIs #12, #4 and #10, respect- ively) to Fn fragments prepared by thermolysin treatment as described in ref. 51.
  • Columns labelled 1-5 indicate adhesion to: 1) NH2-terminal 30-kDa domain; 2) the 55-kDa gelatin- binding domain; 3) the 110-kDa cell attachment domain; 4) the 29-38-kDa heparin binding domains; and 5) the 20-kDa COOH- terminal domain.
  • Open columns represent adhesion in the absence of D-mannose; solid columns represent adhesion in the presence of D-mannose.
  • Mean ⁇ S.E.M. (n 4) ;
  • Fig. 5 is a composite figure illustrating comparison of amino acid sequences of FimH adhesins and active restriction frag ⁇ ments of fimH genes.
  • the published nucleotide and deduced amino acid sequence of the PC31 fimH gene and gene product (ref. 27) serve as prototype.
  • Numbered amino acid residues shown above the model of the PC31 FimH represent residues that are different in other FimH subunits due to amino acid substitution or deletion. Standard one-letter code applies and residues in the other FimH sequences that are different are indicated.
  • Deleted amino acids are indicated by ⁇ .
  • residue 176 is not arginine as published previously (ref. 27) for the PC31 FimH, but proline.
  • Regions of the FimH subunits conferring change in adhesive phenotype were determined by functional assays performed on chimeras between the "classic" mannose-specific PC31 fimH gene present in HB101(pPKL4) and the above described genes. Residues predicted to be key in conferring receptor specificity are circled. Approximate positions of unique restriction sites used to create chimeras are indi ⁇ cated along the bottom of the model;
  • Fig. 6 illustrates plasmid pPKL4 which is a derivative of pBR322 (thick line) carrying the entire fim operon (FimA-H) including the regulatory genes fimB and fimE (not shown) , and the promoter region with the SnaBI site.
  • this plasmid an 8mer linker with an Bgrlll site was inserted in the SnaBI site to create pPKL83;
  • Fig. 7 illustrates the construction of plasmid pSM1314; the vector pVLT33 is a derivative of the broad host range replic ⁇ on RSF1010.
  • Plasmid pPKL83 was digested with Bgrlll and pVLT was digested with Ba ⁇ iHl; the two were ligated and pSM1314 was the resulting plasmid in which expression of the fimA-H cluster is under the control of the tac promoter;
  • Fig. 8 illustrates plasmid pLPA22 and derivatives hereof as used in this study.
  • the triangles indicate the position of translational stop-linkers in the fimH gene in plasmid pPKLH5.
  • the positions of heterologous inserts are indicated (black boxes) .
  • Small triangles indicate signal-peptide encod ⁇ ing sectors.
  • Fig. 9 illustrates plasmids pLPA29, pLPA30, pLPA36, pLPA58, pLPA59 and pLPA98;
  • Fig. 10 shows immuno-electron microscopy with colloid gold labelling of E. coli HB101 cells containing plasmids pLPA22 plus pPKL115 (a) , pLPA37 plus pPKL115 (b) , pLPA38 plus pPKL115 (c) , using anti-pre-S2 monoclonal antiserum. Bar, 0.1 ⁇ m.
  • Yeast Mn a polymannosylated glycoprotein isolated from Saccharo yces cerevisiae cell walls, was obtained from a commercial source (Sigma Chemical Co, St. Louis, MO, U.S.A.). Mannan is composed of an N-linked backbone of ⁇ l, 2-linked mannopyranose units with ⁇ -linked mannopyranose side chains (ref. 38) . The majority of the carbohydrate of human plasma Fn is composed of N-glycosidic complex-type biantennary glycans and no high mannose-type or hybrid-type N-glycans have been described (refs. 30, 45, 54) . Human plasma Fn and Fn fragments were purified as described previously (refs.
  • the saccharide content of the four substrata was characterized using two lectins, concanavalin A (ConA) , well known to react with terminal and internal mannosyl residues, and the Calanthus nivalis agglutinin (GNA) , which recognizes only terminal Man ⁇ l-3Man, Man ⁇ l-6Man and Man ⁇ l- 2Man sequences (E. Y. Laboratories, San Mateo, CA) . Immobi ⁇ lized Mn and Fn both reacted with ConA, whereas GNA bound only to Mn. These results are consistent with the known structures of the oligosaccharide moieties of these two compounds. Neither lectin reacted with immobilized FnSpl. Periodate treatment (ref. 51) of Mn or Fn eliminated lectin reactivity.
  • ConA concanavalin A
  • GNA Calanthus nivalis agglutinin
  • the CSH-50 strain (1ambda “ ,F” araA (l a c -pro) rspL thi fimE: :IS1) is a Cold Spring Harbor K12-derived strain (ref. 35).
  • the E. coli strain MG 1655 (CGSC6300; K12 derivative, lambda “ ,F “ ) and a derivative strain AAEC191A (MG1655 recA Afim were generously provided by Dr. Ian Blomfield (Bowman Gray University, Winston-Salem, NC) .
  • AAEC191A has had the entire fim gene cluster deleted by allelic exchange (ref. 8) .
  • CIs Clinical isolates
  • the 12 CIs used in this study were selected on the basis of MS agglutination of yeast cells after growth in broth, a classic test for type 1 fimbriae.
  • coli strain PC31 K12-derivative, gal tonA phx ara
  • encoding for the expression of fully func ⁇ tional type 1 fimbriae in HB101 (supE hsdS recA ara proA lacY galK rspL xyl mtl AfimBE)
  • pPKL114 is a recombinant plasmid derived from pPKL4, but with a translational stop-linker inserted into the Kp ⁇ l site in the fimH gene. No transcriptional effects of the
  • stop-linker are to be expected.
  • Antibiotics were used at the following final concentrations: ampicillin (50 ⁇ g/ml) , kanamycin (60 ⁇ g/ml) and chloramphenicol (30 ⁇ g/ml) .
  • Oligonucleotide primers were designed using the published sequence for the fimH gene in pPKL4 (ref. 27) .
  • the 5' primers copied regions 13 and 49 bp upstream from the fimH gene and were extended on the 5' end by an Apall restriction site and a GC clamp:
  • Primer 1 5' -GGGGG-GTGCAC-ACC TAC AGC TGA ACC CGG-3' (SEQ ID NO:3)
  • Primer 2 5'-GGGG GTGCAC T CAG GGA ACC ATT CAG GCA-3' (SEQ ID NO:4).
  • the 3' primers copied 18 bases of the bottom strand of the fimH gene that encode for the 6 terminal amino acids of fimH and were extended by an Fspl or Sphl site and a GC clamp: Primer 3: 5'-GGG TGCGCA TTA TTG ATA AAC AAA AGT CAC - 3' (SEQ ID NO:5); Primer 4: 5'-GGG GCATGC TTA TTG ATA AAC AAA AGT CAC-3' (SEQ ID NO:6) . Primer 1 and 3 were used for Cl #10 and pPKL4, primer 1 and 4 were used for Cl #4 and CSH-50 and primer 2 and 4 were used for Cl #s 7 and 12 to generate PCR products from plasmid or chromosomal DNA prepared from E.
  • the PCR reaction mixture consisted of template DNA, primer pairs, dNTPs, and Taq DNA polymerase in PCR buffer.
  • the PCR was performed in a Perkin-Elmer Cetus automatic thermal cycler with denaturation at 96°C for 1 min., primer annealing at 55°C for 1 min., and primer extension at 72°C for 2 mins. for a total of 40 cycles. All of the PCR products migrated similarly in agarose gels. Purification, restriction and ligation of DNA was performed using standard procedures (refs. 39, 48). All primers for PCR and for nucleotide sequencing were produced by the Molecular Resources Center, UT, Memphis.
  • PCR products from CI#10 and from pPKL4 were cut with respective restriction enzymes and ligated into the Apall and Fspl restriction sites of plasmid pACYC177 (New England Biolabs, Beverly, MA, U.S.A.) which is compatible with the pBR322-based pPKLH4 to be used in complementation experi ⁇ ments, creating plasmids pGBl and pGB2, respectively (Fig. 1) .
  • pACYCl77-based plasmids because of a high frequency of appearance of sponta ⁇ neous Km r in the AAEC191A host strain.
  • Fig. 2 Unique restriction sites (Fig. 2) were used to construct chimeric fimH genes between the prototypical MS pPKL4 fimH gene, used as genetic background, and restriction fragments obtained from the newly described fimH genes. Fragments were purified from agarose gels and ligated into restriction "spaces" generated in the pPKL4 fimH gene present in pACYCl'84 (pGB2-24) . Each chimera was analyzed by restriction mapping and the nucleotide sequences of bridging segments were deter- mined to ensure proper constructions.
  • the plasmids containing chimeric fimH genes were transformed into AAEC191A(pPKL114) and clones were tested for agglutination of yeast cells and for adhesion to Mn, Fn and FnSpl.
  • the nucleotide sequences of fimH genes were determined by the dideoxynucleotide chain termination method of Sanger (ref. 49) using a Sequenase II ® kit (U.S. Biochemical Corp., Cleve ⁇ land, Ohio) and [ ⁇ - 35 S]dATP (800 to 1,000 Ci/mmol) according to the manufacturer's suggestions.
  • the amino acid sequences were deduced from nucleotide sequences using MacVector ® protein and DNA analysis software (Eastman Kodak, Rochester, NY) . To ensure fidelity of the PCR amplification, selected fimH genes were re-amplified, cloned, tested for activity and re-sequenced.
  • E. coli were tested for their ability to aggregate yeast cells.
  • Commercial baker's yeast, Saccbaromyyces cerevisiae was suspended in PBS (5 mg dry weight/ml) .
  • E. coli were washed in PBS, resuspended to an OD 530 of 0.4, and mixed with the yeast cell suspension in PBS with or without 1% D- mannose. Aggregation was monitored visually and the titer recorded as the last dilution giving a positive aggregation reaction.
  • Adhesion assays were performed as described previously (ref. 51) . Briefly, microtiter assay wells were coated with receptor molecules as indicated in the text and figure leg ⁇ ends. After the wells were washed two times with PBS, 100 ⁇ l bacterial suspensions were added in 0.1% BSA-PBS. After incubation at 37°C for indicated times, wells were washed three times with PBS and adherent bacteria were detected by using rabbit anti-F. coli serum. Antibody binding was detected using peroxidase-conjugated goat anti-Rabbit IgG.
  • Reaction product generated from the 5-aminosalicylic acid substrate was measured at 405 nm after 10-15 minutes by using an automatic microplate reader (Molecular Devices, Inc., Menlo Park, CA) . Values reported are corrected for background reaction using BSA coated plates as control.
  • type 1 fimbriae of E. coli CSH-50 and HB101(pPKL4) differ func- tionally in their pattern of adhesion to Mn, Fn, periodate- treated Fn and a synthetic peptide, FnSpl, immobilized on plastic (ref. 51). Since CSH-50 and HB101(pPKL4) are labora ⁇ tory strains, we tested 12 clinical E. coli isolates (CIs) obtained from human urine for adhesion to these four substra- ta. All of the CIs agglutinated yeast cells in a MS fashion. Five of the twelve CIs adhered only to Mn.
  • HB101(pPKL4) and of Cl #12 are shown as examples of this class, which we have tentatively designated as M class (Fig. 3A) .
  • M class Three of the 12 CIs adhered to Mn and Fn, but not to periodate-treated Fn or to FnSpl.
  • the adhesive activities of Cl #s 4 and 7 are shown as examples of this class, designated as MF class.
  • the adhesive activities of CSH-50 and Cl #10 are shown as examples of this class, desig ⁇ nated as MEP class.
  • Adhesion of strains representing these three classes to Fn fragments further illustrates the distinct differences between the three classes.
  • the M class Cl #12 does not adhere to any of the Fn fragments (Fig. 4) .
  • the MF class Cl #4 adheres to the 40-kDa gelatin-binding fragment.
  • the MFP class Cl #10 adheres, with only slight differences, to all 5 frag ⁇ ments of Fn tested. Periodate treatment eliminated binding of Cl #4 to domain 2, but had no effect on the binding of Cl #10 to any of the Fn domains (data not shown) .
  • fimH genes were amplified from chromosomal (or plasmid, for pPKL4) DNA and the genes were cloned into pACYC177 and subcloned into pACYC184 under control of the /S-lactamase promoter of pACYC177, according to Materials and Methods (Fig. 1)
  • E. coli K-12 strain AAEC191A (Afim) was first transformed with plasmid pPKLH4, which contains an intact fi gene cluster but with a translational stop-linker inserted into the last gene, fimH. This deriva ⁇ tive produces morphologically normal fimbriae that are non- adhesive due to absence of the FimH subunit. Plasmids har ⁇ bouring cloned fimH genes were transformed into E. coli AAEC191A(pPKL114) and the resultant strains were tested for their ability to adhere to Mn, Fn, periodate-treated Fn and to FnSpl (Fig.
  • FimH amino acid sequences of the following clinical isolates of F. coli are shown: KB21 (SEQ ID N0.-27) , KS54 (SEQ ID NO:35), U221-3 (SEQ ID NO:36), MJ#9-3 (SEQ ID NO:37), M #31-3 (SEQ ID NO:38), MJ#ll-2 (SEQ ID N0:39), MJ#2-2 (SEQ ID NO:l) and F-18 (SEQ ID N0:34). Standard one-letter code applies. Deleted amino acid residues are indicated by ⁇ s. M, M L , MF, MFP, and M R indicate the adhesin class as defined above.
  • the Apall - Tthllll fragment of the Cl #7 fimH gene confers MF class activity in the CI#7/PC31 fimH chimera. Since the asparagine 16 -threonine 16 substitution is within the leader sequence and thus not represented in the mature protein, the histidine 33 - asparagine 33 substitution must be of functional importance for the MF class Cl #7 FimH. Comparison of the active regions of the MF class Cl #4 and the M class Cl #12 FimH subunits suggests the importance of the glutamic acid 73 - glycine 73 substitution for MF class activity of the Cl #4 FimH.
  • histidine 33 , arginine 58 , glutamic acid 37 and deleted glycine 116 -isoleucine 119 appear to be key residues in the functional activity of FimH subunits of Cl #7, CSH-50, Cl #4 and Cl #10, respectively, but a more precise demonstration 48 of which residues are involved and how they affect the ligand-binding cleft(s) remains to be performed.
  • FimH mediated, mannose-sensitive pro ⁇ tein-binding activity of type 1 fimbriae is the most surpris- ing of the adhesive phenotypes described here.
  • protein-binding activity of FimH (i.e. PilE) subunits was noted earlier in a study characterizing mutT-induced muta ⁇ tions in the fimH (pilE) gene (Harris et al., ref. 22) How ⁇ ever, the protein-binding activity described by Harris et al. was not mannose-sensitive.
  • nucleotide and deduced amino acid sequences of the pPKL4 fimH gene are identical to that reported previously, except that residue 176 is not an alanine residue as previously reported, but a proline residue. Independent re-amplifica- tion, re-cloning and re-sequencing confirmed this sequence for the pPKL4 fimH gene. Sequencing was also repeated on independently amplified and cloned isolates of the Cl #10 and Cl #7 fimH genes to confirm sequence fidelity and no errors were found.
  • nucleotide and deduced amino acid sequences of the other fimH alleles described in this Example are > 98% conserved, but there is more than one amino acid residue difference in all but one of the new fimH sequences when compared to the published pPKL4 sequence.
  • advan ⁇ tage of unique restriction sites were taken (Fig. 2) to construct chimeric fimH genes. Multiple restriction fragments covering the entirety of each of the sequenced fimH genes were exchanged with corresponding fragments in the prototypi- cal fimH gene of E.
  • the functional heterogeneity which is described above must be due entirely to allelic variants of the fimH gene.
  • the only variables in the recombinant strains which are described in this Example are the fimH genes; all other genes necessary for fimbrial subunit synthesis, transport and assembly are the same in each case. Since the ratios of the various genes to the ligand-binding cleft(s) and it will ultimately be necessary to determine the 3-dimensional structure of FimH or FimH fragments crystallized in the presence of ligand to fully understand structure/function relationships.
  • the three adhesive classes of type 1 fimbriae identified above may understate the functional heterogeneity of type 1 fimbriae.
  • the group of CIs that has been tested in this Example is small and only a few substances have been tested as potential receptors. A larger group of isolates tested against additional receptor candidates might yield additional functional classes.
  • Preliminary studies with MS Enterobacter aerogenes and Klebsiella pneumoniae strains exhibiting MF class and MFP class activity suggest that heterogeneous receptor specificities will also be found among other type 1 fimbriated enterobacterial species.
  • adhesins from some fimbriae responsible for mannose-resistant hemagglutina- tion or adhesion are structurally related to FimH, but with sequence alterations that eliminate sensitivity to mannose.
  • the possibility that the MS lectin-like properties of FimH might be eliminated while retaining other adhesive properties of FimH (e.g. pellicle formation) has been shown previously in a study characterizing mutT-induced mutations in the fimH (pilE) gene (ref. 26) . At the minimum, it is believed that tests for type 1 fimbriation should include additional func ⁇ tional characterization.
  • type 1 fimbriae-mediated adhesion which have been described in this Example is mannose-sensitive, it is not all mannose- or even saccharide- specific as has commonly been thought. Further studies of type 1 fimbriae as a virulence factor must be able to distin ⁇ guish among the various functional classes.
  • G adhesins of P fimbriated uropathogenic E. coli also results in different functional classes, but the requirement for the Gal ⁇ l-4Gal sequence within isoreceptors is maintained (refs. 52, 53) .
  • These differences in G adhesin receptor specificity appear to be rather subtle, at least superficially, when compared to the differences in FimH receptor specificities. Yet there is significantly greater sequence homology among the fimH genes than among the G adhesin genes, some of which share less than 50 percent homology.
  • the G adhesin receptor specificities affect host susceptibility, due in large part to host-speci ⁇ fic expression of glycolipid isoreceptor variants.
  • FimH family of adhesins bears a similar relationship to host susceptibility or tissue tropism remains to be deter ⁇ mined.
  • the G adhesin family could exhibit additional receptor specificities not restricted to the Gal ⁇ l-4Gal sequence.
  • the lectin-independent affinity of P fimbriae for immobilized Fn is not dependent on the G adhesin, but on two other minor subunits, E and F, neither of which bear significant homology to FimH (refs. 56, 57).
  • the fim operon of E. coli comprises a cluster of genes covering about 8 kb of DNA. This operon has been isolated and cloned on plasmids in its entirety.
  • the promoter upstream of the fi A gene is located within an invertible DNA sequence, which in E. coli leads to a switch on/switch off situation for fimbrial synthesis. In one orientation of the invertible sequence the promoter is directed towards the fim genes, and the cell is fimbriated; in the other orientation the promoter is directed in the opposite direction, and the cell is non- fimbriated.
  • Plasmid pPKL83 is a derivative of pPKL4 (ref. 27) carrying the entire fim operon in pBR322, in which the promoter has been destroyed by inserting a Bg ll linker in the SnaB site located in the promoter sequence. There is a second Bgrlll site in plasmid pPKL83 upstream of the fim operon (Fig. 6) .
  • Plasmid pVLT33 (Fig. 7) is a kanamycin resistant derivative of the broad host range plasmid RSF1010, carrying the lacl 1 gene and the tac promoter placed upstream of a multiple cloning site in which a unique BamHl site is placed.
  • this fusion plasmid will express fimbriae in the presence of IPTG due to the fusion between the fimbrial genes and the lac promoter.
  • the correct orientation of the fusion plasmid pSM1314 was verified by transforming it into a strain of E. coli which carries a deletion of the fim operon.
  • Production of fimbriae was assayed in two ways: 1) Cell aggregation with fimbrial antibodies and 2) ELISA assay of whole cells.
  • the former analysis is rather simple: to a small volume (10 ⁇ l) of an outgrown or IPTG-induced culture of the cells to be tested is added a small volume (2 ⁇ l) of antibodies raised against fimbriae, on a glass slide. After mixing the samples, fimbriated cells begin to show cell aggregates which are easily observed directly as clumps or under a microscope.
  • Plasmid pSM1314 also carries a mob site which allows it to be transferred to other gram negative bacteria provided a helper plasmid is introduced. This type of transfer is most easily performed in "triparental" matings in which a donor strain
  • E. coli carrying pSM1314 a helper strain ⁇ E. coli carrying a plasmid with conjugation genes
  • a recipient strain carrying a selectable marker not present in any of the two other strains are mixed on a plate (directly or on a fil ⁇ ter) . After some growth (often overnight) this mixture is spread on selective plates with antibiotics that only allow the recipient carrying the desired plasmid to grow and form colonies.
  • E. coli strains MC1000 (pSM1314) and MC1000 (pRK2013) and (as recipient) Enterobacter cloacae strain A50 Nal r (ref. 67), were mated.
  • This recipient strain is resistant to nalidixic acid.
  • the resulting clones were grown in liquid medium and assayed for the pres ⁇ ence of fimbriae in the absence/presence of IPTG. The cell aggregation assay was employed.
  • the plasmid pSM1314 in E. coli HB101 was deposited on 26 January 1994 with DSM, the Deutsche Sammlung von Mikroor- ganismen und Zellkulturen GmbH, (German Collection of Micro- organisms and Cell Cultures) , Mascheroder Weg IB, D-38124 Braunschweig, Germany, under the accession number DSM 8922.
  • Heterologous sequences mimicing the pre-S2 region of the hepatitis B viral surface antigen and a neutralizing epitope of the cholera toxin B chain were inserted in two different positions in the FimH adhesin of type 1 fimbriae. This was carried out by introduction of restriction site handles (Bgrlll-sites) in the fimH gene, followed by in-frame inser- tion of heterologous DNA segments encoding the foreign epito ⁇ pes. In the selected positions such insertions did not sig ⁇ nificantly alter the adhesive function of the FimH protein, since hosts producing hybrid fimbriae that contained the chimeric adhesins exhibited adhesion phenotypes and were normally fimbriated.
  • restriction site handles Bgrlll-sites
  • the heterologous inserts of 52 and 15 amino acids, respectively, residing in the chimeric FimH proteins were recognized by specific sera on the surface of the fimbriae on bacterial hosts.
  • the results illustrate the possibility of using bacterial adhesins as general presenters of foreign antigens and epitopes.
  • the Escherichia coli 12 strain HB101 was used in this study as a host for expression of chimeric fimbriae.
  • This strain is phenotypically Fim " due to a deletion in the fim gene cluster (ref. 8) .
  • Cells were grown on solid medium or in liquid broth supplemented with appropriate antibiotics.
  • gene expression from the lac promoter residing in front of the fimH gene in plasmid pLPA22 and its derivatives, was induced by the addition of IPTG (isopropyl thiogalacto- pyranoside) to the growth medium.
  • Plasmids pPKL4 (comprising the entire, functional fim gene cluster) and pPKL114 (comprising the fimH gene) have been described previously.
  • pPKL115 which is a plasmid containing the entire type 1 fim gene cluster with a stop linker insertion in the fimH gene (i.e. this plasmid expresses all the proteins necessary for the production of fimbriae except the FimH protein) was constructed in two steps:
  • plasmid pPKL4 (refs. 27, 28) was digested with Kpnl which recognizes a unique restriction site in the fimH gene.
  • the staggered end of the linearized plasmid was made blunt and ligated with the synthetic piece of DNA below (SEQ ID NO:7) containing stop codons in all three reading frames, resulting in plasmid pPKL114:
  • Plasmid pSM782 (generously provided by S. Molin, Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby) containing the pre-S2 and S encoding regions of the hepatitis B viral genome, was made from plasmid ⁇ -HBVl (ref. 72) by subcloning a EcoRI-Pral fragment into pBR322.
  • Plasmid pLPA22 was constructed by inserting a 1018 bp PvuII- Aflul fragment containing the fimH gene from pPKL4 into plasmid pUC18. The insert was positioned downstream and in a expression compatible orientation to the lac promoter resid- ing on the vector part of the plasmid (Fig. 8) . Expression in E. coli HB101 cells of functional FimH protein was monitored by complementing pLPA22 in trans with pPKL115 and testing for MS adhesion upon induction with IPTG.
  • Plasmids pLPA29 and pLPA30 were made by inserting 9-mer asymmetric Bgrlll-linkers into the BsaAI and Hindi sites, respectively, in the fimH gene of plasmid pLPA22. At six different positions in the pLPA22 fimH gene a Bg ll site was introduced without changing the reading frame, resulting in plasmids pLPA98, pLPA36, pLPA58, pLPA30, pLPA29 and pLPA59 (Fig. 10) . This was done either by inserting a Bgrlll linker into an appropriately treated restriction enzyme site, or by changing 1-3 basepairs using PCR and thereby creating a Bgrlll site.
  • the plasmid pLPA36 was prepared by opening the pLPA22 fimH gene with the restriction enzyme Tthllll and making the ends blunt using Klenow polymerase and ligating using an 8 mer Bgrlll linker (SEQ ID N0:8):
  • Plasmids pLPA58 and pLPA59 were made by Bgrlll site-creating site-directed mutagenesis of pLPA22 using standard PCR and plasmid pLPA98 was constructed by opening the fimH gene, making the ends blunt with T4 DNA polymerase and ligating with the below 10 mer Bgrlll linker (SEQ ID NO:9) :
  • Plasmid pLPA29 has a 9 bp long symmetrical Bgrlll linker inserted into the BsaAI site 66 bp upstream of the stop codon for the fimH gene, while plasmid pLPA30 has the same 9 bp
  • the plasmids pLPA37 and pLPA38 were constructed by inserting a 162 bp DNA fragment encoding the pre-S2 region of the Hepatitis B virus surface antigen into the Bgrlll sites in pLPA29 and pLPA30, respectively.
  • This DNA fragment was crea- ted by a standard polymerase chain reaction (PCR) using the synthetic primers: (i) 5' -GGAGATCTAATTCCACAACCTT-3' (SEQ ID NO:11) and (ii) 5' -GGAGATCTGTTCAGCGCAGGGT-3' (SEQ ID NO:12) , and plasmid pSM782 as a template.
  • PCR polymerase chain reaction
  • a fragment of plasmid pLPA38 comprising the inserted heterologous sequence encoding the pre-S2 region of hepatitis B surface antigen is shown in the below table wherein the heterologous sequence is underlined and the numbers indicated correspond to the positions of the amino acid residues in the ma.ture FimH protein.
  • the plasmids pLPA95 and pLPA93 were then made by inserting the below 51 bp synthetic double stranded DNA segment encoding amino acids 50-64 (comprising an epitope) of the B subunit of the cholera toxin into the Bgrll sites on pLPA30 and pLPA29, respectively (SEQ ID NO:10) : 5' -GATCTGTTGAAGTTCCGGGTAGTCAGCATATCGATAGTCAGAAAAAAGCTG -3' 3' - ACAACTTCAAGGCCCATCAGTCGTATAGCTATCAGTC I ⁇ I'I'CGACCTAG-5' .
  • a fragment of plasmid pLPA93 comprising the heterologous sequence encoding the above DNA segment of the B subunit of i the cholera toxin is shown in the below table wherein the heterologous sequence is underlined and the numbers indicated correspond to the positions of the amino acid residues in the mature FimH protein.
  • PCR Polymerase chain reactions
  • Reactions were set up as 100 ⁇ l volumes containing 200 ⁇ M each of dATP, dCTP, dGTP and dTTP, 0.2-1.0 ⁇ M of each of the two primers, 2 mM MgCl 2 , 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 2.5 units of AmpliTaq DNA polymerase and 0.1-0.2 ⁇ g of plasmid template.
  • the reactions were run for 25-30 cycles each consisting of 1 min. at 94°C, 1 min. at 40°C, and 1 min. at 72°C.
  • the capacity of bacteria to express a D-mannose binding phenotype was assayed by their ability to agglutinate guinea pig erythrocytes on glass slides. Aliquots of liquid cultures grown to an optical density of 3.0 and 5% erythrocytes were mixed, and the time until agglutination occurred was measured.
  • Electron microscopy and immuno-electron microscopy was carried out essentially as described previously (ref. 61) .
  • a 25 ⁇ l aliquot of bacterial suspension was placed on a carbon-coated, glow discharged grid for 30 seconds. Grids were washed in 2 drops of PBS, dehydrated for 5 min in each of the following concentrations of ethanol: 25%, 50%, 75% and 96%, blotted dry and shadowed with tungsten wire at an angle of 30°.
  • a monoclonal antibody directed against the pre-S2 region was used diluted 1:5 as the primary antibody and rabbit anti-mouse serum conjugated with 10 nm gold particles (Dako) was used in dilution 1:20 as the secondary antibody.
  • two potentially optimal positions for insertions of heterologous sequences in the C-terminal domain of the FimH protein were selected. These correspond to positions 225 and 258 in the mature protein predicted to be situated in a surface-exposed part of the FimH protein.
  • the fimH gene was subcloned into the pUC18 vector resulting in plasmid pLPA22. Subsequently a Bgrlll site was introduced in-frame into positions 225 and 258, respectively. This was carried out by site-directed mutagenesis employing synthetic oligomers resulting in plasmids pLPA30 and pLPA29, respect- ively (Fig. 9) .
  • the introduced Bgrlll sites resulted in a codon change from a Leu to a Phe codon in position 225 and addition of codons for the sequence Arg-Ser-Ser, in the case of plasmid pLPA29, and addition of codons for the sequence Arg-Ser-Gly after posi- tion 258 in the case of plasmid pLPA30.
  • Sequence analysis of the entire modified fimH genes in plasmids pLPA29 and pLPA30 confirmed that no other changes had occurred.
  • Host cells which in addition to plasmid pLPA29 or pLPA30 also contained plasmid pPKL115 (fimH) , showed wild-type phenotypic charac- teristics with regard to adhesion and fimbriation as judged by such criteria as hemagglutination (Table 3) and immuno- fluorescence microscopy. 3.2.3. Engineering heterologous DNA-sequences encoding the pre-S2 domain of hepatitis B surface antigen and a cholera toxin epitope into fimH.
  • the pre-S2 region of the hepatitis B surface antigen and a well characterized region of the B subunit of cholera toxin were selected.
  • the pre-S2 region is known to contain immunologically important (and protective) antigenic determinants (ref. 76) .
  • this region is disulphide bond-independent and apparently more immunogenic than the major S protein.
  • the cholera toxin segment consists of residues 50-64 of the B subunit and has previously been shown to elicit antibodies that bind to and neutralize cholera toxin (ref. 77) .
  • a DNA segment of 162 nucleotides encoding 52 of the 55 amino acids of the pre-S2 region was amplified by PCR technology using plasmid pSM782 as template and primers that provided the amplified sequence with flanking Bgrlll sites. Following restriction with Bgrlll and purification the amplified frag- ment was inserted into the Bglll sites of plasmids pLPA29 and pLPA30 resulting in plasmids pLPA37 and pLPA38, respectively (Fig 9) . Subsequent sequence analysis confirmed that the inserts were correctly oriented and that the reading frame of the chimeric fimff-pre-S2 genes was correct.
  • a synthetic DNA segment encoding the cholera epitope was made by annealing two complementary 51 bp oligonucleotides which were designed to result in a double stranded DNA fragment with a Bgrlll overhang in one end, a BairiHI overhang in the other and an internal Clal site.
  • the epitope-encoding segment was inserted into the Bgrlll site in the fimH gene in plasmids pLPA29 and pLPA30, resulting in regeneration of a Bgrlll site at only one end of the insert. This feature was used to identify plasmids with correct orientation of the insert.
  • the presence of the Clal site was used for initial screening for clones containing the insert. Sequence analysis of plasmid pLPA93 and pLPA95, both harbouring the epitope-encoding segment confirmed the orientation and conservation of the reading frame in the chimeric fimH-cholera genes (Fig 8) .
  • the combination of plasmids pLPA37 (pre-S2 in position 258 in FimH) and pPKL115 resulted in weaker, but detectable, hemagglutination (Table 3) .
  • such cells were also shown by electron microscopy to have essen ⁇ tially normal fimbriation (Fig.
  • Genotype and phenotype of plasmids (A. B or U. respectively indicate pACYC184. PBR322 or pUC18 based vector) used in this study, position of inserts and hemagglutination ti er. H l l B
  • the plasmids pLPA22, pLPA29, pLPA30, pLPA37, pLPA38, pLPA93, pLPA95 and pPKL115 in E. coli HB101 were deposited on 26 January 1994 with DSM, the Deutsche Sammlung von Mikro- organismen und Zellkulturen GmbH, (German Collection of Microorganisms and Cell Cultures) , Mascheroder Weg IB, D- 38124 Braunschweig, Germany, under the accession numbers DSM 8915, DSM 8916, DSM 8917, DSM 8918, DSM 8919, DSM 8920, DSM 8921 and DSM 8923, respectively.
  • FnSPl EAQQMVQPQSPVAVSQSKPGCYDNGKHYQI (SEQ ID NO:13)
  • CB-II-G EEGKRGARGEBGAAGPVGPBGERGARGNR (SEQ ID NO:14)
  • RVFPRGTVENPC SEQ ID N0:16
  • sM5(134-163) QESKENEKALNELLEKTVKDKIAKEQENKE (SEQ ID NO:21)
  • SM5(117-146) DLTKELNKTRQELANKQQESKENEKALNEL (SEQ ID NO:22)
  • Example 1 The following clinical isolates of E. coli were tested for adhesion class according to the methods described in Example 1: KB-23, KS-54, U221-3, MJ#9-3, MJ#31-3, MJ#ll-2, MJ#2-2. The results of these experiments are illustrated in Fig. 5. As explained above, the isolate KB-23 showed the M L type of adhesion, and the isolate U221-3 expressed a M class adhesin showing a mannose-resistant type of adhesion and accordingly, this strain was classified as having a M R class adhesin.
  • Table 5 shows the nucleotide sequences of the fimH genes of selected fimH genes disclosed in Example 1 [CI#3 (SEQ ID NO:50), CI#4 (SEQ ID N0:44) , CI#7 (SEQ ID N0:51), CI#10 (SEQ ID NO:48) and CI#12 (SEQ ID NO:54)] and as the reference that of the E. coli K12 strain PC31 as it was originally disclosed by Klemm et al. (ref. 27) as the top sequence designated PC31a and the sequence as it was determined recently (PC31b) . Additionally, the nucleotide sequences of the following clinical isolates of E.
  • coli are shown: KS54 (SEQ ID N0:52) , U221-3 (SEQ ID NO:53), MJ#9-3 (SEQ ID NO:46) , MJ#31-3 (SEQ ID N0:47), MJ#ll-2 (SEQ ID NO:43), MJ#2-2 (SEQ ID N0:45) and F- 18 (SEQ ID NO:42) .
  • PC31a AAT TTT TCC GGG ACC GTA AAA TAT AGT GGC AGT AGC TAT CCA TTT CCT ACC ACC
  • PC31a AGC GAA ACG CCG CGC GTT GTT TAT AAT TCG AGA ACG GAT AAG CCG TGG CCG GTG
  • PC3 a ATT CCA GCG AAT AAC ACG GTA TCG TTA GGA GCA GTA GGG ACT TCG GCG GTG AGT
  • K -casein is the glycosylated isoform of bovine casein con ⁇ sists of a single polypeptide chain containing 169 amino acid residues the sequence of which has been determined (ref. 68) .
  • Bovine i-casein does not contain N-glycosidic linkages, but up to six 0-linked oligosaccharides are present in the C- ter inal region of the molecule (refs. 68, 69) .
  • the sacchari- de moieties are heterologous and also vary as a function of time after parturition. Of significance for the present study is the fact that D-mannose is not present in the bovine K - casein.
  • Adhesion tests were performed to verify the inability of recombinant strains carrying the fimH gene from E. coli strain PC31 to adhere to immobilized K -casein.
  • the E. coli strain used, KBIOOI is HBlOl containing plasmids pPKL115 and pLPA22 (ref. 70) .
  • the adhesion assay was performed using microtiter plates coated with 30 ⁇ g/ml K -casein in 0.1 M sodium bicarbonate (pH 9.6) for 30 minutes, followed by blocking any remaining binding sites with a subsequent in- cubation with 0.1% bovine serum albumin (BSA) in PBS.
  • BSA bovine serum albumin
  • bacteria were diluted to equivalent concentrations (5 x 10 7 cells/100/ ⁇ l) in PBS containing 0.1% BSA, added to coated microtiter wells for 30 minutes at 37°C. After washing the wells thoroughly to remove unbound bacteria, BHI broth was added and the bacteria were allowed to grow at 37°C on a rotating platform (150 rpm) until the optical density could be measured (2-2.5 hours). Comparisons can be made of optical densities obtained in the test wells to those obtained in standard curves developed from the plating of known numbers of bacteria under similar conditions, allowing extrapolation to absolute numbers of bound bacteria (ref. 70) .
  • the KBIOOI strain comprising the fimH gene from PC31 bound to immobilized mannan in significant numbers, but there was substantially no measurable adhesion to immobilized K -casein.
  • cells of KBIOOI were allowed to interact with K-casein immobilized on microtiter wells. After thorough washing to remove non-adhering bacterial cells, cells adhering to the wells were collected and grown over ⁇ night in BHI broth. These "enriched" bacterial cultures were again allowed to interact with immobilized K -casein, the plates were washed and adhering cells collected in nutrient broth. This enrichment cycle was repeated up to ten times.
  • Non-enriched KBIOOI 0.043 + 0.018 pPKL115 + pLPA22
  • plasmid preparations of pLPA22 were made from en ⁇ riched and from non-enriched strains and used to transform E. coli HBlOl containing the auxiliary plasmid pPKL115. Randomly selected transformants resistant to ampicillin and chloram- phenicol were tested for adhesion to K -casein, and several of the transformants harbouring plasmids from enriched cultures adhered in significantly increased numbers relative to plas ⁇ mid-containing cells of the non-enriched parent strain (Table 6.2) .
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Abstract

La présente invention concerne des adhésines bactériennes sélectionnées ou recombinées pour présenter la capacité de se fixer spécifiquement à des récepteurs inanimés ou animés prédéterminés et sélectionnés. L'invention concerne également l'utilisation de telles adhésines ou de bactéries exprimant ces adhésines pour cibler des composés et/ou des bactéries utiles à des cellules et des surfaces sélectionnées.
EP95906910A 1994-01-27 1995-01-27 Adhesines bacteriennes specifiques de recepteurs et leur utilisation Withdrawn EP0738325A1 (fr)

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CA2330699A1 (fr) * 1998-04-30 1999-11-11 Gyre Ltd. Proteines d'adhesine multifonctionnelles et affichage de ces proteines dans des cellules microbiennes
GB9819484D0 (en) * 1998-09-07 1998-10-28 Univ Bristol Therapeutic agents
DE19850718C1 (de) 1998-11-03 2000-05-18 Hildt Eberhardt Zellpermeabilität-vermittelndes Polypeptid
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WO2002004496A2 (fr) * 2000-07-07 2002-01-17 Medimmune, Inc. Proteines du type adhesines fimh et leur procede d'utilisation
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