WO2001090309A2 - Peptides that stimulate mycobacterial growth - Google Patents

Abstract

Compositions are disclosed comprising peptides which may be derived from Mycobacterium tuberculosis, which compositions stimulate the growth of mycobacteria that are at cell densities too low to grow in the absence of the composition. The preferred peptides are ERGVT SEQ ID NO:1 and LVTLVFV [SEQ1D NO: 2] or a mixture thereof. Methods for making culture media that support the growth of mycobacteria under such conditions and methods for stimulating cell growth and rescuing cells from stress using the above peptides and for inducing the promoter of the M. tuberculosis homolog of the acr gene are disclosed.

Description

PEPTIDES THAT STIMULATE MYCOBACTERIAL GROWTH

BACKGROUND OF THE INVENTION

Field of the Invention

This invention in the fields of microbiology and biochemistry relates generally to improved methods of culturing mycobacteria, and in particular to peptide compositions and methods for stimulating growth, particularly of Mycobacterium tuberculosis (hereinafter, M. tuberculosis or Mtb). Methods for isolating such growth-stimulatory peptides and for their use as growth promoters are provided.

Description of the Background Art Tuberculosis (TB) continues to be a public health problem throughout the world. Two to three million deaths every year are directly related to TB, and the number of new cases is about 15 million. The recent increase in TB is attributable in large part to the growing HIV/ AIDS epidemic. TB remains the most frequent infectious disease in developed countries, and developing countries, it constitutes the principal source of human loss related to a single disease. This resurgence of TB has magnified the need to understand the molecular pathogenesis of the organism responsible for the disease, M. tuberculosis (also referred to herein at "Mtb"). Mtb is a facultative intracellular pathogen that establishes respiratory infection following inhalation of bacilli into the alveoli of the lungs. The tubercle bacilli establish themselves intracellularly in monocytes, macrophages and other reticuloendothelial cells. Cell-to-cell spread promoted by lysis infected host cells. Additionally, the organism is able to survive and grow in the extracellular spaces of lung tissue.

Despite the huge toll in life and resources taken by Mtb, universally useful vaccines and diagnostic tests for active tuberculosis are currently unavailable. The development of more appropriate vaccines and effective diagnostic tests is critical in view of the current rapid world- wide spread of the disease.

From studies in the 1950s examining latent tuberculous lesions in human lung tissue following surgical removal (Auerbach, O et al., 1955. J. Thorac. Surg. 29:109-132; Wayne, LG, I960. Am. Rev. Respir. Dis. 82:370-377; Wayne, L et al., 1956, Am. Rev. Tuberc. Pulm. Dis. 74:376-387), it became apparent that viable bacilli were present in blocked airways for years following conversion to a sputum-negative status and that these bacteria could be cultured but were not actively growing. Work in animal TB models suggested that the initial event in infection involved entry and multiplication in unactivated macrophages (Dannenberg, AM et al, 1994, p. 459-483. In B. R. Bloom (ed.), Tuberculosis: pathogenesis, protection, and control.

American Society for Microbiology, Washington, DC; Rook, GAW et al, ibid at p. 485-501).

Following this rapid growth phase, infected Mtb-carrying macrophages are surrounded and walled off by newly recruited activated macrophages to form the characteristic "caseous granuloma" (Dannenberg, AM, Jr., 1993, Hosp. Pract. Jan. 15:51-58). Granuloma formation creates an environment uniquely capable of restricting the growth of mycobacteria through a process involving apoptosis of infected monocytes (Molloy, A et αl, 1994, J. Exp. Med. 180:1499-1509). It is within these primary granulomas, following exponential growth, that bacterial dormancy probably occurs. Although the environment inside the granuloma has not been elucidated, many factors, including oxygen deprivation, nutrient depletion, low pH, toxic oxygen species, and other adverse conditions could contribute to the induction and maintenance of dormancy. Stationary phase in the tuberculosis complex bacteria has only begun to be explored. To characterize metabolic changes which allow Mtb to remain viable for such extended periods of time, Yuan, Y. et αl, J. Bαcteriol, 1996, 178:4484-4492, characterized proteins which were specifically synthesized during late exponential and stationary phase growth in vitro. The authors defined stationary phase as commencing at the end of log-phase growth and showed that it was associated with differential expression of at least seven proteins whose functions may be related to maintaining cell viability. Though physical and metabolic changes accompanying stationary phase are largely unknown, such bacteria are more resistant to antimycobacterial agents, have altered glyoxylate metabolism, and are able to resume growth synchronously (Wayne, LG, 1994. Eur. J. Clin. Microbiol. Infect. Dis. 13:908-914.). This study demonstrated that the "16-kDa antigen" of Mtb was expressed selectively during the transition to stationary phase. The slow initial growth rate observed in both recombinant organisms suggested that the lag phase associated with growth of organisms of the Mtb complex may be related to the necessity to dilute or degrade the 16-kDa antigen in order to achieve maximal log-phase growth rates. Also observed was 16-kDa antigen-related inhibition of growth on solid media. Wayne and colleagues (supra) developed an in vitro system for anaerobic or microaerophilic Mtb cultures in which a population of bacilli is induced to grow in unagitated suspension culture. In this model, cells growing in the layer nearest the surface are thought to grow aerobically and then to slowly adapt to anaerobic conditions as they settle through an oxygen gradient. The relationship between organisms grown in submerged culture and organisms characterized as stationary phase in Yuan et al. study was important to consider. Wayne and Diaz reported that a sudden shift of log-phase cultures from aerobic growth to anaerobic submerged culture resulted in death of the culture (38). Thus, under these conditions, production of this protein is presumably not protective, possibly because a high enough protein concentration is not achieved before cell death. Their experiments with aging cultures and overexpressing organisms suggested that production of this protein at some concentration is protective. These conditions (shifting from aerobic to anaerobic submerged cultures) are similar to those found to induce expression of the 16-kDa antigen. These induction conditions were obviously not a precise mimic for the transition to stationary phase observed in aging cultures. Comparison of the 2D gel profiles of induced proteins revealed that although 16-kDa antigen induction was common to the two conditions, many "induced" proteins were not induced in stationary cultures.

Although anoxia may represent part of the environmental signal resulting in stationary phase, additional environmental changes may be involved. The shift to lower oxygen may occur through the production of cell wall material which renders the organism less permeable or as a result of clumping of multiple organisms, with the interior of these clumps becoming increasingly microaerophilic or anaerobic. Alternatively, the 16-kDa protein may be induced in response to a gradual shift to anaerobic conditions or an unknown condition which occurs during the transition to stationary phase in aerated culture. The 16-kDa antigen is related to the broad family of sHSPs based on amino acid identities ranging from 27 to 31% in comparison with, for example, the sHSP from soybean. In fact, examination of the α-crystallin domain of the Mtb homolog revealed that only 21 of 32 highly conserved residues were present or were conservatively substituted (Caspers, G et al, 1995, J. Mol. Evol. 40:238-248.). Although this sequence homology is not high, the mycobacterial sHSP was shown to possess functional similarities with the proteins in this family. The defining characteristic of proteins in this family lies in their chaperone function, which was first demonstrated through their ability to suppress the thermally induced denaturation of other proteins (Horwitz, J, 1992, Proc. Natl. Acad. Sci. USA 89:10449-10453.). This chaperone activity confers thermoresistance as well as increased resistance to lethal hydrogen peroxide treatments when cells express sHSPs (Mehlen, P et al, 1993, Eur. J. Bioche . 215:277-284; van den Ijissel, PRLA et al, 1994, FEBS Lett. 355:54-56). Mtb overexpressing this protein were more resistance to autolysis following the end of log-phase growth. In addition, in vitro results with purified protein suggested functional homology to the sHSP family. The Yuan et al. (supra) results suggest that the protein forms high-molecular- weight aggregates, characteristic of other members of the sHSP family, and that this aggregation may be responsible for the association with the cell wall. Other α-crystallin proteins have a peripheral association with wall proteins and that this family of proteins is thought to function primarily through the interactions of exposed hydrophobic surfaces with other proteins in a denatured or partially denatured state (Das, KP et al, 1995, FEBS Lett. 369:321-325; Raman, B et al, 1995, FEBS Lett. 365:133-136).

The ability to persist following log-phase growth is common in bacterial metabolism. Many studies have been done in gram-negative organisms such as E. coli where cells conduct an orderly shut-down of metabolism so to preserve the ability to grow after environmental conditions change (Siegele, DA et al, 1992, J. Bacteriol. 174:345-348). In addition to exhibiting changes in metabolic rate, starved or stationary-phase cells undergo numerous changes in cell wall structure, surface properties, and chromosome structure. Some bacteria, e.g., Bacillus subtilis, form fully differentiated spores which survive extended periods without nutrients (Setlow, P, 1981. p. 13-28. In H. S. Levinson et al, (eds.), Sporulation and germination. American Society for Microbiology, Washington, DC). These diverse strategies have common characteristics: the use of developmentally linked programs of protein expression in the face of a shut-down of bulk protein synthesis. Many of the proteins produced during stationary phase, including the proteins involved in carbon starvation, the catalase HPII, exonuclease III, and others (29), are dependent on KatF for their expression. Recently, a possible functional equivalent of KatF was identified in tuberculosis and named SigF. This sigma factor was shown to be strongly induced during the transition from log to stationary phase (DeMaio, J. et al, Proc. Natl. Acad. Sci. USA 93:2790-2794). The 16-kDa antigen is probably not solely regulated by the recently identified mycob acterial

The diversity of this protein family among many eukaryotes and prokaryotes further suggests that the relevant function may be "general" (Caspers et al, supra). The protective effect of this protein may not be directly related to the immediate stress which triggers its synthesis. Instead, Yuan et al, 1996, proposed that this protein serves as a general cellular protectant through an enhanced stability of the proteins required for revivability. Oxygen tension may simply serve as the signal to produce proteins, such as the 16-kDa antigen, required for long-term survival and persistence.

In sum, little is known about the mechanism by which Mtb survives and propagates within the harsh environment of the human lung. The organism may be capable of signaling to its near neighbors about the status of the environment at any given time which in turn contributes to the cells' decisions whether or not to divide. Mtb may employ small molecules to achieve such signaling. These small molecules can readily diffuse across intercellular spaces and carry vital information for the cell. These small molecules could therefore also be used to bring the bacteria out of a latent state into an actively dividing state whereby they would be rendered highly vulnerable to the action of antibiotics.

"Quorum sensing" is a term used to describe a mechanism by which microorganisms keep a close 'head-count' on the numbers or density of its members. (See, for example, Fuqua

WC et al, J Bacteriol. 1994;176:269-275; Swift S et al, Trends Biochem Sci. 1996; 21:214-219; Hanzelka BL et al J Bacteriol. 1996;178:5291-5294; Schaefer AL et al, Proc Natl Acad Sci USA, 1996; 93:9505-9509; Swift S et al, Trends Microbiol, 1996; 4:463-466; Pesci EC et al, Trends Microbiol, 1997; 5:132-135; Kleerebezem M et al. Mol Microbiol, 1997; 24:895-904.) It is hypothesized that any reduction/increase in the total number of viable cells alerts the colony to the external environment, triggering mechanisms for an appropriate response such as dormancy, latency, sporulation, virulence, "sexual" behavior, etc. This form of extracellular "intelligence" is vital to microbial survival. It is mediated by small diffusible molecules that have been termed quorum-sensing molecules or autoinducers. Examples are acyl-homoserine lactones in the case of gram-negative pathogens and peptides in the case of gram-positive microbes. Density-dependent signals can be either directly secreted by the cells or produced by the extracellular action of proteases. Signals can be received on the outside of cells by sensors/receptors coupled to a response regulator systems or by entry of the signal molecule into the cells, either by passive diffusion or active transport (e.g.,, with the aid of peptide permeases) followed by and interactions with other cellular effector molecules.

Known extracellular receptors are histidine kinases (e.g., Piazza F et al, J Bacteriol

1999 Aug; 181 :4540-4548) which are autophosphorylated upon binding of a peptide signal, after which these molecules can donate the phosphate group to cytoplasmic proteins. Some peptides are taken up directly, typically competence factors of B. subtilis, that bind to and inactivate intracellular phosphatases leading to upregulation of a signalling pathway (Dahl MK et al, J Biol Chem 1992; 267:14509-14514; Perego M et al, Proc Natl Acad Sci USA 1996 20; 93:1549-1553; Nanamiya H et al, Biochem Biophys Res Commun 2000; 279:229-233; Jiang M et al, J Bacteriol 2000;182:303-310).

Solomon JM et al, described an extracellular peptide factor that affects competence and sporulation pathways in B. subtilis, (Genes Dev 1996; 10:2014-2024). This factor (CSF), purified from conditioned medium (culture supernatant) based on its ability to stimulate expression of srfA (comS) in cells at low cell density, had the sequence Glu-Arg-Gly-Met-Thr (ERGMT) which are the C-terminal 5 amino acids of the 40-amino-acid peptide encoded by the phrC gene.

Investigators have reported short peptide signaling molecules in a number of gram positive microorganisms but, with one exception (discussed below) not in M. tuberculosis. Bosne-David S et al, Antimicrobial Agents and Chemotherapy 41: 1837-1839 (1997), disclosed that heterologous mycobactins and the synthetic FR160 [N -nonyl,N ,N -bis(2,3-dihydroxybenzoyl) spermidine hydrobromide (C₃oH₄₆N₃O₆-Br)] promoted growth in Mycobacterium aurum in low concentrations.

Mukamolova, GN et al, 1998, Proc Natl Acad Sci USA 95:8916-8921 reported that viable cells of Micrococcus luteus secrete a factor, which promotes the resuscitation and growth of dormant, nongrowing cells of the same organism. The responsible moiety, a protein, was purified to homogeneity with a molecular mass of about 19 kDa. This protein increased the viable cell count of dormant M. luteus cultures at least 100-fold and stimulated growth of several other high G+C Gram-positive organisms, including Mycobacterium avium, Mycobacterium bovis (BCG), Mycobacterium kansasii, Mycobacterium smegmatis, and Mycobacterium tuberculosis. This document provided no evidence that mycobacteria can or do produce or secrete this protein. Moreover, this protein shows no homology to the peptides of the present invention

The following table lists quorum sensing peptides from other gram positive bacteria and the functions with which they have been linked: PEPTIDE SEQ ID ORGANISM FUNCTION NO:

ADPITRQWGD 3 B. subtilis SPORULATION ERGMT 4 B. subtilis COMPETENCE

YSTCDFΓM 5 S. aureus VIRULENCE

LNTLNFN 2 E. faecalis CONJUGATION LFSLNLAG 1 E. faecalis CONJUGATION

M. Malski reported (Medycyna Doswiadczalna Mikrobiologia, 1989, 41:9-14) tested ten di- or tripeptides containing asparagine, glutamic acid, leucine or alanine on growth of Mtb. (using AS medium free of peptones). Bacterial suspensions from patient samples were plated and the number of colonies and rapidity of colony formation under an influence of peptides was tested. The "best" combination was reported to be mixture of 0.01% glutathione, 0.002% Gly- Asn and 0.0033% Leu-Gly, which resulted in an very modest increase of 46% in the number of colonies compared to control medium. Addition to this peptide combination of 0.1% Bacto Tryptone (Difco) caused an increase of 127% of colony number and their growth was hastened by 1.7 days compared to growth on medium + peptides (without the tryptone). Because of the very high concentrations used (mM) and very modest growth stimulation, the present inventors believe these three peptide compounds are acting merely as nutritional supplements (i.e., providing an addition source of amino acids to the bacteria). It may also be related to the stimulation of amino acid and dipeptide uptake such as that induced by Glu. (Lev, M., J. Bacteriol, 1980 143:753-60). The Lev study showed that uptake of a number of amino acids and dipeptides by cells and spheroplasts of Bacteroides melaninogenicus was stimulated by glutamine (peaking at 50 mM). These foregoing phenomena (Malski, supra; Lev, supra) certainly would not fit the biological role of "quorum sensing" as described herein, do not approach the levels of growth stimulation by the peptides of the present mvention (several orders of magnitude vs 1.5-2-fold), and require concentrations of the active compounds 3-5 orders of magnitude higher to achieve a significant biological effect.

It has been reported that spent culture supernatants from early-stationary-phase Mtb (strain H37Ra) cultures acted to (a) increase viability of stationary phase bacilli from aged cultures and (b) allow small inocula to initiate growth in liquid culture (Sun Z et al, J Bacteriol (1999) 181 -.7626-7628). While this brief publication did not identify any specific peptide, it concluded that the "resuscitation factor" was acid labile, heat stable, and had a molecular mass <1,375 Da. The present inventors are the first to discover specific individual peptides which are characterized by their growth stimulatory action on low density inocula of Mtb as well as by their induction of the promoter of the α-crystallin gene (Acr; also termed hsp∑). One of the two active peptides disclosed herein is derived from Mtb, the other from E. faecalis. Thus, the peptide of the present invention may be stringently species-specific, so that it is not possible to predict a priori the existence or activity of similarly acting peptides in different bacterial genera or species.

SUMMARY OF THE INVENTION

The present inventor discovered a novel peptide molecule having the sequence Asp-Arg-Gly-Val-Thr (ERGVT) [SEQ TD NO:l] that regulates the growth of M. tuberculosis. Also described is a second peptide, previously known as a quorum sensing peptide involved in conjugation of E. faecalis, Leu-Val-Thr-Leu-Nal-Phe-Val (LNTLNFV) [SEQ ID ΝO:2] which has never been shown to be associated with cell growth of any organism or to have any action on Mtb. Thus, in a preferred embodiment, the present invention is directed to a composition useful for stimulating the growth of M. tuberculosis bacteria in culture, comprising an isolated peptide selected from the group consisting of Asp-Arg-Gly-Val-Thr (ERGVT) [SEQ ID NO:l] and Leu-Val-Thr-Leu-Val-Phe-Val (LVTLVFV) [SEQ ID NO:2] or a mixture of ERGVT and LVTLVFV, which peptides stimulate the growth of the bacteria when the bacteria are in a dormant state or inoculated into culture at a cell density that is too low for growth. The composition may comprise, either one or both of these peptides.

In the above composition, the bacteria are inoculated preferably into culture at a concentration of less than about 100 CFU/milliliter medium.

Also provided is the above compositions supplemented with an additional purified peptide having a molecular mass (M+H^4") of 1097.2 Da which is derived from culture medium that includes casein hydro lysate, wherein the 1097.2 Da peptide is characterized in that it alone stimulates the growth of the bacteria when added to a tuberculosis culture. hi another embodiment, the present invention is directed to a composition useful for stimulating the growth of M. tuberculosis bacteria which composition: (a) comprises a peptide; (b) stimulates growth of the bacteria when the bacteria are in a dormant state or inoculated into culture at a cell density that is too low for growth; and

(c) induces the promoter of the M. tuberculosis homologue of the Acr gene to express any DNA coding sequence operatively linked thereto. In the foregoing composition the peptide preferably has a molecular mass of <2 kDa, more preferably <1 kDa, even more preferably < 800 Da and most preferably <600 Da.

The above composition is preferably one characterized in that it stimulates the growth of an inoculum of M. tuberculosis having fewer than or about 100 CFU per milliliter to about 10⁵ CFU/ml in a period of about 3 weeks in standard mycobacterial culture medium. Alternatively, the composition stimulates the growth of an inoculum of M. tuberculosis having between 100 and about 1000 CFU per milliliter to about 10⁵ CFU/ml in a period of about 3 weeks in standard mycobacterial culture medium. Preferably, the peptide of the above composition stimulates the growth at concentrations between about 10 pM and about 10 nM, preferably at concentrations less than or equal to about 1 nM. In a preferred embodiment of the above composition the peptide is not glutathione, Gly-

Asn or Leu-Gly (or a combination thereof).

The foregoing composition may be supplemented with an additional purified peptide having a molecular mass (M+H^4-) of 1097.2 Da which is derived from culture medium that includes casein hydrolysate, wherein the 1097.2 Da peptide is characterized in that it alone stimulates the growth of the bacteria when added to a M. tuberculosis culture.

This invention also is directed to a method for stimulating the growth of M. tuberculosis bacteria in culture comprising adding to a suitable culture of the bacteria an effective amount of the above composition.

Also provided is a method for resuscitating M. tuberculosis cells from a dormant state into a growth state, comprising

(a) obtaining a population of M. tuberculosis cells from a stressed environment;

(b) inoculating the cells into a standard mycobacterial culture medium; and

(c) contacting the cells in the medium with the above composition.

In this method, the cells may be obtained from the stressed environment in vivo or in vitro. Preferably, the stressed environment is characterized by the presence of low pH, nutrient deprivation, low oxygen tension or reactive oxygen intermediates. One example the presence of hydrogen peroxide at a concentration that inhibits cell growth. The present invention also provides a method of producing a growth stimulatory culture medium for M. tuberculosis bacteria characterized in that the medium stimulates the growth the bacteria when they are inoculated into culture at a concentration of less than or equal to about 10 CFU/milliliter medium, which method comprises adding to a mycobacterial culture medium a composition as described above. In one embodiment, the bacteria are inoculated into culture at a concentration of less than or equal to about 10² CFU/milliliter medium.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing growth of Mtb beginning at different concentrations of organisms. Inocula labeled as 1 : 10 is a dilution containing between 10⁴ and 10⁵ colony forming units (CFU)/ml. Inocula labeled as 1:500 dilutions contained between 10² and 10³ <CFU/ml. Inocula labeled as 1:1000 dilutions contained about 10² CFU/ml. The results show a dependence of growth on initial cell density

Figure 2 shows the stimulation of growth of low inoculum cultures (starting at about 10 CFU/ml ) by "growth active filtrates" (GAF) which were low molecular weight (<1 OkDa) fractions (labeled "3/10 Frn") of spent medium of Mtb cultures at late logarithmic or early stationary phase.

Figure 3 is a schematic representation of the purification/enrichment of a "quorum sensing" growth activating molecule from spent medium of Mtb cultures Figure 4 shows the purification of the growth activating molecule. Shown at left is a thin layer chromatograms of fractions eluted from a BioGel P-2 column. The fractions that were active in stimulating growth (shown by arrows) were recovered from the TLC spot, extracted with water and again assayed for biological activity. Activity was attributable to material in single, ninhydrin staining spots shown on the TLC slide at right. Figure 5 shows the mass spectrogram (using electrospray mass spectroscopy (ES-MS) of the TLC-purified growth promoting molecule. Different charge stages are shown. The most abundant species was a 561.5 Da (M+H)⁺ product (singly charged ion) that shows up at half that molecular mass when doubly charged (281.3 Da (M+H)²⁺ peak). Also shown are peaks representing various sodium adducts to the right of the 561.5 Da peak. The larger molecular mass peaks represent non-covalently complexed dimers of the 561.5 Da peptide, which are formed because of the polarity of this peptide. Figure 6 shows a mass spectrogram (MS/MS) performed on the fly of the 561.5 m/z ion shown in Figure 5, with the collision energy set at 40%. The MS -MS fragmentation data was used to perform de novo sequencing by calculating the y and b series fragment ions. This led to the elucidation of the sequence of the growth activating factor as ERGVT [SEQ ID NO:l]. Figure 7 is a graph showing dose response growth curves of Mtb stimulated by four different concentrations of ERGVT (ranging from 5 pM to 4 nM. Concentrations between 50 pM and 4 nM stimulated growth (at least 3 logs) of cultures having an initial cell density of about 100 CFU/ml.

Figure 8 is a graph analyzing growth stimulation of Mtb low inoculum cultures by five known quorum sensing peptides and ERGVT. Only ERGVT and LVTLVFV (SEQ ID NO:2] (which has been shown to be associated with conjugation function in Enterococcus faecalis) were active.

Figure 9 is a histogram showing induction of the promoter of the Mtb hsp∑ (heat shock protein X) gene which is the Mtb homologue of the α-crystallin gene- (αcr). Induction was tested in a reporter system wherein the hspX promoter was fused to the E. coli β-galactosidase (lαcZ) gene, β-galactosidase enzymatic activity was measured by conversion of ONPG, a chromogenic substrate, to a colored product which was measured at 420 nm wavelength. Specific activity was defined as nmoles of substrate converted/minute mg protein

Figure 10 is a graph showing that ERGVT can rescue Mtb cells that are in a state of stress induced by treatment with lOmM H₂O₂ hydrogen peroxide. Growth was monitored once every 24 hours by a turbidimetric measurement at 600 nanometers (not shown) and by determining the number of colony forming units. 10 mM hydrogen peroxide strongly inhibited growth. This model was used to test for the ability of the peptide added at 15, 17 and 20 days after the hydrogen peroxide challenge, to resuscitate the stressed cells. ERGVT added to peroxide treated cultures at a concentration of 1 pmol/ ml (1 nM final) on days 15, 17 or 18 (when the CFU numbers were declining exponentially) rescued the cells and resulted in logarithmic growth. Figure 11 shows two chromatograms of reverse phase HPLC separation of the gel filtration peaks. The top panel shows a peak eluting at 48.51 minutes that is characterized by

ES-MS in Figure 12. The lower panel shows several peaks at the right end that have growth promoting activity. The peak eluting at 77.04 is the 561.5 Da (M+H)⁺ peptide (ERGVT) further characterized in Figures 5 and 6.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has become increasingly evident that prokaryotes produce extracellular signaling molecules that mediate cell-to-cell communication. Such intercellular communication, involved in the regulation of a wide range of physiological activities events, has been termed "quorum sensing." In gram-positive bacteria, most of the signaling molecules engaged in these activities are peptides or modified peptides that are biologically active in nanomolar concentrations. The phenomenon of quorum sensing has heretofore been relatively unexplored in Mycobacterium spp. The present inventors observed that in vitro growth of M. tuberculosis in broth culture is highly dependent on the size of the initial inoculum (see Figure 1). This property has been exploited by the present inventors to test various preparations derived from Mtb for their ability to act as growth signals to Mtb.

Lowering the inoculum used to seed a liquid culture of Mtb resulted in a lag phase and growth kinetics that could not be explained merely as a direct consequence of the number of viable cells in the culture. In accordance with the notion of quorum sensing, the "quorum" of cells in the culture was below a threshold that needs to be crossed for active cell division. The observed growth was not proportional to an exponential decrease in the number of viable cells. The present inventors postulated the existence of a mechanism wherein Mtb cells were monitor their numbers; when the numbers were too low (irrespective of how this is sensed), the Mtb cells produce and/or release a chemical signal or signals,

The term "low inoculum" (LI) as used herein refers to a cell density from less than about 10² colony forming units (CFU) per ml of medium up to and including a range of between about 10²-10³ CFU/ml. Conventional techniques and procedures were used herein to assay for bacterial growth.

When small amounts of sterile M. tuberculosis culture filtrate harvested from Mtb cells grown to the late log phase or early stationary phase are subjected to ultrafiltration to exclude molecules greater than about 10 kDa in molecular mass, and are added to LI cultures of Mtb, a significant increase in the growth of these cells occurs. The present inventors' initial experiments indicated that the activity was protease-sensitive, indicating that the active factor was most likely a proteinaceous entity. Initial ultrafiltration analyses suggested a size <5 kDa. The growth promoting fraction was then isolated, partially purified, and characterized by conventional gel filtration chromatography and thin layer chromatography.

When this material was further subjected to BioGel P-2 size exclusion chromatography it was found that fractions have a molecular mass range of about 400-1100 Da retained this growth-promoting activity.

Positive fractions were subjected to thin layer chromatography; ninhydrin positive spots were scraped, eluted in water and analyzed by electrospray mass spectrometry (ES-MS). This identified a single peak having a molecular mass (M+H)⁺ of 561.5 Da which had the ability to stimulate growth of Mtb inoculated at low densities (e.g., approximately 10² cfli/ml or between about 10² and about 10³ cfu/ml). Sequencing of this peptide by MS/MS (see Figure 6) yielded the sequence ERGVT.

The entire genome of M. tuberculosis has been sequenced (Cole, ST et al, Nature 393:531-544 (1998), and the sequence deposited in GenBank, followed by a direct submission on 11 -June 1998, Parkill, J et al, on behalf of the Mycobacterium tuberculosis sequencing and mapping teams, Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 ISA, United Kingdom, and the Unite de Genetique Moleculaire Bacterienne, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France. (Details of M tuberculosis sequencing at the Sanger Centre are available on the World Wide Web at the URL

(http://www.s___iger.ac.uk/Projects/M_tuberculosis/). This permitted the present inventors to scan the entire Mtb genome for genes or open reading frames (ORF's) that included the amino acid sequence ERGVT. This sequence was identified in the following six Mtb genes/ORFs:

Rv3004 Low MW Secreted Protein

Rv0332 Large Hyp. Protein

Rvl338 Murl

Rv2904c Mas

Rv2996c SerA

Rv2737c RecA

Table 1 below provides more detail on these ORFs. To the inventors' knowledge, this is the first report of a specific quorum sensing peptide active on Mtb, and thereby is a major advance in the understanding of the mechanisms underlying latency in this microorganism. The method of this invention are based on the discovery of a peptide, which alone or as part of a mixture with other peptides described herein, is capable of exerting an advantageous and unexpected stimulating effect on the growth of Mtb cells in culture at low inoculum densities under standard growth conditions. Cell growth is preferably an increase in the number of CFU/ml of at least about 10-fold, preferably about 100-fold, more preferably about 1000-fold or more. This growth preferably occurs over a period ranging from about 14 to about 30 days.

The preferred peptide of this invention, ERGVT appears to act in a highly species specific manner for Mtb. It is common among such autoinducer molecules that the molecule produced by one species acts only on cells of the same species. Indeed a crude culture filtrate of Mycobacterium smegmatis in this same size range was not able to stimulate Mtb growth.

Standard growth conditions include the use of any of a number of well-known culture media that support growth of Mtb and other mycobacteria. Examples of preferred growth media are described in the following references, each of which is incorporated by reference in its entirety, are: Gomes, MS et al, Tuber. Lung Dis., 1999, 79:321-328; Biketov, S. et al, FEMS Immunol. Med. Microbiol, 2000. 29:233-240; Ostrovskii, DN et al, Dokl Biol Sci., 2000, 374:543-545; Mariani, F et al, Gene. 2000, 253:281-291; James BW et al, J. Appl Microbiol, 2000, 55:669-677; Petricevich, VL et al, Braz. J. Med. Biol Res., 2001, 34:81-92; Hanscheid, T et al, Am JClin Pathol., 2001, 775:615-617.

Table 1

Mycobacterium tuberculosis genome Open Reading Frames (ORFs) that include the amino acid sequence ERGVT

For example, an Mtb culture have an A₆₅o =1.4 culture ofM. tuberculosis is diluted 1000 fold in 100 ml of 7H9, GAS media or Sauton's broth. The starting culture may optionally be kept frozen until use. Or refrigerated overnight. The test substance may be added in a concentration of 0.1 micro gram (crude material, dry weight estimate) per 100 ml culture either at the start of the growth or as appropriate. Cultures are preferably grown at 37°C with constant aeration, and A₆₅o as a measure of culture turbidity and therefore cell number may be recorded every 24 hours, e.g., on a Spectronic 1001 spectrophotometer (Bausch and Lomb, Stanford, CA).

For the routine assay of fractions, e.g.,, from reverse phase HPLC or size exclusion chromatography, a microtiter plate assay format is preferred. Cell growth is carried out as usual and turbidity (A₆₅o) is measured in a microplate reader (e.g.,, Biorad 0101A) coupled to a computer workstation. The microtiter plate assay is scaled down from the higher volume assay appropriately to the volume of 200 μl in each well vs. 100 ml flasks).

The present studies showed no significant differences in the time required to achieve visible growth among the various assay formats. Growth curves can be obtained using both either 5 ml sealed tube formats as well as 100 ml flasks.

Figure 1 depicts the effect of dilution of the inoculum on growth of Mtb, indicating that the lower the inoculum size, the less the cells grow over the subsequent several weeks. As shown in Figure 2, a size fraction of an Mtb spent culture supernatant (< 10 kDa), that contains the ERGVT peptide of the present invention, stimulates 2- 3 logs of growth in the LI culture started at a density of about 100 CFU/ml.

Even without a precise understanding of the mechanism(s) by which ERGVT signals Mtb cells, the present invention permits improved culturing of Mtb by reducing the lag phase. This improvement in our ability to culture this bacterium facilitates (a) improved, more rapid detection assays, (b) more efficient growth of Mtb in the diagnostic laboratory setting, (c) improvements in the pace of development and testing of TB vaccines and other mycobacterial vaccines.

It is a general property of Mycobacterial species, and other related organisms that grow intracellularly, that optimal in vivo growth and, similarly, in vitro growth, depends strictly on the size of the initial inoculum. One of the primary biological effects attributable to an autoinducer or quorum-sensing type of molecule is a marked enhancement of growth upon addition of the compound to a bacterial culture in which growth is limited because of the size of the initial inoculum.

Accordingly, the present invention provides a new peptide which is naturally produced by Mtb and which is isolatable from Mtb culture filtrate, though it is preferably produced as a chemically synthesized peptide which permits the generation of large quantities for use in accordance with this invention. One skilled in the art will readily appreciate the host of uses to which this invention can be applied in the field of TB research, diagnosis and therapy. The present peptides can be used to stimulate Mtb growth so that even dilute clinical specimens having fewer than 100 cells/ml can be grown for analysis, etc. A large proportion of active tuberculosis cases arise not from initial infection with actively growing bacilli but from reactivation of previously "implanted" organisms that have remained dormant or have grown at exceedingly low rates within the host (Wayne, LG. (1994)

Eur. J. Clin. Microbiol. Infect. Dis. 13:908-914; Dolin, PJ (1994) Bull. WHO 72:213-220).

This asymptomatic stage of the disease can last for decades during which time the bacilli are resistant to available chemotherapies (McCune, RM et al. (1966) J. Exp. Med. 123: 445^.68;

McCune, RM et al. (1966) J. Exp. Med. 123: 469^186; Wayne, LG et al (1994) Antimicrob.

Agents Chemother. 38:2054-2058; de Wit, D et al (1995) Tubercle Lung Dis. 76:555-562).

Examination of surgically removed human lung tissue from tuberculous lesions has demonstrated that bacilli found in surgically excised human lung tissue from TB lesions axe present in blocked airways of patients for years without being metabolically active (Wayne, L.

G. (1960) Am. Rev. Respir. Dis. 82, 370-377). Maintenance of this nonreplicating state is thought to be mediated by reduced oxygen tension and nutrient limitation within a caseous granuloma (Wayne, 1984, supra; Dannenberg, A. M., Jr. (1993) Hosp. Pract. 15, 51-58).

Cessation of Mtb metabolic activity can be induced in vitro by a variety of stress conditions including simple aging of logarithmically growing cultures or growth of unagitated submerged cultures with the attendant oxygen limitation (Wayne, LG et al, (1996) Infect. Immun. 64,

2062- 2069; Wayne, LG et al, (1961) J. Bacteriol. 93, 1374-1381). Acr Protein and its Induction

The Acr family of small heat shock proteins act as ATP-independent chaperones and play an important role in, for example, maintaining transparency of vertebrate eyes (Horwitz, J.,

(1992) Proc. Natl Acad. Sci. USA 89, 10449-10453; Groenen, PJ et al (1994) Eur. J. Biochem.

225, 1-19). Bacterial homologues are involved in spore formation in Bacillus subtilis (Henriques, AO et al. (1997) J. Bacteriol. 179, 1887-1897) and are induced in response to various acute stresses in other microorganisms (Jobin, MP et al. (1997) Appl. Environ. Microbiol. 63, 609-614). Mtb Acr homologue was first described as a major membrane protein (Lee, B-Y et al, (1992) Infect. Immun. 60, 2066-2074), but more recently has been shown to be a potent, ATP-independent, chaperone (a trimer of trimers) (Yuan, Y et al, (1996) J. Bacteriol. 178, 4484-4492; Chang, Z et al. (1996) J. Biol Chem. 211, 7218-7223). For the sake of brevity, the Acr homologue will also be referred to herein as Acr.

Yuan et al, supra, showed that a homologue of the 16-kDa α-crystallin (Acr) (Hspl6.3) protein (encoded by the acr gene), undetectable during logarithmic growth of Mtb, is strongly induced in viable old, stationary phase cultures (and in fact becomes the predominant protein in such cells). Acr is expressed in the stationary phase. The Acr protein is found only in mycobacteria of the closely related tuberculosis complex. The Mtb acr homologue is expressed during human infection with virulent strains - 85% of sera from patients with pulmonary TB recognized this protein (Lee et al, supra; Verbon, A et al. (1992) J. Bacteriol. 174, 1352-1359). Immuno gold-labeling studies showed an association of Acr with the mycobacterial cell wall and other macromolecular structures (Cunningham, AF et al. (1998) J Bacteriol. 180, 801— 808). It is likely that a differentially expressed antigen unique to resting bacilli characterized in the low-oxygen model of mycobacterial persistence and dormancy (Wayne, LG et al, (1979) Infect. Immun. 24, 363-370) is the same protein. Acr overexpression during log-phase growth of Mtb slows both the growth rate of the bacteria and the post-stationary phase autolysis often occurring in aging cultures. Therefore, Acr has been considered as a potentially important component for facilitating survival of Mtb during latent human infection (Yuan et al, supra; Cunningham et al, supra). Yuan et al. (1998) Proc Natl Acad Sci USA 95:9578-9583, examined environmental signals that trigger Acr expression in order to better understand the role of the Acr protein in intracellular Mtb growth in tuberculosis. They found that expression of acr is rapidly and powerfully induced in vivo upon entry of Mtb into macrophages or in vitro under conditions of limited oxygen.

Fusion proteins of Acr and a detectable protein such as luciferase have been useful as reporters of Acr regulation in vivo and in vitro. The robust (>100- fold) induction of acr- luciferase fusions on infection of cultured macrophages was similar in magnitude to the maximum achievable under hypoxic conditions in vitro, suggesting that the oxygen concentration within these macrophages is low (Meylan, PR et al. (1992) Am. Rev. Respir. Dis. 145, 947-953). These findings were contrary to the dogma that low-oxygen is unique to the caseous granuloma. Rather, growth under oxygen limitation may be the norm for tubercle bacilli and in vitro propagation techniques carried out at atmospheric oxygen concentrations may be atypical. Such information is useful for the present invention which contemplates using the growth-inducing peptides disclosed herein to promote growth of Mtb when it is obtained from in vivo tissues in which cell growth is arrested due to conditions of stress as described above, including suboptimal oxygenation, relative absence of nutrients, and the presence of reactive oxygen species or nitrous oxide. In Mtb as well as other systems studied, Acr functions as a chaperonin, protecting other cellular proteins from degradation (Horowitz et al, supra; Groenen et al, supra). However, the hypoxic conditions that induce Acr expression seem unlikely to result in protein instability. Oxygen limitation may instead serve as a signal of a hostile intracellular environment, in which Acr expression protects against other stresses inherent in that environment. It is therefore odd that the knockout strain is not more sensitive to oxidative stress (e.g. , hydrogen peroxide treatment) and that stresses such as low pH, nutrient deprivation or oxidative stress fail to induce Acr expression (Yuan et al, 1996, supra).

Acr product exacts a toll from organisms expressing it in vitro - a lower growth rate. Hence, it can be surmised that Acr production plays some essential role in intracellular Mtb growth. Repressed Acr expression under atmospheric oxygen levels suggests that this regulatory mechanism may have been maintained to promote rapid growth in a highly oxygenated environment. Such an environment might occur in situ in alveolar macrophages or it may occur later in the TB infectious cycle when caseous granulomas undergo liquefaction and the lung wall disintegrates (Dannenberg, supra). As described in the Examples below, a second bacterial peptide that promotes growth of

Mtb was found by its activity in inducing the Acr promoter. This peptide, Leu-Val-Thr-Leu-Val-Phe-Val (LVTLVFV) [SEQ ID NO:2] was previously known to be involved in bacterial conjugation in Enterococcus faecalis.

As mentioned above, the major difficulty in working with mycobacterial cultures is the extended lag period combined with a generation time, generally of the range of 22-24 hours. This difficulty is equally insurmountable for clinicians involved in TB diagnosis as well as for researchers studying the physiology of the microorganism. The present invention can significantly reduce the turn-around time for culture recovery for identification as well as for further research purposes. Accordingly, one skilled in the art will appreciate that any study requiring large scale growth of the microorganism will clearly benefit from the present invention, especially when the availability of the initial inoculum is limiting. The particular advantages of the availability of a growth promoting substance as provided herein that significantly reduces the lag period of mycobacterial growth, are many. In a clinical setting, time to diagnosis will be reduced significantly, permitting faster therapeutic intervention. In a research laboratory setting, cells will be obtainable more rapidly. This is important in the case where the initial inoculum has been derived from a patient isolate and hence is limiting in quantity. The present compositions and methods can contribute to drug discovery by increasing the throughput for screening of drug candidates in a shorter time than would otherwise be possible. In summary, this invention offers a number of solutions to an almost century-old problem in working with mycobacteria, the difficulty in achieving predictable growth in a defined period of time. Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLE 1 Detection of Growth Promoting Activity in M. tuberculosis Culture Snpernatants

Low inoculum (LI) cultures of Mtb showed no or very little growth over almost 30 days of culture. Figure 1 indicates that a culture starting at a density of about 100 CFU/ml did not grow beyond several hundred cells.

Figure 2 shows that when spent medium from a 14-day-old culture was recovered, the cells filtered out and the resulting filtrate fractionated by ultrafiltration using a 10 kDa cutoff membrane, the low molecular weight fraction (which contained the peptide of the present invention) stimulated cell growth in a LI culture.

For initial determination of whether the active factor was a protein or peptide, the fraction was pretreated with proteinase K (8 μg/ml) or pronase E (15 μg /ml) both broad- spectrum proteinases. The proteinases were inactivated for 30 minutes at 80°C, and the materials added to the cultures. Both proteinases destroyed the growth-promoting activity. Even when not inactivated, the presence of either proteinase in the cultures did not influence bacterial growth.

2D gel electrophoresis was carried out on cells stimulated with the growth-promoting fractions and compared to control cells. At least 5 new bands appeared in the treated cell preparation, apparently representing new proteins that had been induced. Of special significance was overexpression of the Acr homologue.

Species specificity was comparing similar <10 kDa fractions from M. tuberculosis, M. smegmatis, M. bovis BCG and M. avium, for stimulation of Mtb growth. The fractions from the slow growing strains of M. avium and M. bovis BCG marginally stimulated the growth of Mtb. while the fraction from the fast growing M smegmatis had no effect on Mtb growth.

EXAMPLE 2

Identification of Growth Stimulating Peptide

The next studies were performed to identify the factors responsible for Mtb cell growth. The strategy is shown schematically in Figure 3 and, along with some results, in Figure 4. Fractionation of Culture Filtrate

5 mg of the total material obtained from the culture filtrate described above by membrane exclusion (0.5-10 kDa) was dried down completely and resuspended in 200 μl of 0.1% trifluoroacetic acid. The suspension was loaded on a 1 x 35 cm Bio gel P-2 column at a flow rate of 30 ml/hour. Fractions of 1 ml were collected and 1 μl of each fraction was spotted on a silica gel TLC plate, and then chromatographed in a mixture of ethyl acetate :pyridine: water: acetic acid (ratio of 50:50:30:10). Visualization was achieved with a 0.2% ninhydrin spray followed by heating at 65-80°C. The fractions were analyzed for growth stimulation by the method discussed herein. In some experiments, the material obtained from the Bio gel P-2 column were subjected to reverse phase HPLC on a C18 matrix, and fractions were assayed for growth.

Electron spray mass spectroscopy (ES-MS) was conducted on the TLC -purified material, to identify the growth activating peptide. A Finnigan LCQ electrospray ion trap mass spectrometer was used. The growth activating factor was dissolved in 20% acetonitrile, 0.5% trifluoroacetic acid. This suspension was infused into the mass spectrometer at a flow of 10 μl per min. The electrospray needle at 3.5 kV with a sheath gas flow of N₂ and a capillary temperature of 100°C.

Figure 5 shows the mass spectrogram. Different charge stages are shown. The most abundant species was a 561.5 Da (M+H)^4" product (singly charged ion) that shows up at half that molecular mass when doubly charged (281.3 Da (M+H)^24" peak). Also shown are peaks representing various sodium adducts to the right of the 561.5 Da peak. The larger molecular mass peaks represent non-covalently complexed dimers of the 561.5 Da peptide, which are formed because of the polarity of this peptide. Figure 6 shows a mass spectrogram (MS/MS) performed on the fly of the 561.2 m/z ion shown in Figure 5, with the collision energy set at 40%. The MS-MS fragmentation data was used to perform de novo sequencing by calculating the y and b series fragment ions. This led to the elucidation of the sequence of the growth activating factor as ERGVT [SEQ ID NO:l].

EXAMPLE 3

Growth Promoting Activity of ERGVT and Other Quorum-Sensing Bacterial Peptides

Figure 7 shows the dose response growth curves of Mtb stimulated by four different concentrations of ERGVT ranging from 5 pM to 4 nM. Concentrations between 50 pM and 4nM stimulated growth (of at least 3 logs) of cultures having an initial cell density of about 100 CFU/ml. This peptide was therefore highly potent, dramatically more so than the peptides described by Malski (1989, supra). Moreover, the sequence of this peptide was distinct from anything disclosed by Malski or by others

Five other quorum-sensing peptides which originate from other bacteria were compared with ERGVT for their ability to promote growth of Mtb LI cultures. Figure 8 shows the results. Two peptides from B. subtilis, ADPITRQWGD (SEQ ID NO:3) and ERGMT (SEQ ID NO:4), which only differed by a single amino acid from ERGVT, showed no activity. A S. aureus peptide , YSTCDFIM (SEQ ID NO:5) and one from E. faecalis, LFSLVLAG (SEQ ID NO:6) were also inactive. Besides ERGVT, only LVTLVFV (SEQ ID NO:2] (which has been shown to be associated with conjugation function in Enter -ococcus faecalis) was active. EXAMPLE 4

Acr-In during Activity of ERGVT and Other Bacterial Quorum-Sensing Peptides

As noted above, Acr production play some essential role in intracellular Mtb growth. Studies were performed to test whether the present peptide acted by inducing the promoter of this gene in cells.

The reporter system obtained from Dr. Clifton E. Barry III, of NIAID, National Institutes of Health. This reporter was a fusion of the acr promoter and the lacZ gene which encodes β- galactosidase (β-gal). The promoter construct was derived from the plasmid pMH66(Rev). The 254-nucleotide gene upstream of the initiator ATG of the acr gene was amplified by PCR and cloned as an Xbal/Hindlll fragment into the pMH66(Rev) to create the acr-lacZ reporter pMH109. These plasmids had hygromycin-resistance and kanamycin-resistance markers for selection.

Mtb were transformed with the above plasmid using standard techniques of spheroplast formation (e.g., Murty MV et al, Ann Microbiol (Paris) 1983 134B:359-65; Udou T et al, Can J Microbiol 1983 Jan;29(l):60-68; Murty MV et al, Ann Microbiol (Paris) 1984 135B:147-54; Murty MV et al, Arm Microbiol (Paris) 1984 135A:367-74). Mtb from midlog phase growth were treated with cycloserine, lysozyme and glycine, as described. Spheroplasts were incubated with 10 ng plasmid DNA ml suspension. After treatment, cells were regrown for four hours and plated on selective medium containing hygromycin to select plasmid-bearing cells. Selected colonies were grown in 5-10 mol cultures. Cultures containing about 10⁵ cells were either lysed by sonication using the standard probe-type sonicator (amplitude setting of 20 μm, 10x30 second pulses with a gap of 30 seconds between pulses) or subjected to chloroform (100 μl/culture) to permit release of the enzyme.

Protein was estimated by the well-known BCA method, β-galactosidase enzymatic activity was measured by a conventional method where 5 μg of culture lysate (or chloroform treated suspension) was incubated at 37°C for 10 minutes with 4mM o-nitrophenyl β-D- galactopyranoside (ONPG), a chromogenic substrate for β-galactosidase. The reaction was terminated and color formed was measured as Absorbance at 420 nm. Specific activity was defined as nmoles of substrate converted/minute mg protein The results are shown in Figure 9 , testing the same 6 peptides as in Example 3. Only two bacterially-derived peptides, ERGVT and LVTLVFV were active. A peptide having a molecular mass of 1097.2 (U.S. application Serial. No. 09/576,044, the present priority application) which is not bacterial in origin but rather is a product of the culture medium, also induced the hspX promoter.

A time course analysis of the induction of the Acr promoter by ERGVT showed a 4.5- fold increase in specific activity from day 11 to day 15, which rapidly declined to base-line levels by day 17.

EXAMPLE 5 Peptides Mediate the Rescue of Stressed Cells

Peroxide and superoxide radicals have been implicated in macrophage-based oxidative killing of microorganisms. Mtb evades this free radical stress in vivo. This was thought to be a result of expression of peroxidases and superoxide dismutases that inactivate the free radicals in addition to an relatively inert and robust cell wall. The ability of ERGVT to act on stressed cultures were tested in a model of peroxide shocked cultures. Bacteria were monitored for growth under prolonged exposure to 5 mM and 10 mM hydrogen peroxide. Once growth kinetics had been established, the peptides were added at time points of 15, 17 and 20 days beyond the hydrogen peroxide challenge, and the ability of the peptide to resuscitate the culture was examined. Results are shown in Figure 10. The peptide was able to resuscitate the cultures in all the sample points tested. In contrast, a growth stimulating exochelin molecule could not revive the peroxide shocked culture beyond Day 15 (not shown), which suggested that some proteinaceous molecules may appear to mimic the effect of the present growth stimulatory peptides, but, in fact, are most likely merely providing nutrition to the cells.

EXAMPLE 6

Growth Activating Peptide having a molecular mass of 1097.2 Da

The present inventors discovered yet another peptide that was originally believed to be of Mtb origin that also stimulated growth of low inoculum cultures (see priority document USSN 09/576,044). As shown in Figure 9, this peptide also induced the Acr promoter. Characterization of this peptide is shown in Figure 10 (Reverse phase HPLC, the peak elutmg at 41.58 minutes) and Figure 11 (ES-MS, peak at 1097.2 Da).

For the results shown in Figure 10, 5 mg of the total material obtained from the culture filtrate by membrane exclusion ( 0.5-10 kDa ) was dried down completely and resuspended in 200 ml of 0.1% trifluoroacetic acid. The suspension was loaded on a 1 cm X 35 cm Biogel P-2 column at a flow rate of 30 ml/h. Fifty 1 ml fractions were collected, completely dried down and 1 μl of each fraction was spotted on a silica gel TLC plate and chromatographed in ethyl acetate:pyridine:water:acetic acid (50:50:30:10 ). Visualization was carried out by 0.2% ninhydrin spray followed by heat application at 65-80°C. Fractions were analyzed for growth stimulation as described above, and active fractions were pooled based on proximity.

For the analysis presented in Figure 12, the pools that stimulated growth were dried down completely and suspended in 1.0 ml of 5% acetonitrile, 0.1 % TFA and applied to a 15 cm X 1 cm C18 microbore reverse phase column ( Vydac, Hesperia, CA ) connected to a Waters 2690 Separation module ( Milford, MA ). The bound material was eluted with an increasing acetonitrile gradient at a flow rate of 25 ml per minute and the reverse phase effluent was introduced directly into a Finnigan LCQ electrospray mass spectrometer where the peptides were analyzed by MS and MS-MS. The ES needle was operated at 6 kV with a sheath gas flow of nitrogen at 40 psi and a capillary temperature of 200oC. The most dominant ion of an MS scan was subjected to MS -MS on the fly with collision energy of 40%. MS of the reverse phase peak at 41.58 minutes revealed a peptide of mass 1097.2 Da.

Further analysis of spent Mtb culture media containing this peptide showed that the peptide originated in the medium, possibly in the casein hydrolysate. The peptide may be a cleavage product of larger polypeptide that is generated by a Mtb-derived a protease. Initial ES- MS analysis of concentrated medium did not reveal the presence of the 1097.2 factor. Also, a peptide with a sequence that would correspond to the characteristics of the 1097.2 Da factor cannot be found in the Mtb genome. Subsequent analysis of the concentrated medium by LC-MS with Selective Ion Monitoring proved that the product was derived from the culture medium derived. However, substances secreted by the Mtb organisms such as proteinases, may play a role in the processing of this compound. The present invention comprises use of the 1097.2 Da peptide in combination with other peptides of bacterial origin described above (albeit, ones that preferably are chemically synthesized) as additives to culture medium to better promote Mtb growth. The references cited above are all incorporated by reference herein, whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

Claims

WHAT IS CLAIMED IS:

1. A composition useful for stimulating the growth of M. tuberculosis bacteria in culture, comprising an isolated peptide selected from the group consisting of Asp-Arg-Gly-Val-Thr (ERGVT) [SEQ ID NO:l] and Leu-Val-Thr-Leu-Val-Phe-Val (LVTLVFV) [SEQ ID NO:2] or a mixture eof ERGVT and LVTLVFV, which peptides stimulate the growth of said bacteria when said bacteria are in a dormant state or inoculated into culture at a cell density that is too low for growth.

2. The composition of claim 1 wherein said peptide is ERGVT.

3. The composition of claim 1 wherein said peptide is LVTLVFV.

4. The composition of claim one that comprises both of said peptides ERGVT and

LVTLVFV.

5. The composition of claim 1 wherein said bacteria are inoculated into culture at a concentration of less than about 100 CFU/milliliter medium.

6. The composition of any of claims 1-5 supplemented with an additional purified peptide having a molecular mass (M+H⁺) of 1097.2 Da which is derived from culture medium that includes casein hydrolysate, wherein said 1097.2 Da peptide is characterized in that it alone stimulates the growth of said bacteria when added to aM tuberculosis culture.

7. A composition useful for stimulating the growth of M. tuberculosis bacteria which: (a) comprises a peptide;

(b) stimulates growth of said bacteria when said bacteria are in a dormant state or inoculated into culture at a cell density that is too low for growth; and

(c) induces the promoter of the M tuberculosis homologue of the Acr gene to express any DNA coding sequence operatively linked thereto.

8. The composition of claim 7 wherein the peptide has a molecular mass of <2kDa.

9. The composition of claim 8 wherein the peptide has a molecular mass of <lkDa.

10. The composition of claim 9 wherein the peptide has a molecular < 800Da.

11. The composition of claim 10 wherein the peptide has a molecular mass of <600Da

12. The composition of any of claims 7-11 characterized in that it stimulates the growth of an inoculum of M tuberculosis having fewer than or about 100 CFU per milliliter to about 10⁵ CFU/ml in a period of about 3 weeks in standard mycobacterial culture medium.

13. The composition of any of claims 7-11 characterized in that it stimulates the growth of an inoculum of M tuberculosis having between 100 and about 1000 CFU per milliliter to about 10⁵ CFU/ml in a period of about 3 weeks in standard mycobacterial culture medium.

14. The compositions of any of claim 7-13 wherein the peptide stimulates said growth at concentrations between about 10 pM and about 10 nM.

15.. The composition of claim 14 wherein the peptide stimulates said growth at concentrations between less than or equal to about 1 nM.

16. The composition of any of claims 7-15 wherein the peptide is not glutathione, Gly- Asn or Leu-Gly.

17.. The composition of any of claims 7-16 supplemented with an additional purified peptide having a molecular mass (M+H⁺) of 1097.2 Da which is derived from culture medium that includes casein hydrolysate, wherein said 1097.2 Da peptide is characterized in that it alone stimulates the growth of said bacteria when added to a M tuberculosis culture.

18. A method for stimulating the growth of M. tuberculosis bacteria in culture comprising adding to a suitable culture of said bacteria an effective amount of the composition of any of claims 1-17.

19. A method for resuscitating M. tuberculosis cells from a dormant state into a growth state, comprising (a) obtaining a population of M. tuberculosis cells from a stressed environment;

(b) inoculating said cells into a standard mycobacterial culture medium; and (c) contacting said cells in said medium with the composition of any of claims 1-17.

20. The method of claim 19 wherein said cells are obtained from said stressed environment in vivo.

21. The method of claim 19 wherein said cells are obtained from said stressed environment in vitro.

22. The method of any of claims 19 -21 wherein said stressed environment is characterized by the presence of low pH, nutrient deprivation, low oxygen tension or reactive oxygen intermediates.

23. The method of any of claims 19-21 wherein said stressed environment is characterized by the presence of hydrogen peroxide at a concentration that inhibits cell growth.

24. A method of producing a growth stimulatory culture medium for M. tuberculosis bacteria characterized in that said medium stimulates the growth the bacteria when they are inoculated into culture at a concentration of less than or equal to about 10³ CFU/milliliter medium, which method comprises adding to a mycobacterial culture medium a composition according to any of claims 1-17.

25. The method of claim 24 wherein said bacteria are inoculated into culture at a concentration of less than or equal to about 10² CFU/milliliter medium.