Activity of DSA against anaerobically adapted Mycobacterium bovis BCG in vitro.

DSA is a beta-sulfonylacetamide with in vitro activity against pathogenic mycobacteria. Although the enzymatic target(s) of DSA has not been identified, studies to date suggest that this class of compounds may interfere directly or indirectly with ATP synthase and other components of the mycobacterial respiratory chain. In this study we further evaluated the in vitro activity of DSA against anaerobically adapted BCG using two established models. DSA killed BCG in the anaerobic Wayne model. Bactericidal activity ranged from >99% to 60%. DSA killed rifampin-tolerant persisters with a reduction in viable counts of 1.5log(10) versus controls. Conclusive identification of the DSA-specific target(s) will permit a better understanding of the unique mechanism of action of this class of compounds against both aerobically growing and anaerobically adapted bacilli in vitro.

Absence of mycobacterium avium subsp. paratuberculosis in Crohn's patients.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) has been suspected of involvement in Crohn's disease (CD). We investigated this potential association by testing whole blood from CD patients and healthy controls for the presence of MAP by culture and molecular methods. In addition, each blood sample was analyzed for polymorphisms in the NOD2/CARD15 gene previously associated with CD. METHODS: Four 4-mL K(2)-EDTA tubes of whole blood were drawn from each subject (n = 260, 130 CD patients and 130 healthy controls). Two tubes of blood were cultured for MAP by the following methods: Mycobacterial Growth Indicator Tube, Herrold's Egg Yolk Agar, BACTEC 460, and Hungate. The remaining 2 tubes of blood were tested for MAP DNA and polymorphisms in the NOD2/CARD15 gene by polymerase chain reaction (PCR). RESULTS: One healthy control patient was positive for MAP via PCR; however, no viable MAP was cultured from this individual. All blood cultures were negative for MAP. One CD patient's blood was culture-positive for M. tuberculosis complex. CD patients exhibited a higher rate of polymorphism in the NOD2/CARD15 gene than healthy control patients. CONCLUSIONS: In this study MAP was not recovered from the blood of CD patients or healthy controls. However, CD patients showed higher mutation rates in the NOD2/CARD15 gene, compared with healthy controls, supporting the findings of other investigators. No correlation between these polymorphisms and MAP bacteremia in CD patients could be identified in this study.

The Mycobacterium tuberculosis SigD sigma factor controls the expression of ribosome-associated gene products in stationary phase and is required for full virulence.

During infection Mycobacterium tuberculosis is exposed to several environmental conditions depending on the stage and severity of the disease. To survive, M. tuberculosis uses alternate sigma factors to regulate its gene expression in response to the changing host environment. In order to better understand the way in which stress response genes are regulated, the extracytoplasmic sigma factor gene sigD was deleted and subsequently complemented in the CDC1551 strain of M. tuberculosis. The DeltasigD mutant strain exhibited an in vitro growth rate in rich medium identical to that of both the sigD-complemented and wild-type CDC1551 strains. Additionally, no differences were observed in short-term intracellular growth between the mutant, complemented, and wild-type bacteria within the J774A.1 macrophage cell line. However, tumour necrosis factor (TNF)-alpha levels in macrophages infected with the DeltasigD mutant were decreased as compared to levels observed in macrophages infected with the wild-type bacteria. In time-to-death studies, C3H mice infected with the DeltasigD mutant exhibited a mortality delay compared to those infected with either the complemented or wild-type strains. Although mice infected with the DeltasigD mutant died at a reduced rate, the bacillary loads in the lungs and spleen of these mice were comparable to those seen in mice infected with either the complemented or wild-type strains. Microarray analysis of the DeltasigD mutant relative to wild type revealed that SigD directs the expression of a small set of ribosomal genes and adenosine triphosphate transporters whose expression is normally induced during stationary phase growth in vitro. Altered expression of a subset of these genes was confirmed by quantitative reverse transcription polymerase chain reaction analysis. Promoter-like elements resembling the consensus sequence AGAAAG-N16-20-CGTTAA were found upstream of 19 of the genes underexpressed in the DeltasigD mutant suggesting this may be the recognition sequence for the M. tuberculosis SigD-holoenzyme, EsigmaD. These data indicate that the M. tuberculosis SigD sigma factor governs the expression of a small set of ribosomal genes typically expressed in stationary phase during in vitro growth and that loss of sigD reduces macrophage TNF-alpha secretion as well as the lethality of M. tuberculosis infection in mice.

Effect of n-octanesulphonylacetamide (OSA) on ATP and protein expression in Mycobacterium bovis BCG.

OBJECTIVE: To determine the effect on BCG of n-octanesulphonylacetamide (OSA), a novel compound of the class beta-sulphonylcarboxamides, which has potent in vitro activity against pathogenic mycobacteria. METHODS AND RESULTS: The effect of OSA in BCG was examined using two-dimensional protein electrophoresis. Treatment of BCG with OSA resulted in overexpression of two proteins identified as the b-subunit of ATP synthase (Rv1306) and a 17 kDa heat shock protein (Rv0251c). [35S]Methionine pulse-labelling revealed that overexpression occurred within as little as 3.5 h post-exposure. These results were confirmed by RT-PCR. ATP levels decreased in OSA-treated BCG at 5 min, and 1, 3 and 24 h, with a 64%, 45%, 54% and 73% reduction in ATP, respectively. Only dicyclohexylcarbodiimide (DCCD), a known ATP synthase inhibitor, had a similar effect. No appreciable difference in ATP level was observed in BCG treated with standard antimycobacterial drugs, additional respiratory chain inhibitors or a fatty acid synthase inhibitor at a comparable time-point. Protein synthesis decreased within 5 min of exposure to OSA (56%), DCCD (74%) and thenoyltrifluoroacetone (TTFA) (77%). Ethanol (2.3%) potentiated the activity of OSA. In contrast, no synergic effect was observed with streptomycin and ethanol. Mycolic acid levels decreased 79% with DCCD, 46% with TTFA, a complex II inhibitor, and 43% with OSA compared with untreated controls. CONCLUSIONS: Our results suggest that OSA may interfere directly or indirectly with ATP synthase and possibly other components of the mycobacterial respiratory chain. These effects may hinder energy production, leading to interruption in the synthesis of large macromolecules including proteins and mycolic acids.

Mycobacterium tuberculosis ECF sigma factor sigC is required for lethality in mice and for the conditional expression of a defined gene set.

Bacterial alternative RNA polymerase sigma factors are key global adaptive response regulators with a likely role in Mycobacterium tuberculosis pathogenesis. We constructed a mutant lacking the sigma factor gene, sigC, by allelic exchange, in the virulent CDC1551 strain of M. tuberculosis and compared the resulting mutant with the isogenic wild-type strain and complemented mutant strain. In vitro, compared to the wild-type and complemented strains, the mutant was found to have similar ability to survive in both murine bone marrow-derived macrophages and activated J774 macrophages. In time-to-death experiments in the mouse model, the DeltasigC mutant was significantly attenuated, causing no death in infected mice whereas the wild-type and complemented strains caused 100% mortality within 235 days after aerosol infection with a median time to death of 170 days. Mouse organ bacterial burdens indicated that the mutant proliferated and persisted at the same level as the wild-type and complemented strains in lung tissue and was able to persist in mice without causing death for > 300 days. A complete genomic microarray study demonstrated that SigC modulates the expression of several key virulence-associated genes including hspX, senX3 and mtrA, encoding the alpha-crystallin homologue, a two-component sensor kinase and a two-component response regulator respectively. Altered expression of a subset of these genes was confirmed by quantitative RT-PCR analysis. Analysis of genes modulated by SigC also revealed a putative consensus DNA recognition sequence for SigC of SSSAAT-N(16-20)-CGTSSS (S = C or G). Promoter recognition for one of these genes was confirmed by in vitro transcription analysis after purification of recombinant SigC and reconstitution of an Esigma(C) RNA polymerase holoenzyme. These data indicate that the M. tuberculosis transcription factor SigC governs expression of an important M. tuberculosis regulon and is essential for lethality in mice, but is not required for bacterial survival in this species. These observations place the DeltasigC mutant in a class of M. tuberculosis mutants which persist in tissues but are attenuated in their ability to elicit lethal immunopathology.

Attenuation of late-stage disease in mice infected by the Mycobacterium tuberculosis mutant lacking the SigF alternate sigma factor and identification of SigF-dependent genes by microarray analysis.

The Mycobacterium tuberculosis alternate sigma factor, SigF, is expressed during stationary growth phase and under stress conditions in vitro. To better understand the function of SigF we studied the phenotype of the M. tuberculosis DeltasigF mutant in vivo during mouse infection, tested the mutant as a vaccine in rabbits, and evaluated the mutant's microarray expression profile in comparison with the wild type. In mice the growth rates of the DeltasigF mutant and wild-type strains were nearly identical during the first 8 weeks after infection. At 8 weeks, the DeltasigF mutant persisted in the lung, while the wild type continued growing through 20 weeks. Histopathological analysis showed that both wild-type and mutant strains had similar degrees of interstitial and granulomatous inflammation during the first 12 weeks of infection. However, from 12 to 20 weeks the mutant strain showed smaller and fewer lesions and less inflammation in the lungs and spleen. Intradermal vaccination of rabbits with the M. tuberculosis DeltasigF strain, followed by aerosol challenge, resulted in fewer tubercles than did intradermal M. bovis BCG vaccination. Complete genomic microarray analysis revealed that 187 genes were relatively underexpressed in the absence of SigF in early stationary phase, 277 in late stationary phase, and only 38 genes in exponential growth phase. Numerous regulatory genes and those involved in cell envelope synthesis were down-regulated in the absence of SigF; moreover, the DeltasigF mutant strain lacked neutral red staining, suggesting a reduction in the expression of envelope-associated sulfolipids. Examination of 5'-untranslated sequences among the downregulated genes revealed multiple instances of a putative SigF consensus recognition sequence: GGTTTCX(18)GGGTAT. These results indicate that in the mouse the M. tuberculosis DeltasigF mutant strain persists in the lung but at lower bacterial burdens than wild type and is attenuated by histopathologic assessment. Microarray analysis has identified SigF-dependent genes and a putative SigF consensus recognition site.

Growth, Congo Red agar colony morphotypes and antibiotic susceptibility testing of Mycobacterium avium subspecies paratuberculosis.

OBJECTIVE: Mycobacterium avium subspecies (subsp.) paratuberculosis (MAP) is the causative agent of Johne's disease in ruminants and has been associated with Crohn's disease in humans. We sought to test growth rates and susceptibilities of various strains of MAP in two available growth media. DESIGN: Paired comparison design. METHODS: Using the BACTEC macrobroth radiometric growth system and Congo Red-staining agar media, we determined inherent differences in growth characteristics of three bovine and two human strains of MAP and compared susceptibility results obtained in each growth system. RESULTS: Significant differences were observed in growth rate as well as mycobactin J dependence between strains and between a laboratory-adapted isolate of the same strain in the macrobroth system. Similarly, colonial morphology and Congo Red staining on agar media were observed. Two strains, one human and one bovine, demonstrated a 100% rough transparent colony with white coloration on Congo Red agar, while one bovine isolate exclusively grew as a smooth opaque colony with red coloration on Congo Red agar. The remaining strains exhibited mixtures of these two colonial morphotypes on agar media. Comparative susceptibility results between the BACTEC radiometric macrobroth method and the agar proportionality method showed good correlation for most antibiotics/inhibitors tested. However, erratic or poor growth in the macrobroth system prevented minimal inhibitory concentration determinations for two bovine strains by this method. CONCLUSION: This study demonstrates the variability in the colonial morphology of MAP on Congo Red agar as well as the correlation of antibiotic susceptibility results between the BACTEC macro broth method and the agar proportionality method. This study also emphasizes the need for the development of improved, standardized culture and susceptibility test methods for MAP.

Deletion of Mycobacterium tuberculosis sigma factor E results in delayed time to death with bacterial persistence in the lungs of aerosol-infected mice.

The stress-induced extracytoplasmic sigma factor E (SigE) of Mycobacterium tuberculosis shows increased expression after heat shock, sodium dodecyl sulfate treatment, and oxidative stress, as well as after phagocytosis in macrophages. We report that deletion of sigE results in delayed lethality in mice without a significant reduction of bacterial numbers in lungs.

Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative sigma factor, SigH.

The pathogenesis of tuberculosis involves multiple phases and is believed to involve both a carefully deployed series of adaptive bacterial virulence factors and inappropriate host immune responses that lead to tissue damage. A defined Mycobacterium tuberculosis mutant strain lacking the sigH-encoded transcription factor showed a distinctive infection phenotype. In resistant C57BL/6 mice, the mutant achieved high bacterial counts in lung and spleen that persisted in tissues in a pattern identical to those of wild-type bacteria. Despite a high bacterial burden, the mutant produced a blunted, delayed pulmonary inflammatory response, and recruited fewer CD4(+) and CD8(+) T cells to the lung in the early stages of infection. In susceptible C3H mice, the mutant again showed diminished immunopathology and was nonlethal at over 170 days after intravenous infection, in contrast to isogenic wild-type bacilli, which killed with a median time to death of 52 days. Complete genomic microarray analysis revealed that M. tuberculosis sigH may mediate the transcription of at least 31 genes directly and that it modulates the expression of about 150 others; the SigH regulon governs thioredoxin recycling and may be involved in the maintenance of intrabacterial reducing capacity. These data show that the M. tuberculosis sigH gene is dispensable for bacterial growth and survival within the host, but is required for the production of immunopathology and lethality. This phenotype demonstrates that beyond an ability to grow and persist within the host, M. tuberculosis has distinct virulence mechanisms that elicit deleterious host responses and progressive pulmonary disease.

Evaluation of a whole-blood interferon-gamma release assay for the detection of Mycobacterium tuberculosis infection in 2 study populations.

A whole-blood interferon-gamma release assay (IGRA) is being evaluated for its potential to replace the tuberculin skin test (TST) for detecting Mycobacterium tuberculosis infection. To test the assay in a population in which tuberculosis is highly endemic and in another population that is representative of an urban United States population, 253 volunteers from Ethiopia and 175 volunteers from Baltimore were studied for responsiveness on IGRA compared with a simultaneously performed TST. The agreement between the 2 tests, beyond that due to chance, was 68% among subjects from Baltimore and only 35% among those from Ethiopia. IGRA had a sensitivity of 71%, compared with 95% sensitivity for the TST, among 21 subjects who had undergone treatment for culture-confirmed tuberculosis. The specificity was 85% for IGRA and 96% for TST among 52 subjects with no known history of exposure to tuberculosis. In its current form, with purified protein derivative used as the stimulation antigen, the IGRA was found to perform poorly in comparison to the TST in diagnosing M. tuberculosis infection.