GROWTH-ENHANCING MEDIA FOR MYCOBACTERIA

Abstract: This invention provides a method for enhancing the growth of acid-fast microorganisms in culture comprising growing the acid-fast microorganisms in the presence of an aliphatic organic acid of 8-10 carbon atoms. The diagnosis of tuberculosis and many other mycobacterial diseases has been hampered by the slow growth of these organisms using currently available culture methods. The addition of octanoic acid to currently available agar and broth media formulations utilized for the cultivation of pathogenic mycobacteria results in a marked reduction in generation time with an increase in growth yield. The growth promoting activity of octanoic acid offers the potential for significantly more rapid diagnosis, and treatment of these diseases.

Description: GROWTH-ENHANCING MEDIA FOR MYCOBACTERIA BACKGROUND OF THE INVENTION Field of the Invention: This invention is directed to methods of enhancing the growth of acid-fast bacteria and culture media for use in such methods. Review of Related Art The pathogenic mycobacteria are slow-growing bacteria with a generation time of approximately 24 hours, despite a wide range of metabolic enzymes (Wayne 1994). In contrast to other prokaryotes, the mycobacteria possess a complex, lipophilic cell wall composed predominantly of a variety of unusual lipids comprising approximately 10% of the dry weight of the organism (Dhariwal, K. et al., "Alterations In Lipid Constituents During Growth of Mycobacterium smegmatis, CDC46 and Mycobacterium-phlei," Microbios., 16:169-182, 1976). It has been suggested that the synthesis of these complex lipids may be a rate limiting factor for growth (Wheeler, P. et al., "Metabolism of Mycobacterium tuberculosis " Tuberculosis: Pathogenesis, Protection and Control, edited by B. Bloom, ASM Press, Washington, DC, p. 359, 1994; Ratledge, C, "Lipids: Composition, Fatty Acid Biosynthesis," Biology of the Mycobacteria, edited by C. Ratledge and J. Stanford, Academic Press, Inc., London 1:53-94. 1982). Early investigators determined that glycerol was necessary for the in vitro cultivation of mycobacteria and stimulated growth (Wayne, L., "Cultivation of Mycobacterium tuberculosis for Research Purposes," Tuberculosis: Pathogenesis, Protection and Control, edited by B. Bloom, ASM Press, Washington, DC, pp. 74-77, 1994). Glycerol promotes lipid and carbohydrate synthesis (Tepper, B., "Differences in the Utilization of Glycerol and Glucose by Mycobacterium phlei", J. Bacteriol. 95:1713-1717, 1968) and increases oxygen consumption (Wayne, 1994). A number of other potential substrates for optimal growth in vivo have been suggested (Wheeler, P., et al., "Control and Location of Acyl-hydrolysing Phospholipase Activity in Mycobacteria," J. Gen. Microbiol, 138:825-830, 1992), but one of the most unusual metabolic characteristics of mycobacteria is their ability to utilize aliphatic hydrocarbons. Due to the inert nature of hydrocarbons, the ability to utilize these substrates is uncommon and found only among a relatively limited number of organisms, particularly the ability to grow on short-chain hydrocarbons such as ethane, propane, butane, and N-alkanes up to Cg (Gottschalk, G., "Catabolic Activities of Aerobic Heterotrophs," Bacterial Metabolism, edited by G. Gottschalk, Springer-Verlag, New York, pp. 141-177, 1985). Previous investigators have demonstrated the ability of M. tuberculosis to synthesize long-chain fatty acids through direct condensation of shorter chain length acyl-CoA's, specifically octanoyl-CoA, rather than by a step-wise elongation mechanism (Kanemasa, Y., et al., "Direct Incoφoration of Octanoate into Long- chain Fatty Acids by Soluble Enzymes of Mycobacterium tuberculosis " Biochem. Biophys. Ada., 98:476-485. 1965). The authors suggest the reaction may involve "head-to-tail" condensation with the activation of the ω-carbon of the acyl-CoA as the initial step. Lipoic acid is known to be a vital enzymatic cofactor in central metabolism: as an acyl carrier in both the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes, the glycine cleavage system, and the branched chain, α- keto acid dehydrogenase complex (Jordan, S., et al, "A New Metabolic Link: The Acyl Carrier Protein of Lipid Synthesis Donates Lipoic Acid to the Pyruvate Dehydrogenase Complex in Escherichia coli and Mitochondria," J. Biol. Chem., 272:17903-17906, 1997; Fujiwara, C, et al, "Lipoylation of Acyltransferase Components of Alpha-Ketoacid Dehydrogenase Complexes," J. Biol. Chem. 271(22): 12932-12936, 1996; Macherel, D., et al, "Expression, Lipoylation and Structure Determination of Recombinant pea H-protein in Escherichia coli. " Eur. J. Biochem., 236:27-33, 1996). Investigators have also suggested that lipoic acid, derived from octanoic acid precursors, may play a functional role in the mechanism of oxidative phosphorylation, serving as a cofactor for the ATP synthase complex (Griffith, D., "Studies of Energy-Linked Reactions: Net Synthesis of Adenosine Triphosphate by Isolated Adenosine Triphosphate Synthase Preparations: A Role for Lipoic Acid and Unsaturated Fatty Acids," Biochem J, 160:809-812, 1976). Fatty acyl-CoA esters have been implicated as regulators of cellular metabolism in both eukaryotic and prokaryotic cells (Shrago, E., et al., "Fatty Acyl- CoA Esters as Regulators of Cell Metabolism," Prostaglandins, Leukotrienes and Essential Fatty Acids, 52: 163-166, 1997; Faergeman, N., et al., "Role of Long-Chain Fatty Acyl-CoA Esters in the Regulation of Metabolism and in Cell Signaling," Biochem. J, 323:1-12, 1997; Raman, N., et al., "Characterization of the Fatty Acid- Responsive Transcription Factor FadR.," J. Biol. Chem., 272:30645-30650, 1997). In E. coli, FadR is a global regulator of lipid metabolism involving more than 10 genes and operons (Shrago, 1997). The FadR global transcription regulator is controlled via modulation by acyl-CoA's. SUMMARY OF THE INVENTION The diagnosis of tuberculosis and many other mycobacterial diseases has been hampered by the slow growth of these organisms using currently available culture methods. The addition of octanoic acid to currently available agar and broth media formulations utilized for the cultivation of pathogenic mycobacteria results in a marked reduction in generation time with an increase in growth yield. The growth promoting activity of octanoic acid offers the potential for significantly more rapid diagnosis, and treatment of these diseases. It is an object of this invention to exploit the growth promoting activity of octanoic acid on mycobacteria and other acid-fast bacteria. In one embodiment, this invention provides a method of enhancing the growth of cultured acid-fast microorganisms comprising growing the acid-fast microorganisms in the presence of a growth-enhancing compound (GEC). Suitable acid-fast microorganisms include members of genera selected from Mycobacterium, Corynebacterium, Gordona, Nocardia, Rhodococcus, and Tsukamurella. Preferred acid-fast microorganisms include nonpathogenic Mycobacterium bovis BCG, the pathogenic and non-pathogen M. avium complex, M. tuberculosis and M. leprae. Preferably, the GEC is an aliphatic organic acid, such as octanoic acid. Alternatively, the organic acid compound may be a branched or straight chain organic acid which may be saturated or unsaturated having from 8-10 carbon atoms. Suitable organic acid compounds include octanoic acid, nonanoic acid, decanoic acid, methyl nonanoic acid, and 10-methyloctadecanoic acid. In another embodiment, this invention provides a medium for growing acid- fast microorganisms characterized in that the medium comprises a GEC. Preferably, the GEC is selected from the group consisting of octanoic acid, nonanoic acid, decanoic acid, methyl nonanoic acid, and 10-methyloctadecanoic acid. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows bacterial growth of Mycobacterium avium complex in the presence and absence of octanoic acid. Figure 2 shows a comparison of 14 C labeled acetate, octanoate, and palmitate incoφoration into the non-polar (B) and saponifiable (A) lipids of M. bovis BCG. Figure 3 shows [l- 14 C]-octanoate incoφoration into various lipid fractions of M. bovis BCG in the presence of cold (unlabeled) acetate. Figure 4 shows [l- 14 C]-palmitate incoφoration into various lipid fractions of M. bovis BCG in the presence of cold octanoate Figure 5 shows a comparison of 2-dimensional protein profiles of soluble proteins from control versus octanoic acid treated (100 μg/ml) M. bovis BCG at 48 hours post-treatment. Molecular size standards (in kilodaltons) are indicated on the left of each gel; the first dimension isoelectric focusing is run from acidic on the right to basic on the left (bottom). Arrow indicates an upregulated protein identified as AcpM with a pi of approximately 3.0 to 3.4 and a molecular weight of around 12 kDa. Figure 6 shows the effects of octanoic acid supplementation on protein synthesis in Mycobacterium bovis BCG. Figure 7 shows the lipoic acid content of octanoic acid treated M. bovis BCG. DETAILED DESCRIPTION OF THE EMBODIMENTS The discovery of the growth enhancing activity of octanoic acid in mycobacteria grew out of work on the mechanism of action of N-octanesulfonylacetamide (OSA), a novel antimycobacterial compound designed to inhibit fatty acid synthesis. Experiments were designed to evaluate the ability of short chain fatty acids to alleviate or "rescue" mycobacteria from the inhibitory effects on fatty acid synthesis by OSA. The inventors observed that octanoic acid not only rescued mycobacteria from OSA mediated inhibition but also appeared to enhance the growth of both Mycobacterium bovis BCG and the Mycobacterium avium complex. Biochemical studies suggest that octanoic acid has global effects on pathogenic mycobacterial metabolism. These include both qualitative and quantitative changes in both protein and lipid synthesis when compared to mycobacterial cells grown in the absence of octanoic acid. Based on the structure of octanoic acid and current knowledge of mycobacterial metabolism its growth enhancing effects are potentially mediated through lipoic acid and mycolic acid synthesis. Preliminary studies have suggested at least three potential mechanisms of growth potentiation by octanoic acid. The first suggestion is "head-to-tail " condensation with the activation of the ω-carbon of the acyl-CoA as the initial step, which is consistent with the ability of mycobacteria to utilize short chain hydrocarbons. The second possibility is the function of octanoic acid as a precursor in lipoic acid synthesis. Finally, the dramatic effect of octanoic acid on 2- dimensional protein profiles suggests that this particular fatty acid may play a critical regulatory role in mycobacterial metabolism. This molecular mechanism, regulation of gene expression by acyl-CoA's, may be extrapolated to the present example of octanoic acid in mycobacteria. In view of the slow growth generally observed for acid-fast bacteria, there is a significant need for methods which enhance the growth rate of these bacteria in culture. The observations of the present inventors suggest that adding octanoic acid or analogs of octanoic acid to the culture medium for acid-fast bacteria will address this problem. Analogs of octanoic acid for the puφoses of this invention include growth-enhancing compounds (GEC) which may be selected by testing the effect of candidate GECs on growth of acid-fast microorganisms, such as M. bovis BCG, and preferably by testing the ability of candidate GECs to effect increases in the level of one or more of (1) acyl carrier protein M (acpM), (2) lipoyl-ligase (LipB), and/or (3) lipoic acid in a culture of acid-fast microorganisms. Setting up test cultures is within the skill of the art. Briefly, an acid-fast microorganism, including, but not limited to nonpathogenic Mycobacterium bovis BCG, the pathogenic and non-pathogen M. avium complex, M. tuberculosis and M. leprae, is cultured in any suitable medium (see, e.g., the Examples below). The culture is divided and an effective amount (or a range of amounts) of the candidate GEC is added to one portion, while the other serves as a control. After a suitable incubation (typically a few hours or overnight), the cultures are tested for the levels of one or more of (1) acyl carrier protein M (acpM), (2) lipoyl-ligase (LipB), and/or (3) lipoic acid. Detection methods for these components are described in the literature or in the present Examples. Therefore, the present invention is directed to improved methods of culturing acid-fast bacteria by including one or more GECs in the culture medium. Candidate GECs include organic acids which effect the desired enhancement in levels of the detected components. Typically the GEC according to this invention is a branched or straight chain organic acid, which may be saturated or unsaturated, having from 8-10 carbon atoms. Preferably, the culture medium contains octanoic acid, nonanoic acid, decanoic acid, methyl nonanoic acid, and/or 10-methyloctadecanoic acid. Higher molecular weight acids which are metabolized into one of the preferred acids may be added to the culture medium, in the alternative. The culture medium may contain a single GEC or a combination of GECs according to this invention. The GEC(s) may be added at any stage in the culture, although typically they will be added at or before the time of inoculating the culture to maximize the growth-enhancing effect. The GECs which are organic acids may be added as free acids or as salts or esters. In particular, lower alkyl esters, such as methyl, ethyl, isopropyl, etc., may be added. Alternatively, other esters, such as glycerol esters may be added (e.g., mono- or diglycerides, or even triglycerides, such as milk fat or coconut oil which contain esterified fatty acid residues of the desired carbon number). Suitable media for growth of acid-fast bacteria are well-known in the art, and enhanced growth may be achieved by adding GECs to any of them. Selection of suitable medium components is easily within the skill of the art; examples of culture media for acid-fast bacteria are provided in Atlas,R.M., and L.C. Parks,. 1997, "Microbiological Media," CRC Press, Inc., Boca Raton, Fla., Power, D.A., and P.J. McCuen, 1988, "Manual of BBL Products and Laboratory Procedures," 6th ed., Becton Dickinson Microbiology Systems, Cockeysville, Md., Remel Microbiology Products, 1997, "Technical Manual," Remel Microbiology Products, Lenexa, Kansas, Difco Laboratories, 1985, "Difco Manual," 10th ed., Difco Laboratories, Detroit, Mich., all of which are incoφorated herein by reference in their entirety. Liquid media, solid media or semi-solid media are all within the contemplation of this invention. One or more GECs according to this invention are added to the culture medium at a suitable level up to saturation, preferably in the range from 100 μg/ml to lmg/ml. Alternatively, media containing one or more GECs according to this invention may be prepared as single strength liquid media or concentrates and stored prior to use. Acid-fast bacteria are cultured in media according to this invention using standard culture conditions well-known in the art for these bacteria. Acid-fast bacteria which may be cultured in media according to this invention include members the genera Mycobacterium, Corynebacterium, Gordona, Nocardia, Rhodococcus, and Tsukamurella. In particular, this invention is suitable for enhancing growth in culture of nonpathogenic Mycobacterium bovis BCG, the pathogenic and non-pathogen M. avium complex, M. tuberculosis and M. leprae. EXAMPLES In order to facilitate a more complete understanding of the invention, a number of Examples are provided below. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for puφoses of illustration only. Example 1. Growth with octanoic acid: solid media. The growth stimulating activity of octanoic acid was determined using solid agar (M7H10-ADC agar) for two species of mycobacteria. M. bovis BCG (BCG) (Pasteur strain, ATCC 35734) and a clinical isolate of the M. avium complex (MAC) were used. A stock solution of octanoic acid (Matreya, Pleasant Gap, Pennsylvania) was prepared in sterile distilled water (10 mg/ml), spread on the surface of M7H10-ADC agar plates (final concentration = 100, 250 or 500 μg/ml), and allowed to air dry for 1 hour at room temperature. BCG and MAC were grown in M7H9-ADC-Tw broth to stationary phase (OD = A 600 1.0). Subsequently, a 1:100 dilution of each culture was made and 0.1 ml applied to agar plates containing either diluent alone (sterile distilled water) or the previously described concentration of octanoic acid. Plates were incubated at 37°C with 5% CO 2 and checked daily for growth. BCG and MAC were grown on M7H10-ADC agar plates containing either diluent alone (sterile distilled water) or varying concentrations of octanoic acid. Plates were incubated at 37°C with 5% CO 2 and examined daily for evidence of growth. Typically, these conditions produce visible colonies in a minimum of 2 to 3 weeks. In this study, MAC and BCG controls required 14 and 16 days, respectively, for visible lawns to form. However, in plates supplemented with octanoic acid, complete lawns were observed in as little as 9 days for both mycobacterial species tested. This effect varied between mycobacterial species with the concentration of octanoic acid used. For example, in BCG the optimal octanoic acid concentration appeared to be 500 μg/ml, with more variable results obtained with each of the two lower amounts used. In MAC, accelerated growth was achieved with a lower concentration of octanoic acid (100-250 μg/ml), above which less than optimal results were observed. Example 2. Growth with octanoic acid broth media. Cultures of the Mycobacterium avium complex were grown to an OD of 1.0 (A 600 ) in M7H9-ADC-Tw. Subsequently, cultures were diluted in fresh M7H9- ADC-Tw to an OD of 0.02 (A 6 oo) and separated into 50 ml aliquots in 125 ml Erlenmeyer flasks. A stock solution of octanoic acid (10 mg/ml) in distilled water was prepared and filter sterilized through a 0.22 μm Millipore filter. Octanoic acid was added to respective cultures (final concentration = 100 μg/ml) which were incubated on a rotary shaker at 37°C. At 24 hour intervals, 1 ml aliquots were removed an OD's read as A 0 o- Improved growth of the M. avium complex is shown in Figure 1. The effects of octanoic acid on lipid and protein synthesis were evaluated to determine the metabolic mechanisms of growth enhancement. Example 3. Lipid pulse-labeling with 1 C-Octanoate. BCG was grown in M7H9-ADC-Tw (Difco, Detroit, Michigan) to mid-log phase (OD - A 6θo 0.4). From this a 10% inoculum was prepared in 50 ml fresh broth (M7H9-ADC-Tw), followed by aeration and incubation at 37°C for 24 hours (approximately 1 generation time). Subsequently, [l- 14 C]-Octanoic acid or [1, 2- 14 C]-acetate were added to individual flasks (final concentration = 1 μCi/ml) (American Radiolabeled Chemicals, St. Louis, Missouri), and incubated an additional 24 hours at 37°C. Cells were harvested by centrifugation (3000 x g), washed once in distilled water and frozen at -70°C (approximate yield = 500 mg wet weight). Preparation of extractable and saponifiable mycobacterial lipids. Extractions were performed as previously described (Dobson, G., et al., "Systematic Analysis of Complex Mycobacterial Lipids," Chemical Methods in Bacterial Systemics, edited by M. Goodfellow and D. Minnikin, Academic Press, London, pp. 237-265, 1985. Briefly, 50 ml cultures of M. smegmatis, BCG, or MAC were harvested by centrifugation at 3000 x g for 10 minutes. Equal volumes of cells (100-150 mg wet weight) were suspended in methanolic saline [methanol-0.3 % aqueous NaCl (100:10 v/v) (2ml)] and extracted three times with petroleum ether [(bp 60 - 80°C] (1 ml) to yield non-polar extractable lipids. The remaining cells and residual aqueous phase were then boiled for 5 minutes, cooled for 5 minutes at 37°C and extracted once with a mixture of monophasic chloroform-methanol-0.3% NaCl (90:100:30v/v) and twice using chloroform-methanol-0.3%) NaCl (50:100:40 v/v) with mixing for 30 minutes. The residue containing the defatted cells was saved. Polar lipids were extracted from the aqueous supernatants by adding chloroform-0.3% NaCl (1 : 1), mixing for 5 minutes and centrifuging. The lower phase, containing polar extractable lipids, was dried under N 2 (20°C). Defatted cells were subjected to alkaline hydrolysis in methanol (1 ml), 30% KOH (1 ml) and toluene (0.1 ml) at 75°C overnight and subsequently cooled to room temperature. The mixture was acidified to pH = 1 with 3.6%> HC1 and extracted 3 times with diethyl ether. Combined extracts were dried under N 2 . Fatty acid methyl esters of mycolic acids and other long chain compounds were prepared by mixing dichloromethane (1 ml), a catalyst solution (1 ml) (Gyllenhaal and Ehrsson, 1975), and iodomethane (25 ml), for 30 minutes, centrifuging, and discarding the upper phase. The lower phase was dried under N 2 Analysis of mycobacterial lipids. Incoφoration of C-acetate, C-palmitate and C-octanoate into lipid fractions was determined by scintillation counting (Beckman LS6500 multi-puφose scintillation counter). Extracted samples were dissolved in chloroform and dried under N 2 . Equal counts of each of the three lipid classes were loaded onto thin-layer chromatography plates (20 X 20 cm silica gel g, 250 gm, analytical plates Analtech, Newark, Delaware) and subjected to a panel of five different 2-dimensional solvent combinations. Spots were identified according to Dobson, et al (1985). Solvent systems used were as follows in order of increasing polarity (First dimension; second dimension): system 1, for the isolation of triglycerides, petroleum ether (bp 60-80°C)-ethyl acetate (98:2, v/v, 3 times); petroleum ether (bp 60-80°C)-acetone (98:2, v/v, once); system 2, for the separation of non-polar extractable lipids, was petroleum ether (bp 60-80°C)-acetone (92:8, v/v, 3 times): toluene-acetone (95:5, once); system 3, for glycopeptidolipids, was chloroform-methanol-water (100: 14:0.8, v/v); chloroform-acetone-methanol-water (50:60:2,5:3, v/v); system 4, for phospho-, glyco-, and other polar lipids, was chloroform methanol-water (60:30:6, v/v); chloroform-acetic acid-methanol-water (40:25:3:6, v/v); system 5 for mycolic acids, consisted of petroleum ether (bp 60-80°C)-acetone (95:5, v/v 3 times); toluene-acetone (97:3, v/v, once). Visualization and comparison of thin-layer chromatograms was done using either a Molecular Dynamics (425E, Sunnyvale, California) or a Fugi systems (Fujix BAS 1000) phosphorimager. Spot intensities were quantified using either Imagequant (Molecular Dynamics v. 3.3) or NIH Image (v. 1.57, National Institutes of Health, Bethesda, Maryland) software programs and were expressed as a percentage of the total phosphorimage signal for the particular TLC plate. Relative intensities were then calculated by normalizing cerulenin treatment values to controls. Comparison of lipid pulse-labeling with [1,2 14 C]-acetate, [1 14 C]-octanoate, and [1 14 C]-palmitate. Comparison of the non-polar extractable and saponifiable lipids of BCG following exposure to three different radiolabeled substrates was done using 2-dimensional thin-layer chromatography. Equivalent amounts of radioactivity were added to each of the [l,2- 14 C]-acetate and [1- 14 C] -octanoate containing cultures, while half that amount was added to the [l- I4 C]-palmitate culture due to a limited quantity of this particular label. Octanoate was incoφorated at a higher level into both lipid fractions in comparison with either palmitate or acetate (TLC's not shown). However, incoφoration of palmitate was still greater than that observed with acetate. These differences are quantitatively reflected in Figure 2 which demonstrates the level of incoφoration of each of the labels into the respective lipid fractions for a given period of time. Interestingly, these differences appeared to be primarily restricted to extractable non-polar and saponifiable lipids, with little change observed in polar extractable lipids (data not shown). Example 4. Additional labeling experiments in the presence of cold substrates. Additional labeling experiments in the presence of cold substrates. As mentioned in the preceding section, [l- 1 C] -octanoate incoφoration into both extractable non-polar and saponifiable lipids, including mycolic acids, was greater than that associated with 14 C-labeled palmitate, a finding reaffirmed by this assay. To determine if this activity could be affected by the addition of cold substrates, we examined [1- 14 C] — octanoate incoφoration in the presence of an equal amount of unlabeled acetate. Lipid profiles of BCG were examined using combinations of labeled and unlabeled substrates in equal amounts such as [ 1, 2- 14 C] -acetate (Amersham, Arlington Heights, Illinois) and [l- 14 C]-palmitate (Amersham) alone or in combination with cold octanoate. Unlabeled acetate (Sigma, St. Louis, Missouri) and octanoate were prepared in sterile distilled water (10 mg/ml) and filter sterilized through a 0.22 μm Millipore filter (Millipore Coφoration, Bedford, Massachusetts). BCG cultures (OD 60 o = 0.2) were used to make a 10% inoculum into 50 ml fresh broth (M7119-ADC-Tw) and incubated overnight on a rotary shaker at 37°C. Subsequently, labeled and unlabeled substrates were added simultaneously to respective cultures as follows: [1- C]-palmitate alone or with cold octanoate (final concentration of each =0.2 μg/ml, or [1,2- C] acetate alone with cold octanoate (final concentration of each 1.5 μg/ml). Incoφoration of labeled octanoate decreased with the addition of cold acetate into both non-polar extractable and saponifiable lipids, whereas no appreciable differences were noted in incoφoration into polar extractable lipids (Figure 3). A similar finding was observed with [l- 14 C]-palmitate and cold octanoate on incoφoration of label into non-polar extractable lipids, although this difference was not as great as that observed with the previous group (Figure 4). In addition, like the labeled octanoate group, no significant difference in 14 C-palmitate incoφoration into polar extractable lipids followed the addition of unlabeled octanoate. However, this finding was also observed for saponifiable lipids (Figure 4). This is in contrast to the difference in incoφoration of label into this particular lipid fraction observed with labeled octanoate and cold acetate. Example 5. Protein profiles of octanoic acid treated BCG. To investigate the potential effect of octanoic acid supplementation on protein synthesis, 2-dimensional gel electrophoretic protein profiles of treated BCG were examined following a 48 hour incubation post exposure. BCG cultures (20 ml) were grown at 37°C on a rotary shaker to mid-log phase (OD = A 6 oo 0.4), at which time either sterile distilled water (diluent) or octanoic acid (100 μg/ml) was added to control and treated cultures, respectively. Following additional incubation for 24 hours under the same conditions, 2 ml aliquots of cells were harvested by centrifugation (13,000 x g for 30 seconds), the supernatant removed, and the process repeated once using 1 X PBS. The cellular contents of each tube was divided into 2 to 3 eppendorfs (roughly 3 to 5 OD units per tube), and 250 μl of a lysis solution containing 3M urea (Sigma), 0.5% Triton X-100 (Sigma), 500 mg dithiothreitol (DTT) (Gibco BRL, Life Technologies, Gaithersburg, Maryland) and 500 μl Pharmalyte (Pharmacia Biotech, Piscataway, New Jersey) was added. Subsequently, phenylmethylsulfonyl fluoride (PMSF) (Sigma) (100 μg/ml) and leupeptin (2 μg/ml) (Sigma) were also added. Mixtures containing cells and lysis solution were beadbeaten at maximum speed for 60 seconds in a Biospec minibeadbeater using 200-300 μm glass beads (Sigma). This process was repeated three times per sample with 30 second intervals on ice between beatings. The contents of each tube was centrifuged (13,000 x g) a minimum of 30 seconds and the protein containing supernatant removed. Protein concentration was determined using a standardized colorimetric assay (Coomassie Plus, Pierce, Rockford, Illinois) and BSA standards (Pierce) in a Shimadzu UV-1201 spectrophotometer. Quantitated samples were aliquoted and frozen at -70°C. Protein assays: 2-dimensional gels. Approximately 250 μg of each protein sample was mixed with a solution containing 8M urea, 0.5% Triton X-100, Pharmalyte 1-10, DTT, and a few grains of Bromophenol blue (Sigma) (240 μl total volume/sample). This mixture was used to rehydrate commercially prepared acrylamide pH strips (gradient 3-10) overnight at room temperature using manufacturer's standard protocols (Pharmacia Biotech). Completely rehydrated strips were subsequently subjected to a 2-dimensional protein gel system according to standardized protocols (Pharmacia Biotech) (first dimension - 16 hours at 20°C, followed by equilibration of individuals gel strips in a solution containing Tris-HC I (pH 6-8), urea (8M), glycerol (30%), SDS (1 mg/ml) (Sigma) and either DTT (0.3%) or iodoacetamide (Sigma), respectively. Following equilibration, strips were applied to commercially prepared acrylamide gels (245 x 110 x 0.5 mm, gradient 8-18%), Pharmacia) and run for 2 hours at 15°C following manufacturer's standard protocols (Pharmacia). Molecular weight standards (size range: 14 kD to 200 kD) were purchased from Gibco BRL, Life Technologies. Visualization of proteins was done by Coomassie blue (Sigma) staining of gels overnight. Excess stain was removed with 2 to 3 washes of methanokwater: acetic acid (9:9:2, Sigma). Initial experiments revealed striking differences in overall protein profiles between unsupplemented controls and cultures exposed to 100 μg/ml of octanoic acid. As shown in Figure 5, multiple protein species appear to be downregulated with the addition of octanoic acid relative to controls, while one protein species is markedly upregulated. This particular protein has an estimated molecular weight of 12 to 14kD and an acidic pi of approximately 3.0 to 4.0. Example 6. Individual protein species upregulated following octanoic acid exposure. BCG cultures (20 ml) were grown at 37°C on a rotary shaker to mid-log phase (OD = A 600 0.2), at which time either sterile distilled water (diluent) or octanoic acid (100 μg/ml) was added to control and treated cultures, respectively. Following additional incubation for 24 hours under the same conditions, cells were harvested by centrifugation, the supernatant removed, and the process repeated once using sterile distilled water. Protein extraction was carried out by beadbeating followed by centrifugation. Two-dimensional PAGE was done according to established protocols (Pharmacia Biotech). Proteins were visualized by Coomassie staining of gels overnight or silver staining for 4 hours. Initial coomassie staining revealed the upregulation of an acidic (pl~3.8), small (MW ~ 12 kD) protein species in the presence of octanoic acid. This protein, (arrow, Figure 5) was isolated from multiple gels and the amino acid sequence determined by Harvard Microchem (Cambridge, Massachusettes). Alignment of the obtained sequence with the newly completed mycobacterial genomic database identified the protein as AcpM, an acyl carrier protein known to be involved in mycolic acid synthesis (Mdluli et al., 1998, "Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid." Science, 280:1607-10). Subsequently, a more sensitive silver stain, showed the upregulation of a second protein species following exposure to octanoic acid. This protein species (~35 kD, pi -9.1) is consistent in both size and isolelectric point with LipB according to the annotated mycobacterial genomic database. LipB, known to play a role in lipoic acid metabolism, encodes a lipoyl- ligase. Evidence suggests that this ligase is responsible for the attachment of endogenously synthesized lipoyl groups to enzymes requiring lipoic acid as a cofactor (Morris et al., 1995, "Lipoic acid metabolism in Escherichia coli: the lplA and LipB genes define redundant pathways for ligation of lipoyl groups to apoprotein." J. Bacteriol., 177:1-10.). Example 7. Effect of octanoic acid on protein synthesis. Stock Mycobacterium bovis BCG (500 ml) was grown at 37°C, shaking, to mid-log phase (OD = A 6 oo 0-4) and split into 150 ml aliquots. At this time, either sterile distilled water or octanoic acid (100 μg/ml) was added to respective control and treated cultures followed by incubation and aeration at 37°C for 1 hour. Subsequently, 12.5 μCi/ml of 35 S-methionine was added to each culture with additional incubation for 30 minutes under the same conditions. Aliquots (20 ml) were removed from each culture at hourly intervals during the first 4 hours post addition of diluent or octanoic acid, with extra timepoints at 16, 24 and 48 hours. Cells were harvested by low speed centrifugation, washed once in sterile distilled water, and frozen at -70°C. Proteins were extracted, quantitated, and aliquots removed for scintillation counting. As can be seen in Figure 6, octanoic acid treatment in BCG significantly increased S-methionine incoφoration into proteins. This effect was noted in as little as 2 hours post addition of octanoic acid. Example 8. Lipoic acid levels in the presence of octanoic acid. Cultures of M. bovis BCG (500 ml) were grown to mid-log phase (OD = A 6 oo 0.4-0.5) at which time octanoic acid (100 μg/ml) or diluent (sterile, distilled water) was added. Cultures were incubated an additional 24 hours on a rotary shaker at 37°C and harvested by low-speed centrifugation. Lipoic acid was extracted by autoclaving the cells in 6N H 2 SO 4 for 2 hours. Samples were cooled to room temperature and adjusted to neutral pH with 4N NaOH. Remaining particulate matter was removed by filtering samples through glass wool. Prior to use, lipoic acid containing filtrates were sterilized using 0.22 μm Millipore filters. Determination of the lipoic acid concentration of treated versus control BCG cultures was accomplished via a specific bio-assay (Herbert and Guest, 1975, "Lipoic acid content of Escherichia coli and other microorganisms." Arch. Microbiol, 106:259-266). Lipoic acid concentration was correlated to the original dry weight of cells for each respective culture. As shown in Figure 7, lipoic acid levels increased following exposure to octanoic acid in M. bovis BCG. For puφoses of clarity of understanding, the foregoing invention has been described in some detail by way of illustration and example in conjunction with specific embodiments, although other aspects, advantages and modifications will be apparent to those skilled in the art to which the invention pertains. The foregoing description and examples are intended to illustrate, but not limit the scope of the invention. Modifications of the above-described modes for carrying out the invention that are apparent to persons of skill in clinical laboratory medicine, microbiology, and/or related fields are intended to be within the scope of the invention, which is limited only by the appended claims. All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incoφorated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference.

Claims: We claim: 1. A method of enhancing the growth of cultured acid-fast microorganisms comprising growing said acid-fast microorganisms in the presence of a compound, wherein said compound, upon addition to a culture of Mycobacterium bovis BCG, stimulates an increase in said culture in the level of a component selected from the group consisting of acyl carrier protein M, lipoyl- ligase LipB, and lipoic acid. 2. The method of claim 1, wherein said compound is an organic acid. 3. The method of claim 1, wherein said acid-fast microorganisms are members of a genus selected from Mycobacterium, Corynebacterium, Gordona, Nocardia, Rhodococcus, and Tsukamurella. 4. The method of claim 1, wherein said acid-fast microorganism is selected from the group consisting of nonpathogenic Mycobacterium bovis BCG, pathogenic and non-pathogen M. avium complex, M. tuberculosis and M. leprae. 5. The method of claim 2, wherein said organic acid compound is present in a growth-enhancing amount. 6. The method of claim 2, wherein said organic acid compound is an aliphatic organic acid. 7. The method of claim 6, wherein said aliphatic organic acid is octanoic acid. 8. The method of claim 2, wherein said organic acid compound is a branched or straight chain organic acid which may be saturated or unsaturated having from 8-10 carbon atoms. 9. The method of claim 2, wherein the organic acid compound is selected from the group consisting of octanoic acid, nonanoic acid, decanoic acid, methyl nonanoic acid, and 10-methyloctadecanoic acid. 10. A medium for growing acid-fast microorganisms comprising a growth-enhancing compound, wherein said compound, upon addition to a culture of Mycobacterium bovis BCG, stimulates an increase in said culture in the level of a component selected from the group consisting of acyl carrier protein M, lipoyl- ligase LipB, and lipoic acid. 11. A medium according to claim 10 for growing acid-fast microorganisms, said medium comprising an organic acid. 12. The medium of claim 11, wherein said organic acid is selected from the group consisting of octanoic acid, nonanoic acid, decanoic acid, methyl nonanoic acid, and 10-methyloctadecanoic acid. 13. The medium of claim 10, wherein the medium is adapted for liquid phase culture or for solid phase culture. 14. The medium of claim 10, wherein acid-fast microorganisms selected from the group consisting of nonpathogenic mycobacterium M. bovis BCG, pathogenic and non-pathogen mycobacteria M. avium complex, M. tuberculosis and M. leprae and cultured in said medium grow more quickly than said microorganisms cultured in the same medium less said organic acid. 15. A method of screening to aid in selecting a test compound which stimulates growth of an acid-fast microorganism in culture comprising culturing said acid-fast microorganism in the presence and absence of a test compound and comparing the level of a component selected from the group consisting of acyl carrier protein M (acpM), lipoyl-ligase LipB, and lipoic acid between the two cultures, an increase in said component in the presence of said test compound indicating that said test compound is likely to stimulate growth of said acid-fast microorganism. 16. The method of screening of claim 15, further comprising comparing the level of said component in cultures of acid-fast microorganisms containing the test compound to the level of said component in cultures of acid-fast bacteria containing a growth-enhancing amount of an organic acid compound.

Inventors: DICK JAMES D, PARRISH NICOLE M, TOWNSEND CRAIG A, DICK, JAMES, D., PARRISH, NICOLE, M., TOWNSEND, CRAIG, A.
Applicants: DICK JAMES D, PARRISH NICOLE M, TOWNSEND CRAIG A, UNIV JOHNS HOPKINS MED, DICK, JAMES, D., PARRISH, NICOLE, M., THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE, TOWNSEND, CRAIG, A.
Application year: 2000
Main classification: C07K14
IPC codes: C12N1, C12Q1, C12R1