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Mycobacterium avium subsp. hominissuis (MAH) is increasingly recognized as a significant cause of morbidity, particularly in elderly patients or those with immune deficiency or underlying lung impairment. Disease due to MAH is particularly difficult to treat, often requiring years of antibiotic therapy. Identification of genes essential for MAH growth may lead to novel strategies for improving curative therapy. Here we have generated saturating genome-wide transposon mutant pools in a strain of MAH (MAC109) and developed a novel computational technique for classifying annotated genomic features based on the in vitro effect of transposon mutagenesis. Our findings may help guide future genetic and biochemical studies of MAH pathogenesis and aid in the identification of new drugs to improve the treatment of these serious infections.

The stringent response enables Mycobacterium tuberculosis ( Mtb ) to shut down its replication and metabolism under various stresses. Here we show that Mtb lacking the stringent response enzyme Rel Mtb was unable to slow its replication rate during nutrient starvation. Metabolomics analysis revealed that the nutrient-starved rel Mtb -deficient strain had increased metabolism similar to that of exponentially growing wild-type bacteria in nutrient-rich broth, consistent with an inability to enter quiescence. Deficiency of rel Mtb increased the susceptibility of mutant bacteria to killing by isoniazid during nutrient starvation and in the lungs of chronically infected mice. We screened a pharmaceutical library of over 2 million compounds for inhibitors of Rel Mtb and showed that the lead compound X9 was able to directly kill nutrient-starved M. tuberculosis and enhanced the killing activity of isoniazid. Inhibition of Rel Mtb is a promising approach to target M. tuberculosis persisters, with the potential to shorten the duration of TB treatment.

Mycobacterium tuberculosis (Mtb) remains a global health challenge due to the limited efficacy of the Mtb vaccine in current use, Bacillus Calmette-Guérin (BCG). To date, there is no available vaccine for immunocompromised individuals. Thus, there is an urgent need to develop a new vaccine candidate which can induce mucosal immunity in hosts with different immune statuses. DnaK (HSP70) has been shown to induce protective immunity against Mtb infection when administered by DNA vaccine; however, the protection is inferior to that induced by the BCG vaccine. In our study, we vaccinated C57BL/6J mice with DnaK protein alone. Subcutaneous or intranasal vaccination with DnaK generated IFNγ-secreting CD4 + T cells in the spleen, but only intranasal vaccination generated IL-17-releasing CD4 + T cells in the lungs, even when circulating CD4 + T cells were diminished. Furthermore, intranasal vaccination with DnaK generated tissue resident CD4 + T cells in the lungs. Vaccination with DnaK alone resulted in protective immunity comparable to BCG vaccination against tuberculosis in mice. Our results demonstrate that intranasal vaccination with DnaK can generate mucosal immunity in immunocompromised or immunocompetent mice and DnaK vaccination can generate protection against Mtb similar to BCG, underscoring its potential utility as an Mtb vaccine candidate in humans.

Aim: There is controversy regarding the potential fitness costs of rifampicin (RIF) resistance-conferring mutations in the Mycobacterium tuberculosis (Mtb) rpoB gene. We characterized the pathogenicity of an Mtb RpoB H526D mutant., Materials & Methods: A mutant containing the RpoB H526D mutation was isolated from wild-type Mtb grown on RIF-containing plates and complemented for determination of in vitro and in vivo fitness costs., Results: The RpoB H526D mutant showed reduced survival relative to control strains during progressive hypoxia and delayed growth following resuscitation from nutrient starvation (p < 0.05), which was associated with reduced expression of the resuscitation-promoting factor genes rpfB, rpfC and rpfE. Relative to the isogenic wild-type strain, the mutant showed significantly attenuated growth and long-term survival as well as reduced inflammation in mouse lungs. Conclusion & future perspective: Our data suggest that RpoB H526D mutation confers a fitness cost during growth-limiting conditions in vitro and in mouse lungs.

The interaction of Mycobacterium tuberculosis (Mtb) with host cell death signaling pathways is characterized by an initial anti-apoptotic phase followed by a pro-necrotic phase to allow for host cell exit of the bacteria. The bacterial modulators regulating necrosis induction are poorly understood. Here we describe the identification of a transcriptional repressor, Rv3167c responsible for regulating the escape of Mtb from the phagosome. Increased cytosolic localization of MtbΔRv3167c was accompanied by elevated levels of mitochondrial reactive oxygen species and reduced activation of the protein kinase Akt, and these events were critical for the induction of host cell necrosis and macroautophagy. The increase in necrosis led to an increase in bacterial virulence as reflected in higher bacterial burden and reduced survival of mice infected with MtbΔRv3167c. The regulon of Rv3167c thus contains the bacterial mediators involved in escape from the phagosome and host cell necrosis induction, both of which are crucial steps in the intracellular lifecycle and virulence of Mtb.

Co-infection with Mycobacterium tuberculosis accelerates progression from HIV to AIDS. Our previous studies showed that M. tuberculosis complex, unlike M. smegmatis, enhances TLR2-dependent susceptibility of CD4+ T cells to HIV. The M. tuberculosis complex produces multiple TLR2-stimulating lipoproteins, which are absent in M. smegmatis. M. tuberculosis production of mature lipoproteins and TLR2 stimulation is dependent on cleavage by lipoprotein signal peptidase A (LspA). In order to determine the role of potential TLR2-stimulating lipoproteins on mycobacterial-mediated HIV infectivity of CD4+ T cells, we generated M. smegmatis recombinant strains overexpressing genes encoding various M. bovis BCG lipoproteins, as well as a Mycobacterium bovis BCG strain deficient in LspA (ΔlspA). Exposure of human peripheral blood mononuclear cells (PBMC) to M. smegmatis strains overexpressing the BCG lipoproteins, LprF (p<0.01), LprH (p<0.05), LprI (p<0.05), LprP (p<0.001), LprQ (p<0.005), MPT83 (p<0.005), or PhoS1 (p<0.05), resulted in increased HIV infectivity of CD4+ T cells isolated from these PBMC. Conversely, infection of PBMC with ΔlspA reduced HIV infectivity of CD4+ T cells by 40% relative to BCG-infected cells (p<0.05). These results may have important implications for TB vaccination programs in areas with high mother-to-child HIV transmission.

Background: Mycobacterium tuberculosis (Mtb) must adapt to various stress conditions during host infection. The two-component regulatory system (2CRS) SenX3-RegX3 is required for Mtb virulence. We showed recently that the senX3-regX3 intergenic region contains promoter activity, driving senX3-independent regX3 expression. In the current study, we tested the hypothesis that RegX3 has a SenX3-independent role in Mtb virulence. The gene expression patterns, growth, and survival of mutants containing transposon insertions in senX3 (senX3::Tn) and regX3 (regX3::Tn) were compared to those of their respective complemented strains and the isogenic wild-type parent strain during axenic growth in nutrient-rich broth, phosphate depletion, nutrient starvation, and in the lungs of BALB/c mice., Results: regX3 expression was reduced in senX3::Tn during phosphate depletion and nutrient starvation, and expression of the phosphate-specific transport gene pstC2 was reduced similarly in senX3::Tn and regX3::Tn during phosphate depletion. Although senX3 and regX3 were each dispensable for Mtb growth in nutrient-rich broth, disruption of senX3 or regX3 caused a similar growth defect during phosphate depletion. Interestingly, senX3::Tn, in which monocistronic regX3 expression is preserved, showed significantly higher survival relative to regX3::Tn after 7 days of nutrient starvation (p <0.01), and in mouse lungs at Day 31 (p < 0.01), Day 62 (p < 0.01), and Day 124 (p = 0.05) after aerosol infection., Conclusion: Our data demonstrate the specificity of the senX3-regX3 2CRS for sensing and responding to low ambient phosphate, but also raise the possibility that RegX3 may function independently of its cognate sensor histidine kinase.

The highly successful pathogen Mycobacterium tuberculosis (Mtb) has evolved strategies to adapt to various stress conditions, thus promoting survival within the infected host. The two-component regulatory system (2CRS) senX3-regX3, which has been implicated in the Mtb response to inorganic phosphate depletion, is believed to behave as an auto-regulatory bicistronic operon. Unlike other 2CRS, Mtb senX3-regX3 features an intergenic region (IR) containing several mycobacterium interspersed repetitive units (MIRU) of unknown function. In this study, we used a lacZ reporter system to study the promoter activity of the 5' untranslated region of senX3, and that of various numbers of MIRUs in the senX3-regX3 IR, during axenic Mtb growth in nutrient-rich broth, and upon exposure to growth-restricting conditions. Activity of the senX3 promoter was induced during phosphate depletion and nutrient starvation, and IR promoter activity under these conditions was directly proportional to the number of MIRUs present. Quantitative reverse transcriptase (qRT)-PCR analysis of exponentially growing Mtb revealed monocistronic transcription of senX3 and regX3, and, to a lesser degree, bicistronic transcription of the operon. In addition, we observed primarily monocistronic upregulation of regX3 during phosphate depletion of Mtb, which was confirmed by Northern analysis in wild-type Mtb and by RT-PCR in a senX3-disrupted mutant, while upregulation of regX3 in nutrient-starved Mtb was chiefly bicistronic. Our findings of differential regulation of senX3-regX3 highlight the potential regulatory role of MIRUs in the Mtb genome and provide insight into the regulatory mechanisms underlying Mtb adaptation to physiologically relevant conditions., (© 2014 The Authors.)

Background: We have previously identified Mycobacterium tuberculosis PknD to be an important virulence factor required for the pathogenesis of central nervous system (CNS) tuberculosis (TB). Specifically, PknD mediates bacillary invasion of the blood-brain barrier, which can be neutralized by specific antisera, suggesting its potential role as a therapeutic target against TB meningitis., Methodology/principal Findings: We utilized an aerosol challenge guinea pig model of CNS TB and compared the protective efficacy of recombinant M. tuberculosis PknD subunit protein with that of M. bovis BCG against bacillary dissemination to the brain. BCG vaccination limited the pulmonary bacillary burden after aerosol challenge with virulent M. tuberculosis in guinea pigs and also reduced bacillary dissemination to the brain (P = 0.01). PknD vaccination also offered significant protection against bacterial dissemination to the brain, which was no different from BCG (P>0.24), even though PknD vaccinated animals had almost 100-fold higher pulmonary bacterial burdens. Higher levels of PknD-specific IgG were noted in animals immunized with PknD, but not in BCG-vaccinated or control animals. Furthermore, pre-incubation of M. tuberculosis with sera from PknD-vaccinated animals, but not with sera from BCG-vaccinated or control animals, significantly reduced bacterial invasion in a human blood-brain barrier model (P<0.01)., Conclusion: Current recommendations for administering BCG at birth are based on protection gained against severe disease, such as TB meningitis, during infancy. We demonstrate that vaccination with recombinant M. tuberculosis PknD subunit offers a novel strategy to protect against TB meningitis, which is equivalent to BCG in a guinea pig model. Moreover, since BCG lacks the PknD sensor, BCG could also be boosted to develop a more effective vaccine against TB meningitis, a devastating disease that disproportionately affects young children.

Tuberculosis is difficult to cure, requiring a minimum of 6 months of treatment with multiple antibiotics. Small numbers of organisms are able to tolerate the antibiotics and persist in the lungs of infected humans, but they still require some metabolic activity to survive. We studied the role of the hypoxia-induced Rv1894c gene in Mycobacterium tuberculosis virulence in guinea pigs, which develop hypoxic, necrotic granulomas histologically resembling those in humans and found this gene to be necessary for full bacillary growth and survival. We characterized the function of the encoded enzyme as a nitronate monooxygenase, which is needed to prevent the buildup of toxic products during hypoxic metabolism and is negatively regulated by the transcriptional repressor KstR. Future studies will focus on developing small-molecule inhibitors that target Rv1894c and its homologs, with the goal of killing persistent bacteria, thereby shortening the time needed to treat tuberculosis.

Multidrug-resistant tuberculosis has emerged as a major threat to tuberculosis control. Phylogenetically related rifampin-resistant actinomycetes with mutations mapping to clinically dominant Mycobacterium tuberculosis mutations in the rpoB gene show upregulation of gene networks encoding secondary metabolites. We compared the expressed proteomes and metabolomes of two fully drug-susceptible clinical strains of M. tuberculosis (wild type) to those of their respective rifampin-resistant, rpoB mutant progeny strains with confirmed rifampin monoresistance following antitubercular therapy. Each of these strains was also used to infect gamma interferon- and lipopolysaccharide-activated murine J774A.1 macrophages to analyze transcriptional responses in a physiologically relevant model. Both rpoB mutants showed significant upregulation of the polyketide synthase genes ppsA-ppsE and drrA, which constitute an operon encoding multifunctional enzymes involved in the biosynthesis of phthiocerol dimycocerosate and other lipids in M. tuberculosis, but also of various secondary metabolites in related organisms, including antibiotics, such as erythromycin and rifamycins. ppsA (Rv2931), ppsB (Rv2932), and ppsC (Rv2933) were also found to be upregulated more than 10-fold in the Beijing rpoB mutant strain relative to its wild-type parent strain during infection of activated murine macrophages. In addition, metabolomics identified precursors of phthiocerol dimycocerosate, but not the intact molecule itself, in greater abundance in both rpoB mutant isolates. These data suggest that rpoB mutation in M. tuberculosis may trigger compensatory transcriptional changes in secondary metabolism genes analogous to those observed in related actinobacteria. These findings may assist in developing novel methods to diagnose and treat drug-resistant M. tuberculosis infections.

Tuberculosis is a worldwide health problem, given that one-third of the world's population is currently infected with Mycobacterium tuberculosis. Understanding the regulation of virulence on the molecular level will provide a better understanding of how M. tuberculosis can establish chronic infection. Using in vivo microarray analysis (IVMA), we previously identified a group of genes that are activated in BALB/c mouse lungs compared to in vitro cultures, including the rv0990c gene. Our analysis indicated that this gene is a member of the heat shock regulon and was activated under other stress conditions, including survival in macrophages or during the late phase of chronic tuberculosis in the murine lungs. Deletion of rv0990c from the genome of M. tuberculosis strain H37Rv affected the transcriptional profiles of many genes (n = 382) and operons involved in mycobacterial survival, including the dormancy regulon, ATP synthesis, respiration, protein synthesis, and lipid metabolism. Comparison of the proteomes of the mutant to those of the wild-type strain further confirmed the differential expression of 15 proteins, especially those involved in the heat shock response (e.g., DnaK and GrpE). Finally, the rv0990c mutant strain showed survival equivalent to that of the isogenic wild-type strain during active tuberculosis in guinea pigs, despite showing significant attenuation in BALB/c mice during the chronic phase of the disease. Overall, we suggest that rv0990c encodes a heat shock protein that plays an important role in mycobacterial virulence. Hence, we renamed rv0990c heat shock protein 22.5 (hsp22.5), reflecting its molecular mass.

Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosis in necrotic lung lesions.

During human latent tuberculosis infection, Mycobacterium tuberculosis likely resides within the nutrient‐starved environment of caseous lung granulomas. The stringent response alarmone (p)ppGpp is synthesized by Rel in response to nutrient starvation, thus enabling tubercle bacilli to restrict growth and shut down metabolism in a coordinated fashion. In this study, we investigated the virulence of a rel‐deficient M. tuberculosis mutant in the guinea pig model. Quantitative reverse‐transcription polymerase chain reaction was used to study the effect of (p)ppGpp deficiency on expression of key cytokine and chemokine genes in guinea pig lungs. The rel‐deficient mutant showed impaired initial growth and survival relative to the wild‐type strain. Loss of Rel was associated with the striking absence of tubercle lesions grossly and of caseous granulomas histologically. The attenuated phenotype of the rel‐deficient mutant was not associated with increased expression of genes encoding the proinflammatory cytokines interferon‐γ and tumor necrosis factor α in the lungs 28 days after infection.

The Mycobacterium tuberculosis dosR gene (Rv3133c) is part of an operon, Rv3134c-Rv3132c, and encodes a response regulator that has been shown to be upregulated by hypoxia and other in vitro stress conditions and may be important for bacterial survival within granulomatous lesions found in tuberculosis. DosR is activated in response to hypoxia and nitric oxide by DosS (Rv3132c) or DosT (Rv2027c). We compared the virulence levels of an M. tuberculosis dosR-dosS deletion mutant (DeltadosR-dosS [DeltadosR-S]), a dosR-complemented strain, and wild-type H37Rv in rabbits, guinea pigs, and mice infected by the aerosol route and in a mouse hollow-fiber model that may mimic in vivo granulomatous conditions. In the mouse and the guinea pig models, the DeltadosR-S mutant exhibited a growth defect. In the rabbit, the DeltadosR-S mutant did not replicate more than the wild type. In the hollow-fiber model, the mutant phenotype was not different from that of the wild-type strain. Our analyses reveal that the dosR and dosS genes are required for full virulence and that there may be differences in the patterns of attenuation of this mutant between the animal models studied.

To characterize the roles of SigB and SigF in sigma factor regulation in Mycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpress sigB and sigF. Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes after sigB induction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression of sigB and sigF. Overexpression of sigB resulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction of sigB did not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of the M. tuberculosis sigma factor regulatory cascade. Analysis of the 5'-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N(14-18)-NNGNNG. This sequence appeared upstream of both sigB (Rv2710) and the gene following it, ideR (Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression of sigF revealed that only the sigC gene was significantly upregulated 6 and 12 h after sigF induction. The previously identified SigF promoter consensus sequence AGTTTG-N(15)-GGGTTT was identified in the 5' UTR of the sigC gene, and SigF-dependent in vitro transcription of the promoter upstream of sigC was confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression of sigB and sigF resulted in reduced rates of M. tuberculosis intracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.

Background: Mouse and guinea pig models have been used to identify Mycobacterium tuberculosis mutants attenuated for survival. However, unlike mice, M. tuberculosis-infected guinea pigs form caseating granulomas, which may simulate human disease more closely., Methods: We used designer arrays for defined mutant analysis, a high-throughput subtractive competition assay, for genotypically defined M. tuberculosis mutants and compared the survival of the same mutant pools in guinea pig and mouse aerosol models. Selected mutants found to be attenuated in either aerosol model were also analyzed in the mouse hollow-fiber model., Results: M. tuberculosis mutants representing 74 genes were tested. Eighteen M. tuberculosis mutants were attenuated for survival in either aerosol model, with 70% of selected mutants also attenuated in the mouse hollow-fiber model. The majority of attenuated mutants in the mouse aerosol model were detected only after 90 days of infection. There was a high degree of concordance between the genes identified by the 2 aerosol models, with detection being significantly earlier in the guinea pig (P<.0003)., Conclusions: We identified M. tuberculosis genes required for survival in mammalian lungs. The majority of mouse late-stage survival mutants were detected significantly earlier in the guinea pig, which suggests that differences in tuberculosis-induced lung pathologic changes may account for this accelerated detection.

Mycobacterium tuberculosis SigF is homologous to stress response and sporulation sigma factors in many bacteria. Consistent with a possible role in mycobacterial survival under stress conditions, M. tuberculosis sigF is strongly induced within cultured human macrophages and upon nutrient starvation, and SigF has been implicated in M. tuberculosis entry into stationary phase. On the other hand, SigF appears to contribute to the immune pathology of tuberculosis (TB), and a sigF-deficient mutant has altered cell membrane properties. Using an M. tuberculosis sigF deletion mutant, we show here that sigF is not required for bacillary survival under nutrient starvation conditions and within activated murine macrophages or for extracellular persistence in an in vivo granuloma model of latent TB infection. Using a chemically inducible recombinant strain to conditionally overexpress sigF, we did not observe arrest or retardation of growth in exponentially growing cultures or reduced susceptibility to rifampin and isoniazid. Consistent with our hypothesis that SigF may mediate TB immunopathogenesis by altering cell membrane properties, we found that overexpression of sigF resulted in the differential regulation of many cell wall-associated proteins, including members of the MmpL, PE, and PPE families, several of which have been shown to influence host-pathogen interactions. The most highly upregulated gene by quantitative reverse transcription-PCR at all time points following sigF induction was Rv3301c (phoY1), which encodes a probable transcriptional regulatory protein homologous to PhoU proteins involved in regulation of phosphate uptake. Using in vitro transcription analysis, we show that SigF directly regulates phoY1, whose proposed promoter sequence is GGATTG-N(16)-GGGTAT.