WO2002102409A1 - Inactivated mycobacterial ompatb and uses thereof - Google Patents

Abstract

This invention relates to the role of the outer membrane protein OmpATb in the virulence of Mycobacteria such as M. tuberculosis. The OmpATb protein is shown to be a virulence factor which allows growth in acidic environments. Mycobacterium OmpATb mutants, particularly mutants of mycobacteria which are members of the Mycobacterium tuberculosis complex, such as BCG and M. tuberculosis, are shown to be useful as immunotherapeutic agents, vaccines, or carriers for use in generating new vaccines, for example in the treatment of a range of disorders, including tuberculosis.

Description

INACTIVATED MYCOBACTERIAL OMPATB AND USES THEREOF

This invention relates to the role of the outer membrane protein OmpATb in the virulence of Mycobacteria such as M. tuberculosis. Mycobacterium OmpATb mutants, particularly mutants of mycobacteria which are members of the Mycobacterium tuberculosis complex, such as BCG and M. tuberculosis, are useful as immunotherapeutic agents, vaccines, or carriers for use in generating new vaccines, for example in the treatment of a range of disorders, including tuberculosis. The OmpATb protein itself is shown to be a potential drug target for use in the development of therapies for these disorders.

Mycobacteria have an outer permeability barrier which, although it is functionally equivalent to the outer membrane of Gram-negative bacteria, is chemically distinct (Brennan, P.J. and Ni aido, H. (1995) Ann. Rev. Biochem. 64: 29-63; Daffe, M. and Draper, P. (1998) Adv. Microb. Physiol. 39:131-203; Draper, P. (1998) Frontiers in Bioscience 3:1253-1261).

The outer membranes of Gram-negative bacteria contain pore- forming proteins called Porins (Nikaido, H. (1994) 269: 3905-3908) . Porins comprise a series of membrane-crossing strands in a β-barrel configuration which form a pore. Typically, such pores form a water-filled channels through which hydrophilic molecules can diffuse. The channels are commonly non-specific, or have weak specificities for e . g. cations, and allow diffusion of substances up to a molecular mass limit which is determined by the diameter of the pore (Jap, B.K. and Walian, P.J. (1996) Physiol. Rev. 76:1073-1088; Schirmer, T. (1998) J. Struct. Biol . 121:101- Several mycobacterial porins have been identified (Trias, J. et al (1992) Science 258:1479-1481; Mukopadhyay, S. et al (1997) J. Bacteriol. 179: 6205-6207; Mobasheri, H. et al (1998) Biophysical J. 74: A320 (abstract); Lichtinger, T. et al (1999) FEBS Letters 454: 349-355; Kartmann, B. et al (1999) J. Bacteriol. 181: 6543-6546), although the peptide sequences of only two of these are known (Senaratne, R.H. et al (1998) Journal of Bacteriology 180: 3541-3547; Niederweis, M. et al . (1999) Mol . Micro. 33: 933-945).

OmpATb is an M. tuberculosis protein which forms pores permeable to hydrophilic substances in both liposomes (artificial membrane vesicles) and lipid bilayers (Senaratne et al . (1998) supra) . Although the protein has some sequence homology with OmpA of Escherichia coli , this homology is confined to the carboxy-terminal regions of the two proteins; this region does, not form part of the membrane-crossing structure in E. coli .

Although it is a major protein of the outer membrane, the function of OmpA of jE. coli is unclear. While OmpA forms pores in experimental membrane systems (Saint, N. et al (1993) Biochimica Biophysica Acta 1145:119-123; Sugawara, E. and Nikaido, H. (1997) Biophysical J. 72: A138

(abstract) ) , the determined crystal structure of a modified form of the protein contains no continuous water-filled channels (Pautsch, A. and Schultz, G.E. (2000) J. Mol. Biol. 298: 273-282). Furthermore, the known membrane-crossing region of OmpA does not share sufficient similarity with that of OmpATb to allow conclusions to be drawn about similar or different functions for these proteins. Although infection with the mycobacterium M. tuberculosis is a major cause of human morbidity and mortality, relatively little is known about its virulence factors and mechanisms of pathogenicity.

Vaccines and medicaments which are able to stimulate an immune response against M. tuberculosis have been developed for the treatment of M. tuberculosis infection. Such vaccines employ non-virulent strains of M. tuberculosis or less virulent related mycobacteria, for example, other ^■ members of the M. tuberculosis complex.

Mycobacterium bovis strain bacille Calmette-Guerin (BCG) is a member of the M. tuberculosis complex which is used as live vaccine against M. tuberculosis infection and has been administered to more than a billion people world-wide (Cohn, D.L. (1997) Am. J. Med. Sci . 6: 372 - 376.). BCG has also been used as a non-specific immunotherapeutic . agent in cancer treatment (Nseyo, U.O., and Lamm, D.L. (1997) Semin . Surg. Oncol . 13: 342 - 349; Patard, J.J. et al (1998). Urol . Res . 26: 155 - 159.).

Members of the M. tuberculosis complex, such as BCG and M. tuberculosis are invasive micro-organisms which infect mammalian hosts. In addition to the production of chemical species such as reactive oxygen metabolites, the phagocytic cells of a mammalian host are able to generate low pH conditions within phagosomes. These conditions damage microbial DNA, proteins, and membranes and present a hostile environment to invasive microorganisms (Hassett, D.J., and Cohen, M.S. (1989) FASEB J. 3: 2574 - 2582). Mechanisms which enable a micro-organism to cope with such conditions are therefore particularly important for the survival and virulence of intracellular pathogens within the body.

Despite its widespread use, BCG is known to cause severe infections in immunocompromised individuals (Steg, A. et al (1989) Eur. Urol . 16: 161 - 164; Stone, M.M. et al (1995) N. Engl . J. Med. 333: 561 - 563.; Hill, A.V. (1998) Annu. Rev. Immunol . 16: 593 - 617; Vesterhus, P. et al (1998) Clin . Infect . Dis . 27: 822 - 825). This indicates that this organism is endowed with residual virulence properties which may manifest in the absence of an effective immune response.

The ability of non-virulent strains of M. tuberculosis, BCG and other mycobacteria to survive for prolonged periods without causing progressive infection in immunocompetent individuals is an important component of the protective properties of these micro-organisms (Bloom, B.R., and Fine, P.E.M. : Bloom, B.R. (ed.) Tuberculosis: pathogenesis, protection and control. ASM Press, New York, 1994 p: 531 - 558; Behr et al (1999) Science 284: 1520-153) and in animal models, the persistence of such strains correlates with protective efficacy. It is therefore important that any mycobacterium used in therapeutic applications is able to survive in an immunized immunocompetent host without causing disease.

The mycobacterial strains presently used in vaccines have several potential drawbacks. They are genetically unstable, ineffective in certain geographical areas and the protection conferred on a vaccinated individual declines over time. There is therefore a need to develop reduced or non-virulent strains of mycobacteria for use as immunotherapeutics The lack of genetic information about the virulence and pathogenicity of mycobacteria such as M. tuberculosis, has hampered the development of such strains.

The present invention relates to characterization of the role of the OmpATb gene and the unexpected discovery that the its inactivation leads to attenuation the virulence of mycobacteria of the M. tuberculosis complex within a host. This has important applications for the generation of new therapeutics against M. tuberculosis and other conditions. Furthermore, this finding evidences the role of OmpATb as a virulence factor and therefore a target for therapeutics.

The term ' . tuberculosis complex cell' as used herein refers to a cell from a mycobacterium which is a member of the M. tuberculosis .complex. Mycobacterium which are members of the M. tuberculosis complex include . tuberculosis, BCG, M. bovis, M. africanum, M. canetti and M. micro ti .

The term ^λ ompATb gene' means the ompATb gene of M. tuberculosis (designated Rv0899 in the M. tuberculosis genome: Cole, S. T. et al (1998) Nature 393; 537-544: SwissProt open reading frame MTCY31.27: SwissProt Ace. No: Q10557) or the equivalent gene in other M. tuberculosis complex mycobacteria.

Mycobacteria of the M. tuberculosis complex have endogenous antigens which are cross-reactive with M. tuberculosis . Antibodies raised against such a cross-reactive antigen will also bind specifically to one or more antigens from M. tuberculosis . These cross-reactive antigens are able to evoke and/or potentiate an immune response against M. tuberculosis in an individual. Inoculation of an individual with a mycobacterium of the M. tuberculosis complex, or one or more antigens therefrom, may therefore prime the immune system to react against challenge with M. tuberculosis and may therefore potentiate a immune response against subsequent M. tuberculosis infection. This may reduce or abolish the symptoms and/or duration of infection and thereby provide a protective effect against such infection.

A first aspect of the present invention provides a cell of a mycobacterium which is a member of the Λf. tuberculosis complex and which has inactivated ompATb function.

A cell of a mycobacterium as described herein may be in isolated and/or puri-fied form, free or substantially free of material with which it is naturally associated or with which it is associated in the course of recombinant production, such as culture medium, other micro-organisms and microbial by-products .

An M. tuberculosis complex cell, particularly an M. tuberculosis cell, as described herein may persist in a host immunized therewith. However, the presence of the ompATb mutation affects the ability of the cell to cause progressive infection in an immunocompetent host i.e. such a cell has reduced, attenuated or decreased virulence, more preferably is non-virulent (i.e. virulence has been abolished) , and does not evoke disease symptoms in an individual. Such a cell may be used in pharmaceutical compositions and vaccines as described herein.

An M. tuberculosis complex cell of the present invention may be used in a range of therapeutic (including prophylactic) or other medical or veterinary applications.

OmpATb function in a M. tuberculosis complex cell may be inactivated, totally or partially, by the inactivation of the ompATb gene. The ompATb gene may be inactivated by a mutation such as an insertion, deletion or frameshift mutation. Any mutation which inactivates or reduces the activity of the ompATb gene may be employed in accordance with the present invention. Mutations may occur in the coding region and affect (i.e. reduce or abolish) the ompATb activity of the expressed protein or in the non- coding region and affect (i.e. reduce or abolish) the expression of active ompATb protein. In some embodiments, OmpATb function may be inactivated partially by a mutation in a cell which reduces, lowers or decreases the activity of the ompATb gene relative to the non-mutated cell, but does not lead to the complete abolition of ompATb activity.

Mutations may, for example, be carried out by- replacing the endogenous ompATb gene of an M. tuberculosis complex cell, such as BCG or Mycobacterium tuberculosis, with a ompATb transgene which carries a mutation which reduces or inactivates the function of the oiηpATjb transgene.

A mutation which reduces or inactivates the function of the ompATb transgene may comprise the replacement of ompATb sequence in a mutant ompATb gene with non-oipATjb sequence. Non-ompi-Tb sequence may include a gene encoding a positive selectable marker. Other suitable mutations are well known to those skilled in the art.

An ompATb gene from a mycobacterium of the M. tuberculosis complex encodes a polypeptide which has the transport and acid response activity of the M. tuberculosis ompATb gene product and may have greater than 70% sequence identity with the ompATb gene of M. tuberculosis, greater than 80% sequence, identity, greater than 85% sequence identity, greater than 90% sequence identity, greater than 95% sequence identity or greater than 98% sequence identity.

Sequence identity is generally defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) . GAP uses the Needleman and Wunsch algorithm t'o align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may_. be used, e.g. BLAST (which uses the method of Altschul et al . (1990) J. Mol . Biol . 215: 405- 410) , FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith- Waterman algorithm (Smith and Waterman (1981) J. Mol Biol . 147 : 195-197), generally employing default parameters.

An OmpATp gene from a mycobacterium of the M. tuberculosis complex may be obtained by amplification of genomic DNA from the mycobacterium using primers PI and P2 as described herein.

The endogenous gene may be replaced by a transgene using homologous recombination, for example, using a method wherein the cell is transformed with a vector comprising a transgene, a positive selectable marker and a counterselectable marker, as described in Sander, P. et al (2001) Infection and Immunity 69: 3562-3568. The transgene may comprise a gene encoding a positive selectable marker.

Using such methods, screening for ompATb mutants is carried out using a two stage selection. In a first stage, transformed cells in which the vector has integrated into the genome are identified by selecting for a positive marker on the vector. In a second stage, transformants in which double cross over recombination, and therefore allelic replacement, has occurred, are then isolated.

Suitable dominant negative selectable markers include rpsL and SacB, which is preferably used in conjunction with an additional counterselectable marker, such as a thermosensitive origin of replication.

Other suitable methods for producing a mutant mycobacterial cell are described in Glickman M. et al (2000) Molecular Cell . 5: 717-727.

A cell of a mycobacterium of the M. tuberculosis complex as described herein, which has attenuated or abolished ompATb function, for example a BCG or ycoJbacterium tuberculosis cell, may be used in a method of treatment of the human or animal body, for example a method of therapeutic treatment. Such a cell may be useful as a vaccine, which may be administered prophylactically, for the treatment of a mycobacterial infection such as tuberculosis'.

A cell of a mycobacterium of the M. tuberculosis complex as described herein may over-express one or more endogenous antigens, for example heat shock protein antigens. Such over-expression may enhance the immunogenicity of the cell and may be useful in therapeutic applications._. Methods of causing the over-expression of endogenous antigens are well known in the art and may, for example, include transforming the cell with an expression vector which comprises one or more nucleic acid sequences encoding the endogenous antigen operably linked to regulatory elements which direct high levels of expression within the cell.

A M. tuberculosis complex cell of the present invention may further comprise a gene encoding a non-mycobacterial or foreign antigen. Expression of such an antigen in an M. tuberculosis complex cell, for example, an BCG cell allows the generation of an immune response in a vaccinated individual against the non-mycobacterial antigen. The cell may therefore be used as an antigen delivery system in the treatment of any disease, such as a pathogenic infection, which is ameliorated by an immune response against a particular antigen.

Suitable antigens include viral, protozoal, tumour cell, bacterial, fungal and other antigens. For example, an antigen from H. pylori , Measles virus (Fennelly G. J. et al (1995) J. Infect . Dis . 172: 698-705), Mumps virus,

Rubeola virus (e.g. OspA: Stover, C.K. et al (1993) J. Exp . Med. 178: 197-209), B. burgdorferi (e.g. protein A: Langermann et al (1994) J. Exp . Med. 180: 2277-2286), Herpesvirus, Papillomavirus, Tetanustoxin, Diphtheriatoxin, Pneumococcus spp (e.g. Surface protein A: Langermann et al

(1994) supra), tumour cells, Leishmania (e.g. surface proteinase gp63 : Connell N. et al (1993) Proc. Natl . Acad. Sci . USA . 90: 11473-11477) or HIV (or SIV: Yasutomi Y. et al (1993) J. Immunol . 150: 3101-3107) may be used. Such an antigen may be useful in the treatment of ulcers, measles, mumps, rubeola, Lyme disease, herpes, cancer, tetanus, diphtheria, cancer, Leishmaniasis or AIDS respectively.

A further aspect of the present invention therefore provides a M. tuberculosis complex cell as described herein which comprises genetic material encoding an antigen or immunogen exogenous or foreign to the mycobacterium. Examples of a suitable non-mycobacterial antigen or immunogen that may be encoded are listed above.

The M. tuberculosis complex cell is able to express the said genetic material upon infection of a host cell, thereby producing the encoded antigen or immunogen, to which an immune response may be generated.

An M. tuberculosis complex cell of the present invention may thereby confer immunity against a pathogen other than the mycobacterium itself (i.e. a pathogen, such as a bacteria, virus or fungus, which is not a mycobacterium of the M. tuberculosis complex) in a susceptible species immunised therewith.

A further aspect of the present invention provides the use of a nucleic acid comprising an inactivated ompATb transgene as disclosed herein in a method of attenuating, lowering, reducing or decreasing the virulence of a M. tuberculosis complex cell, for example, an M. tuberculosis cell.

The inactivated ompATb transgene may be used as described herein to replace the endogenous ompATb gene of the cell.

A further aspect of the present invention provides a method for reducing attenuating, lowering, reducing or diminishing the virulence of a mycobacterial cell of the M. tuberculosis complex comprising inactivating a ompATb gene within the cell.

A related aspect of the present invention provides a method for reducing attenuating, lowering, reducing or diminishing the virulence of a vaccine comprising a mycobacterial cell of the M. tuberculosis complex, comprising inactivating a ompATb gene within the mycobacterial cell.

Inactivating a ompATb gene may comprise replacing an endogenous ompATb gene with an inactive ompATb transgene. The replacement may occur by homologous recombination as described herein.

Methods of the present invention may include isolating and/or purifying a M. tuberculosis complex^' cell after inactivation of the ompATb gene as described herein, culturing and/or formulating such a cell into a pharmaceutical composition, for example, by admixing the cell with one or more of a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.

A M. tuberculosis complex cell of the present invention may therefore be isolated and/or purified and manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, vaccine, pharmaceutical or veterinary composition or drug. These may be administered to individuals.

Individuals include humans and other mammals, including farm animals (e.g. cows) and wild animals (e.g. badgers) which are susceptible to infection with members of the

Mycobacterium tuberculosis complex, such as Mycojbacterium tuberculosis and Mycobacterium bovis .

Pharmaceutical or veterinary compositions and vaccines according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical or veterinary compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Another aspect of the present invention therefore provides a pharmaceutical or veterinary composition or vaccine comprising an M. tuberculosis complex cell and having an inactivated ompATb function as disclosed herein.

An inactivated ompATb function means an abolished or reduced ompATb activity within the cell. This may be achieved by inactivating an endogenous mycobacterial ompATb gene .

Such a pharmaceutical may be an immunotherapeutic agent, vaccine, or carrier of antigenic or immunogenic material and may be used to generate an immune response in the treatment (including prophylactic treatment) of a disorder in an individual in which said response is beneficial. Suitable disorders include disorders in which an immune response against, for example, BCG or M. tuberculosis is beneficial, for example, tuberculosis and cancer.

Another aspect of the present invention provides the use of a M. tuberculosis complex cell as described herein in the manufacture of a medicament for use in the treatment of a disorder in which an immune response against an antigen expressed by the M. tuberculosis complex cell is beneficial, for example, infection by a microorganism which comprises said antigen.

Such disorders include disorders in which an immune response against an endogenous M. tuberculosis cross-reactive antigen expressed by the mycobacterial cell is beneficial, for example, tuberculosis and cancer, and disorders in which an immune response against a foreign (non-tuberculosis, or non- mycobacterial) antigen expressed by a BCG cell is beneficial (for example, ulcers, measles, mumps, rubeola, Lyme disease, herpes, cancer, tetanus, diphtheria, cancer and AIDS).

Another aspect of the present invention provides a method of making a pharmaceutical or veterinary composition comprising admixing such an M. tuberculosis complex cell, particularly an M. tuberculosis cell as described herein, with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other_^ingredients .

Another aspect of the present invention also provides a method comprising the administration of a M. tuberculosis complex cell as described herein to a mammal in need thereof for the treatment of a disorder in which an immune response against the cell is beneficial. Such disorders include cancer and mycobacterial infections such as tuberculosis .

A method of treatment of a disorder described above may include administering a vaccine comprising a M. tuberculosis complex cell as described herein to^' an individual in need thereof . Such a method may have a prophylactic purpose, for example when the individual is a risk of suffering from the disorder, or a therapeutic purpose, for example when the individual is suffering from the disorder.

A M. tuberculosis complex cell as described herein may be used to present foreign antigens as disclosed herein and for the purpose of generating an immune response against the foreign antigen. Such a cell may be used in the treatment of disorders characterised by the presence of a foreign antigen in the body, for example, infection by a pathogen.

Administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount"

(as the case may be, although prophylaxis is encompassed by the term 'therapy'), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course, of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors .

BCG administration is well established throughout the world as a prophylactic treatment for tuberculosis. A skilled person in the field is therefore familiar with the protocols, formulations, dosages and clinical practice associated with the administration of BCG -and other mycobacteria. Such protocols, formulations, dosages and clinical practice are entirely suitable for use with pharmaceutical compositions and vaccines of the present invention. A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

OmpATb is shown by the results set out below to be an important mycobacterial virulence factor. For example, at low pH, OmpATb is shown to be the sole uptake mechanism for small hydrophilic molecules such as serine, glycerol and glucose. In the light of these results, OmpATb thus represents a potential target for therapeutic intervention.

Further aspects of the present invention provide an OmpATb inhibitor for use in a method of treating a mycobacterial infection and the use of OmpATb inhibitor in the manufacture of a medicament for use in the treatment of infection by a mycobacterium.

..J¹ Another aspect of the present invention provides a method of treatment of a condition associated with infection by a mycobacterial cell comprising; inhibiting the OmpATb activity of the cell.

The mycobacterium or mycbacterial cell according to these aspects may be a member of the M. tuberculosis complex, for example M. tuberculosis .

Other aspects of the present invention relate to methods for obtaining inhibitors of OmpATb.

A method of obtaining an inhibitor of OmpATb may comprise; contacting an OmpATb polypeptide with a test compound, and; determining the activity of said OmpATb polypeptide. Activity of the OmpATb polypeptide in the presence of a test compound may be compared with the activity of the OmpATb polypeptide in comparable reaction medium and conditions in the absence of a test compound.

A difference (i.e. an increase or decrease) in activity in the presence of test compound relative to the absence is indicative that the test compound is an agent which is able to modulate the activity of OmpATb . In particular, a decrease in activity in the .presence of test compound relative. to the absence is indicative that the test compound is an inhibitor of OmpATb .

The activity of the OmpATb polypeptide may be determined by determining the cellular uptake of a substrate molecule. A suitable substrate molecule may be selected from the group consisting of glucose, glycerol and serine.

In some embodiments of such methods, the OmpATb polypeptide may be comprised within a cell, for example a bacterial or mycobaterial cell or within a liposome. The OmpATb polypeptide may be endogenous to the cell (i.e. the cell may naturally express the OmpATb polypeptide) or it may be heterologous (i.e. the cell does not naturally express the OmpATb polypeptide) .

Methods for the cloning of genes encoding heterologous polypeptides and their expression in host cells are well- known in the art, see for example Molecular Cloning: a Laboratory Manual: 3rd edition, Sa brook & Russell., 2001, Cold Spring Harbor Laboratory Press and Current Protocols in Molecular Biology, Ausubel et al . (1992) eds . John Wiley & Sons . The OmpATb polypeptide may be contacted with the test compound under acidic conditions (i.e. a pH less that 7), for example pH 6.5 or less or pH 6 or less. In some embodiments, pH 5.5 is used.

A test compound may be a small chemical entity, peptide, antibody molecule or other molecule whose effect on the activity of OmpATb is to be determined. Suitable test compounds may be selected from compound collections and designed compounds, for example using combinatorial chemistry as described below.

Combinatorial library technology (Schultz, JS (1996)

Biotechnol. Prog. 12:729-743) provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide.

The amount of test substance or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.01 to 100 nM concentrations of putative inhibitor compound may be used, for example from 0.1 to 10 nM.

Compounds which may be used may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants which contain several characterised or uncharacterised components may also be used.

Test compounds may also be based on modelling the 3- dimensional structure of a OmpATb polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics-.

An agent identified using one or more primary screens as having ability to inhibit the activity of OmpATb may be assessed further using one or more secondary screens . A secondary screen may involve testing for a biological function of OmpATb .

For example, a method may further comprise determining the ability of said test compound to inhibit the growth and/or virulence of a mycobacterium.

Inhibition may be determined in vi tro, for example in cultured cell lines or in vivo, for example in (non-human) animal model systems . Methods employing animal model systems may comprise the step of sacrificing the animal.

A method may comprise identifying a test compound as a inhibitor of OmpATb ."

A method may further include isolating, purifying and/or manufacturing a compound which inhibits OmpATb.

Optionally, compounds which inhibit OmpATb which were obtained using an method described herein may be modified to optimise activity or provide other beneficial characteristics such as increased half-life or reduced side effects upon administration to an individual.

Methods of the present invention may further include formulating the agent into a composition, such as a medicament, pharmaceutical composition or drug, with a pharmaceutically acceptable excipient as described below. Such a composition may be administered to an individual.

The present invention extends in various aspects not only to a compound which inhibits OmpATb obtained by a method described above, but also a pharmaceutical composition, medicament, drug or other composition comprising such a compound, a method comprising administration of such a composition to a patient, e.g. for treatment (which may include preventative treatment) of conditions associated with mycobacterial infection such as tuberculosis, use of such a compound in manufacture of a composition for administration, e.g. for treatment of a condition associated with mycobacterial infection, and a method of making a pharmaceutical composition comprising admixing such a compound with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients .

Aspects of the present invention will now be illustrated with reference to the accompanying figures described already above and experimental exemplification, by way of example and not limitation. Further aspects and embodiments will be apparent to those of ordinary skill in the art. All documents mentioned in this specification are hereby incorporated herein by reference.

Fig. 1 shows the growth of M. tuberculosis H37Rv (circles) and a mutant lacking functional OmpATb (ΔOmpATb) (squares) in Dubos medium at pH 7 (closed symbols, continuous lines) or in Dubos medium adjusted to pH 5.5 (open symbols, broken lines) .

Fig. 2 shows the growth of wild-type M. tuberculosis H37Rv and the ompATb mutant in macrophages . The wild-type (circles) and the ompATb mutant (squares) strains of . tuberculosis were grown in murine bone marrow-derived macrophages (A) or the human monocytic cell line THP1 (B) . ** denotes that the ompATb mutant differed significantly from the wild-type by the paired student-t test (p<0.05) .

Fig. 3 shows the uptake of small hydrophilic molecules by the ompATb-deleted mutant and by wild-type M. tuberculosis . A: glucose. B: glycerol . C: serine. D: glycine. In each case the closed circles represent wild-type M. tuberculosis and the open squares represent the ompATb mutant . The closed triangles in B represent the single cross-over strain. Each experiment was carried out a minimum of four times; the data shown in A, B, C and D are representative of these replicated experiments. Panel E shows the data for a single time point for each compound (90 days for glucose, serine and glycine, 180 days for glycerol) . Each bar represents the mean .of four experiments with the uptake of the mutant expressed as a percentage of the uptake of the wild-type,- the black bars represent the ompATb mutant and the white bar represents the single cross-over strain (included for glycerol only) . Error bars represent plus or minus the standard error of the mean. ** denotes that the ompATb mutant differed significantly from the wild-type by the paired student-t test (p<0.05).

Figure 4 shows the uptake of serine as shown in figure 3 at pH 7 (left hand panel) and pH 5 (right hand panel) for wild-type M. tuberculosis H37Rv and the ompATb mutant.

Figure 5 shows expression of ompATb by M. tuberculosis exposed to low pH or isolated from macrophages. A: the effect of reduced pH on ompATb expression, measured by real-time quantitative PCR. The amount of ompATb mRNA relative to that of the normalizing gene, sigA, was determined by real-time quantitative RT-PCR. The' values shown are the means; the error bars indicate the standard deviations. B: ompATb expression by M. tuberculosis growing inside the human monocytic cell line THP1 (white bar) , or murine bone marrow-derived macrophages (black bar) measured by real-time quantitative PCR. Normalisation was carried out as in A. The values shown are means; the error bars indicate the standard deviations.

Fig. 6 shows the growth of wild-type M. tuberculosis and the ompATb mutant in mice. BALB/c mice were infected intravenously with approximately 5 x 10⁵ cfu M. tuberculosis. The numbers of cfu per tissue were determined for lungs (A) and spleens (B) at different time intervals. The wild-type is shown by circles and the mutant by triangles. Each point represents the mean of 4 to 5 mice; error bars represent standard errors. In a second experiment (C) the single cross-over strain was included and growth in the lungs at a single time point (65 days after infection) was determined; the black bar represents the wild type, the hatched bar represents the single cross- over and the white bar represents the ompATb mutant. ** denotes that the ompATb mutant differed significantly from the wild-type by the paired student-t test (p<0.05).

Figure 7 shows the expression of ompATb in M. tuberculosis phagocytosed by normal bone marrow macrophages and by normal and activated cells of the macrophage-like line THP- 1, as measured by RT-PCR. Results are expressed as ratios, taking the amount 'of mRNA for ompATb in M. tuberculosis grown in Dubos medium at pH 7 as unity.

Experimental Materials and Methods Construction of ompATb knockout mutant

The ompATb knockout mutant was prepared using a previously published strategy for gene replacement in mycobacteria (Sander, P., and Bδttger, E.C.(1998) Methods in Molecular Biology 101: 207-16; Sander, P. et al (2001) Infection and Immunity 69: 3562-3568). ^'

Transformation of M. tuberculosis H37Rv

M. tuberculosis H37Rv was grown in 2 1 roller bottles containing 400 ml 7H9-OADC-Tween until an OD of 0.6 was achieved. One day before harvesting the cells glycine was added to a final concentration of 1.5 % (v/v) and cells were incubated for an additional 24 hours. All following steps were performed at room temperature. Cells were harvested by centrifugation, washed several times with 10 % glycerol and finally • re-suspended in a volume of 5 ml .

For electroporation 400 μl competent cells were mixed with 1 μg supercoiled plasmid DNA and electroporated in a BioRad Gene pulser II with the following settings: 2.5 kV, 1000 Ohms, 25 μF. After electroporation cells were re-suspended in 4 ml of 7H9-OADC-Tween and incubated for 20 h with vigorous shaking at 37°C. Following incubation appropriate dilutions were plated on selective agar. Single colonies were picked, restreaked and grown in liquid broth when necessary.

Construction of M. tuberculosis ΔompATb Mutants

OmpATb was cloned as described in Senaratne et al (1998) supra. Briefly, open reading frame MTCY31.27 was amplified from genomic DNA of M tuberculosis H37Rv with the following primers :

PI, 5' AGGGAGTCATATGGTGGCTTCTAAGGCGGGTTTG-3' P2, 5' GAAGGATCCCCCCCAGGAACGCCAGCAGGTA-3' .

Primer 1 has an Ndel restriction site; primer 2 has a BamHI site. PCR was performed with the Expand High Fidelity PCR system (Boehringer Mannheim Ltd., Lewes, E. Sussex, United Kingdom) , using the 1.5 mM MgCI₂ Expand buffer supplied with the kit at an annealing temperature of 58°C. The products were separated on a 1% agarose gel, isolated with a QIAquick gel extraction kit (Qiagen Ltd. , Crawley, W. Sussex, United Kingdom) , and digested with restriction endonucleases Ndel and BamHI (Boehringer Mannheim) .

A 4.4 kb BcoRI fragment (H37Rv genomic coordinates 1001133 to 1005566) containing ompATb was cloned at the EcoRI site of vector ptrpA-l-rpsL⁺ using T4 ligase according to standard molecular biology techniques. ptrpA-l-rpsL⁺ is a pBluescript vector carrying the wild-type rpsL gene of M. bovis BCG as a counter-selection marker.

The ompATb gene was then inactivated by replacing a part of the ompATb coding sequence (425 bp fragment located between Bsml and Hpal sites; H37Rv coordinates 1002955 tol003379) with a 1.26 kb Km^r cassette from pUC4K (Amersham Pharmacia Biotech) to construct the suicide vector pompA: :aph-rps, according to the methods described in Sander et al (2001) supra .

This non-replicative plasmid was introduced into streptomycin resistant (Str^R) M. tuberculosis H37Rv (strain 1424) by electroporation and plated on 7H11 agar containing 50 μg ml^"1 kanamycin.

Southern analysis was performed by isolating chromosomal DNA from transformants, digesting it with Nsil and transferring it to a Nylon N+ membrane by vacuum blotting, then hybridising to a 450 bp probe located within the 3' end of the cloned flanking region (H37Rv coordinates 1004610 to 1005059) . The probe was labelled with α-³²P-dCTP using Klenow enzyme and buffer from the Oligolabelling kit (Amersham Pharmacia Biotech Inc, UK) . The hybridisation and washing protocols were carried out under high stringency conditions .

Kanamycin resistant colonies were screened by Southern blot analysis for single crossover recombinants.

Transformants in which the vector was integrated on the chromosome via homologous single crossover were identified by Southern analysis, and subjected to counter selection against the rpsL gene encoded by the vector. To counter select for rpsL, transformants were grown in liquid broth to OD-0.5, and dilutions were plated on 7H11+ kanamycin (50 μg ml^"1^ streptomycin (100 μg ml^"1). After 4 weeks of incubation at 37°C, the efficiency of counter-selection was determined by dividing the number of colonies obtained on plates containing kanamycin plus streptomycin by the number of colonies obtained on kanamycin.

Streptomycin resistant colonies were analysed by Southern blotting to identify recombinants that had undergone second cross-over events resulting in deletion of ompATb. Standard blotting procedures were used and DNA was hybridised to a fragment of pBluescript-ompATb using the digoxygenin system according to the manufacturer's instructions (Boehringer, Mannheim) .

All- DNA manipulations were carried out using standard techniques as described in Molecular Cloning: a Laboratory ^■Manual: 2nd edition, Sambrook et al . , 1989, Cold Spring Harbor Laboratory Press and Current Protocols in Molecular Biology, Ausubel et al.(1992) eds . John Wiley & Sons.

Bacterial Growth in vi tro

M. tuberculosis H37Rv (1424 is a streptomycin resistant strain with a mutation in the rpsL gene , which has been used for insertional inactivation of genes via homologous recombination (Springer et al . , 2001).

Mycobacterium tuberculosis H37Rv and AompATb were grown at

37 °C in Dubos broth (Difco) supplemented with Dubos medium albumin (4%) and glycerol (0.2%) in 100 ml cultures in a roller incubator rotating at 2 r.p.m or on 7H11 agar with Dubos oleic albumin complex supplement (Difco Laboratories) at 37°C. In the case of the ompATb-deleted mutant, 20μg ml^"1 kanamycin was included in the media. Growth was followed by measuring O.D. at 660 nm after diluting cultures 1 in 10.

Medium was acidified, if required, as described below.

Growth of M. tuberculosis at low pH

The wild-type and the ompATb deletion mutant of M. tuberculosis were grown to exponential phase and then inoculated into Dubos medium adjusted to pH 5.5 with HCl to give an OD_S00 of 0.01. The OD_Soo of the cultures was determined at various time intervals following incubation at 37°C. Preparation of Macrophages

THPl cells were cultured as described previously (Ragno et al . , 2001). After expansion the cells were centrifuged at

200g for 5 minutes at room temperature in RPMI medium (Life Technologies) supplemented with 50 nM phorbol 12-myrisate

13 -acetate (Sigma) at a concentration of 1 x 10⁶ cells ml^"1 or with β-mercaptoethanol at 2 μl per 5 ml. 12-well tissue culture plates (Nunc) were inoculated with 1 ml of this suspension per well and incubated at 37 °C in 5% C0₂ for 24 hours. Under these conditions, THPl cells differentiate into macrophages, stop dividing and adhere to the bottom of the wells (Tsuchiya et al . , 1982) . If necessary, THP-1 cells were activated with 100U γ-interferon per 20 ml, 24 h before infection.

Murine bone marrow macrophages were flushed from the femurs of 6 to 8 week old Balb/C mice and suspended in Dulbeccos medium with low glucose (1 g l^"1) and high carbonate (3.7 g l^"1) concentrations (Gibco BRL) and enriched with 10% heat- inactivated foetal calf serum, 10% L-cell conditioned medium and 2 mM glutamine. For the infection assays, mice bone marrow- macrophages were seeded into 12-well tissues culture plates (IxlO⁶ cells per well in a volume of 1 ml) and allowed to differentiate for 6 to 8 days.

Infection of macrophages to determine intracellular growth of M. tuberculosis

THPl cells and murine bone marrow macrophages were infected by removing the medium and replacing it with 1 ml RPMI containing 10⁵ colony forming units (cfu) of M. tuberculosis (equivalent to 1 cfu per 10 macrophages) . Cultures were incubated at 37 °C in a 5% C0₂ atmosphere for 16 hours. The medium was removed and the cells washed twice with 1 ml of warm medium to remove extracellular bacteria. 1 ml of fresh culture medium was added to each well and the plate was re- incubated at 37 °C in a 5% C0₂ atmosphere. Medium was replaced every 48 hours. At different time intervals the medium was removed from three wells and the intracellular bacteria released by lysing the macrophages with 500 μl of 2% saponin. The resulting lysate was immediately serially diluted in sterile saline and plated onto 7H11 agar plates. These plates were incubated at 37 °C for 14 days and colonies counted.

Growth of Bacteria in Mice

The wild type M . tuberculosis and the ompATb-deleted mutant strain {AompATb) were grown in Dubos 7H9 broth for 14 days. Each strain was diluted in phosphate-buffered saline to give a suspension of approximately 10⁶ colony forming units (cfu) per ml and 0.2 ml of these suspensions were inoculated intravenously into 6 to 8 week old female Balb/c mice. The infection was monitored by removing the lungs and spleens of infected mice and homogenising them by shaking with 2 mm diameter glass beads in chilled saline with a Mini-Bead Beater (CP Instruments) . Serial 10-fold dilutions of the resultant suspensions were plated on to Dubos 7H11 agar with Dubos oleic albumen complex • supplement. The numbers of cfu were determined after the plates had been incubated at 37°C for approximately 20 days. In a second experiment the AompATb, or single cross over strain (which contains copies of both the wild-type and the mutant allele) , and the wild type strain were inoculated intravenously into athymic mice (nu/nu^") and the

.infections monitored as described above. Immunoblotting of M. tuberculosis cell lysates Bacterial cell pellets were collected by centrifugation and washed in Tris buffer pH 9.5 and resuspended in Urea/Thiourea buffer [5 M Urea, 2 M Thiourea, 2% (w/v)

CHAPS, 2% (w/v) SB 3-10, 65 mM DTT, 20 mM Tris-pH 9.5, 0.1 mM EDTA and Complete protease inhibitors (Roche) ] . Bacteria were lysed in the presence of glass beads (150-212 microns, Sigma) in a Ribolyser (Hybaid) at a speed setting of 6.5 for 4 X 30 seconds. The tubes were cooled on ice for 5 min between each cycle of disruption. The supernatant was collected by centrifugation, brought to room temperature and filtered through a low-binding Durapore 0.22 μM membrane filter (Ultrafree-MC; Millipore) . An aliquot of the cell extract was used to determine its protein concentration using a Coomassie Plus protein assay reagent (Pierce) .

Cell-free extracts (-30 μg/lane) were resolved by SDS-PAGE (12% gel) and transferred at 60 mA for 1 h to PVDF membrane (Millipore) in a semi-dry blotter (Hybaid) using Tris/glycine/SDS buffer (48 mM Tris, 39 mM glycine, 0.037% SDS and 20% methanol, pH~8.3) . Equal loading of the proteins was confirmed by Coomassie staining of an identical gel and the efficiency of transfer was verified by staining the blot with a solution of 0.1% Ponceau S in 5% acetic acid. The membrane was blocked with 10% non-fat milk in TTBS [20 mM Tris (pH 7.5), 0.5M NaCl buffer containing 0.1% Tween 20] . The primary anti-OmpATb antibody was raised in rabbit against recombinant truncated OmpATb (Senaratne et al . , 1998) and used at 1:2000 dilution. The secondary goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase (Dako, UK) was used at 1:1000 dilution. The blots were washed and developed according to the enhanced chemiluminescence (ECL) detection protocol (Amersham Pharmacia Biotech, UK) .

Uptake of Radioactive Substrates

[¹C] glycerol (5.66 GBq mmol^"1) , L- [U-¹⁴C] serine (5.74 GBq mmol-1) , D- [U-¹⁴C] glucose (11.5 GBq mmol^'1) and [U-¹C] glycine (3.74 GBq mmol^"1) were from Amersham Biosciences . Uptake experiments were performed using 1x10¹⁰ bacteria in 1 ml saline buffer at room temperature (approximately 23° C) , containing either 1.6xl0^"6 M [¹⁴C] glycerol, 1.9xl0^"6 M L- [U- ¹C] serine, 5.1xl0^"6 M D- [U-¹C] glucose or 3.9xl0^"s M [U-¹C] glycine) . Aliquots of 0.1 ml were taken at different times and added onto the top of 0.8 ml of 0.5 M sucrose contained in an Eppendorf centrifuge tube. Bacteria were rapidly sedimented through the sucrose gradient by centrifugation (13,000 g for 1 min) . The supernatant was carefully removed and the radioactivity associated with the bacterial pellet was determined by liquid scintillation counting. Uptake determinations were repeated in at least three independent experiments .

Extraction of RNA

Bacteria were collected by centrifuging and suspended in 1 ml Trizol (Life Technologies) , transferred to a Bead Beater tube containing 500 μl 0.1 mm glass beads (Polylabo) and broken by 3 x 1 min in the Bead Beater running at maximum speed. Tubes were centrifuged at 13,000 x g for 1 min, and the supernatants were transferred to fresh tubes containing 300 μl chloroform: isoamyl alcohol. After centrifuging at 13,000 x g for 10 min the upper, aqueous phase was transferred to a tube containing 270 μl isopropanol. After standing overnight at 4 °C the precipitated RNA was washed with 1 ml 75% ethanol and resuspended in 90 μl diethyl pyrocarbonate-treated water. Contaminating DNA was removed by digestion with DNase I (Ambion) according to the supplier's instructions.

Alternatively, M. tuberculosis RNA was extracted using the Hybaid Ribolyser™ Kit blue (Hybaid) . M. tuberculosis cultures (OD_Soo= 0.6-0.8) were centrifuged at 2000 g for 10 min. The pellet was resuspended in 0.6 ml of Reagent A (chaotropic RNA stabilising reagent) and then transferred to a Hybaid Ribolyser tube containing 300 μl of reagent B (Phenol acid reagent) and 100 μl of reagent C (chloroform: isoamyl alcohol) and silica/ceramic matrix for optimal bacterial lysis. Bacteria were broken by shaking for 2 x 20 seconds in a Ribolyser (Hybaid) at a speed rating of 6. The tubes were centrifuged (13,000 g for 15 min) and the supernatants were transferred to new tubes containing 300 μl of Reagent C. After 10 min of centrifugation at 13,, 000 g, the aqueous phase was transferred to a tube containing 500 μl of reagent D (DEPC- treated isopropanol precipitation solution) . Total RNA was then precipitated overnight at 4°C, and washed with 1 ml 75% ethanol. RNA was resuspended in 90 ml of DEPC treated water. Contaminating DNA was removed by digestion with DNase I according to the manufacturer's instructions (Roche) .

To extract RNA from M. tuberculosis grown in macrophages, THPl cells or murine bone marrow macrophages were infected with M. tuberculosis at a multiplicity of infection of 10 for 8 hours (see below) . The macrophages were lysed with 500 μl of 2% saponin, and the surpernatant containing the bacteria was centrifuged for 10 min at 2000 g and the pellet washed twice with saline buffer. M. tuberculosis RNA was extracted as described above.

Preparation of cDNA with Reverse Transcriptase RNA, 5 μl, 1 μl primer for each mRNA to be copied and water to 12 μl were mixed and held at 65 °C for 10 min, then kept on ice. Reverse transcription was then carried out with a C. therm, poly erase kit (Roche) for 1 h at 60 °C followed by 5 min at 95 °C. The cDNA was diluted with 40 μl water.

Measurement of cDNA by real-time PCR

Real-time quantitative PCR was carried out on the ABI Prism 7700 sequence detection system using the Taqman Universal • PCR Master Mix (PE Applied Biosystems) . The primers and the Taqman probes (carrying both a fluorophore and a quencher) ' were designed using the Primer Express software and obtained from PE Applied Biosystems. The sequences were as follows: oiηpA forward primer: ATGTGCCGACCTGCAATCA; ompA reverse primer: ATTTCATAGTCGGCTGGGATCA; sigA forward primer: TCGGTTCGCGCCTACCT; sigA reverse primer:

TGGCTAGCTCGACCTCTTCCT; ompA FAM-labelled probe: TGGACCCATCGCGTTTGGCAA; sigA FAM-labelled probe: TTGAGCAGCGCTACCTTGCCG) .

RT-PCR experiments were carried out with 1 μg of RNA and

2.5 pmol of specific reverse primers for ompA and sigA in a volume of 8 μl. After denaturation at 65 °C for 10 min, 12 μl of the mixture containing 2 μl of dNTP (25 mM) , 4μl of 4x buffer, 2 μl of DTT, 1 μl of RNAsin (Promega) and 1.5 μl of Superscript II (Invitrogen) was added. Samples were incubated for 60 min a 42 °C, heated at 75 °C for 15 min and then chilled on ice. Samples were then diluted with 30 μl of H₂0 and stored at -20 °C. PCR conditions were identical for all reactions. The 25 μl reactions consisted of 12.5 μl of PCR master mix (Promega), 4 μl of template, 5 pmol of each primer and 2.5 pmol of the appropriate probe . The reactions were carried out in sealed tubes . Results were normalized to the amount of sigA mRNA which was shown to be constant under the conditions used (Manganelli et al . ,

(1999) Mol. Microbiol. 31 375-724; Manganelli et al . ,

(2001) Mol. Microbiol. 41 423-437.)

cDNA was measured in an ABI Prism 7700 Sequence Detector (TaqMan: Applied Biosystems Ltd) using the following mixture: AmpliTaq Gold 0.2 μl, 25 mM dNTP 0.6 μl , proprietary buffer 2.5 μl, MgCl₂ solution (as supplied) 4 μl, and appropriate volume of probe mixture and water to 25 μl . Volumes of probes used were: 0.5 μl for ompATb and sigA; 0.11 μl for gnd . The machine was operated according to the manufacturer's instructions.

Results Inactivation of ompATb by homologous recombination To facilitate isolation of an ompATb knock-out, a streptomycin-resistant strain of M. tuberculosis H37Rv was transformed with the suicide plasmid pompA-aph-rpsL⁺ and the transformants were selected on 7H11 media containing kanamycin and streptomycin (positive and counter selection markers respectively) and on kanamycin alone. The transformants were screened by Southern hybridisation analysis to distinguish knock-outs from merodiploids and the parental strain.

Based on the restriction pattern for Nsil , it was predicted that the probe would hybridise to a 4.7 kb fragment in the wild-type and to a 2.7 kb fragment in the knock-out. Merodiploid strains carrying both the wild-type and knockout alleles were expected to hybridise to two fragments (2.7 kb + 11 kb) in the case of a 3' single crossover and to a 11 kb fragment in case of a 5' single crossover.

Southern analysis of Nsil digested DNA demonstrated that an ompATb knock-out strain arose following a double crossover homologous recombination (a single 2.7 kb band), and a merodiploid strain arose following a 3' -single crossover recombination (a 2.7 kb band and a 11 kb band) .

Allelic exchange in the knock-out strain was also confirmed by Southern hybridisation analysis of Styl-digested DNA and PCR amplification from the flanking regions .

Successful deletion of the ompATb gene was further confirmed by immunoblotting a M. tuberculosis cell lysate with an antibody raised against OmpATb. A single band was observed for the wil,d-type lysate but no band was observed with the knock-out strain.

Growth of Wild type and Mutant M. tuberculosis Mutant M. tuberculosis lacking OmpATb (ΔOmpATb) grew at a similar rate to the wild type in supplemented Dubos broth at pH 7 (Fig. 1) . The growth rate was not affected by addition of 0.2M sucrose or 0.2M NaCl to the medium (to provide osmotic stress) . At pH 5.5 growth of the wild type showed a somewhat prolonged lag phase, followed by growth at the normal rate, but growth of ΔOmpATb was poor throughout the duration of the experiment (Fig. 1) .

The mutant grew at the same rate as the wild type in unstimulated macrophages but was significantly attenuated in normal mice (Fig. 6) . In nu/nu- mice, no such difference was apparent; these mice are effectively lacking in cell- mediated immunity, and extremely susceptible to infections with M. tuberculosis .

OmpATb Activity

The role of OmpATb in increasing the permeability of M. tuberculosis to small hydrophilic molecules was investigated by comparing the uptake of radiolabelled glucose, glycerol, serine and glycine by the mutant and the wild-type (Figure 3) . The mutant was significantly defective in the uptake of glucose (Figure 3A) , glycerol (Figure 3B) and serine (Figure 3C) , indicating that the lack of the protein reduced the permeability of the bacterial cells to hydrophilic substances.

With labelled glycine, however, uptake was increased in the mutant compared to the wild-type (Figure 3D) , indicating that the po-rin formed by OmpATb is not involved in glycine uptake, and that restricted access to other hydrophilic substances may result in a compensatory increase in the uptake of glycine .

The merodiploid strain, carrying both the wild-type and knock-out alleles, was included for the experiments with glycerol and behaved identically to the wild-type (Figure 3B) . Figure 3E shows the mean of four experiments for each molecule, and confirms that the differences observed between the mutant and the wild-type were statistically significant. None of the molecules was totally excluded from the mutant, indicating that while OmpATb may not be the only molecule involved in the transportation of small hydrophilic molecules across the outer permeability barrier . Whilst the OmpATb mutant was observed to take up serine at pH 7, no serine uptake was observed at pH 5.5. This indicates that OmpATb is the only molecule which is involved with the uptake of these molecules at low pH

(Figure 4) . A role of OmpATb may therefore be to transport small hydrophilic molecules in a macrophage environment.

Acid stress response of M. tuberculosis The regulation of expression of ompATb in normal M. tuberculosis was demonstrated using real-time PCR (TaqMan) as described above. The amount of messenger RNA (mRNA) transcribed from a target gene is measured as a ratio to mRNA for a 'housekeeping' gene whose expression is constant with varying conditions .

In present experiments, the transcription factor sigA was used as the reference gene; its expression is known not to be affected by lowered pH (Manganelli, R. et al (1999) Molecular Microbiology 31: 715-724).

A striking increase was observed in transcription of ompATb in M. tuberculosis cultured at low pH, with expression levels correlating with increased acidity as pH was lowered from 7 to, 6.5, 6.0 and 5.5 (Figure 5A) . Because vacuole acidification is known to be involved in macrophages infected with M. tuberculosis, we used a similar approach to investigate the levels of ompATb expression in M. tuberculosis which had been phagocytosed by -different types of macrophages (Figures 5B and 7) .

The results show that expression is significantly increased in M. tuberculosis growing in the human monocytic cell line THPl and in murine bone marrow macrophages. The expression of ompATb was further increased in THP-1 cells activated with γ-interferon (Figure 7) .

We also demonstrated that the increased transcription of ompATb in acidified medium was reflected in an increase in levels of OmpATb protein. Total M. tuberculosis cell lysates were prepared from bacteria grown at pH 7 and then transferred to medium at pH 5.5 for 18 hours; im unoblotting of these lysates with the OmpATb antibody revealed a substantial increase in amounts of OmpATb in the bacteria exposed to low pH.

The growth characteristics of the knock-out mutant in acidified media (Figure 1) strongly support the role of OmpATb in the response of M. tuberculosis to lowered pH.

While the parental strain showed slightly delayed growth at pH 5.5 compared to pH 7, growth of the mutant was significantly impaired, although it eventually appeared to adapt and reached similar optical densities to the wild- type.

The ompATb mutant shows reduced growth in macrophages and in intravenously infected mice.

The finding that ompATb expression is up-regulated in M. tuberculosis growing in macrophages indicates that the protein contributes to intracellular survival of the bacteria. In order to confirm this, cultured murine bone- marrow macrophages (Figure 2A) and the human monocytic cell line THPl (Figure 2B) , were infected with M. tuberculosis; the macrophages were lysed at various time intervals and the number of viable M. tuberculosis determined. In both cell types the mutant showed significantly reduced multiplication compared to the wild type. In order to see if this reduced growth ability was also manifested during infection in vivo, mice were infected intravenously with either the mutant or the parental strain and growth in spleens (Figure 6A) and lungs (Figure 6B) was monitored. The levels of infection achieved by the mutant were significantly reduced compared to those of the wild type. In a second experiment the mutant and wild type were also compared with the ompATb^ ompATb : :Km^r merodiploid single-crossover strain, which carries both a mutant and wild type ompATb (Figure 6), at a single time point. Again the mutant showed significantly reduced growth compared to the both of the control strains .

Interestingly, infection of athymic (nu/nu) mice was similar for both the mutant and wild type implying that the loss of ompATb impaired the ability to resist acquired cell-mediated immunity rather than innate immune mechanisms .

Discussion

The characterization of OmpATb mutants described herein demonstrates that the OmpATb gene plays an important role in the virulence of M. tuberculosis . OmpATb is shown to function as a transport molecule, with significantly reduced uptake of glucose, glycerol and serine being observed in the absence of the protein. The increased uptake of glycine observed probably reflects a reconfiguration of metabolic pathways in the ompATb mutant, as preferred substrates are altered to substances whose permeation is less affected by loss of OmpATb. Glycine may thus be used preferentially when the availability of other substrates is limited due to reduced permeability. Deletion of the main porin in the rapid-growing Mycobacterium smegmatis makes the mycobacteria much less permeable to hydrophilic solutes (Stahl, C. et al (2001) Mol .Microbiol .40 : 451-464), so disruption of M. tuberculosis ompATb was expected to lead to an altered response to osmotic stress. However, no effect on the osmotic stress response was observed for the present M tuberculosis mutants.

Using both quantitative real-time PCR (Figure 2A) and immunoblotting, expression of ompATb wild type M. tuberculosis was shown to be markedly up-regulated at low pH. However, growth of the mutant M. tuberculosis strain was, dramatically inhibited by mild acidity in the medium.

This finding indicates that OmpATb has a second function in enabling M. tuberculosis to grow in acidic conditions which is unrelated to permeability.

Acid exposure is a hazard for an intracellular microorganism, since phagocytic cells acidify phagosome. The ability of such an organism to survive such exposure is important for virulence.

M. tuberculosis is avidly phagocytosed by macrophages, and these cells are its major environment during the infection, despite their usual bactericidal capacities . Live pathogenic mycobacteria suppress the normal lowering of pH inside phagocytic vacuoles (Sturgill-Koszycki, S (1994) Science 263: 678-681, Xu, S. et al . (1994) J. Immunol. 153: 2568-2578) , but this effect is partial and is over-ridden when the macrophages are activated (Schaible et al., (1998) J Immunol. 160 1290-1296). Exposure to the intracellular environment of macrophages results in up-regulation of ompATb expression (Figure 3B) . The reduced ability of the ompATb mutant to grow at slightly lowered pH is consistent with its impaired growth in macrophages. Activation of macrophages is part of the mechanism by which cell-mediated immunity prevents the growth of intracellular pathogens, and so its poor response to lowered pH explains why the ompATb mutant ceases growth in mice at about the time when specific cell-mediated immunity begins to operate (Figure 6) . OmpATb is particular important as a transporter of small hydrophilic molecules at low pH.

The prevention of the acidification of the vacuole by the mycobacterium is also over-ridden in immunologically intact mice when specific cell-mediated immunity is activated during the infectious process. This is demonstrated by the low virulence of the mutant mycobacteria in normal mice, compared with normal virulence in mice which lack cell- mediated immunity.

The reduced virulence of M. tuberculosis which lacks functional ompATb is therefore a consequence of its inability to respond to a normal immunological response. As described above, successful vaccination by live mycobacteria requires that some growth of the vaccine occurs (Collins, F.M. (1971) Infection and Immunity 4: 688- 696) . Such minimal growth is provided by the mutant mycobacterial cells described herein, in which growth is inhibited only after a delay. Such cells therefore offer an excellent basis for vaccines and medicaments for immunotherapy. Furthermore, the important role of the OmpATb protein in the survival of the Mycobacterium within the acid environment of the macrophage makes it a potential target for drugs and other therapies designed to inhibit or block its action.

Claims

CLAIMS :

1. A cell of a mycobacterium which is a member of the M. tuberculosis complex and which has an inactivated ompATb function

2. A cell according to claim 1 which is a Mycobacterium bovis BCG or Mycobacterium tuberculosis cell.

3. A cell according to claim 1 or claim 2 which is non- virulent .

4. A cell according to any one of the preceding claims wherein the ompATb gene of said cell is inactivated by mutation.

5. A cell according to any one of the preceding claims wherein one or more endogenous antigens of said cell are over-expressed

6. A cell according to any one of the preceding claims for use in a method of treatment of the human or animal body.

7. A cell according to any one of the preceding claims for use as a vaccine in the treatment of a condition associated with mycobacterial infection.

8. A cell according to claim 8 wherein said condition is tuberculosis.

9. Use of a cell according to any one claims 1 to 6 in the manufacture of a medicament for use in the treatment of a disorder in which an immune response against an antigen expressed by the M. tuberculosis complex cell is beneficial

10. Use according to claim 9 wherein said disorder is a mycobacterial infection.

11. A method for reducing the virulence of a M. tuberculosis complex cell comprising inactivating a ompATb gene within the cell.

12. A method according to claim 11 comprising replacing the endogenous ompATb gene of said cell with a ompATb transgene which carries a mutation which reduces the function of the ompATb transgene.

13. A method according to claim 11 or claim 12 comprising formulating said cell into a pharmaceutical preparation.

14. A pharmaceutical, composition or vaccine comprising an M. tuberculosis complex cell according to any one of claims

1 to 5.

15. A method of making a pharmaceutical composition or vaccine comprising admixing a cell according to any one of claims 1 to 5 with a pharmaceutically acceptable excipient, vehicle or carrier.

16. A method comprising the administration of a composition or vaccine according to claim 14 to a mammal in need thereof for use in the treatment of a disorder in which an immune response against the cell is beneficial.

17. A method according to claim 16 wherein the disorder is a mycobacterial infection.

18. An OmpATb inhibitor for use in a method of treating a mycobacterial infection.

19. Use of OmpATb inhibitor in the manufacture of a medicament for use in the treatment of infection by a mycobacterium.

20. Use according to claim 19 wherein the mycobacterium is a member of the M. tuberculosis complex.

21. Use according to claim 20 wherein the mycobacterium is M. tuberculosis.

22. A method of treatment of a condition associated with infection by a mycobacterial cell comprising; inhibiting the OmpATb activity of the cell .

23. A method according to claim 22 wherein the mycobacterium is a member of the M. tuberculosis complex.

24. A method according to claim 23 wherein the mycobacterium is M. tuberculosis.

25. A method of obtaining an inhibitor of OmpATb comprising; contacting an OmpATb polypeptide with a test compound, and; determining the activity of said OmpATb polypeptide.

26. A method according to claim 25 wherein the activity of the OmpATb polypeptide is determined by determining the cellular uptake of a substrate molecule.

27. A method according to claim 26 wherein the substrate molecule is selected from the group consisting of glucose, glycerol and serine.

28. A method according to any one of claims 25 to 27 wherein the OmpATb polypeptide is contacting with the test compound under acidic conditions.

29. A method according to any one of claims 25 to 28 comprising identifying a test compound as a inhibitor of OmpATb

30. A method according to claim 29 further comprising determining the ability of said test compound to inhibit the intracellular growth of a mycobacterium.

31. A method according to claim 29 or 30 comprising isolating the test compound.

32. A method according to claim 31 comprising formulating the test compound with a pharmaceutically acceptable excipient .

33. A cell of a mycobacterium which is substantially as described herein.