Search

SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in Mycobacterium tuberculosis, and this sigma factor is required for virulence in animal models of infection. SigH is negatively regulated by RshA, its cognate anti-sigma factor, which functions as a stress sensor and redox switch. While RshA provides a direct mechanism for sensing stress and activating transcription, bacteria use several types of signal transduction systems to sense the external environment. M. tuberculosis encodes several serine-threonine protein kinase signaling molecules, 2 of which, PknA and PknB, are essential and have been shown to regulate cell morphology and cell wall synthesis. In this work, we demonstrate that SigH and RshA are phosphorylated in vitro and in vivo by PknB. We show that phosphorylation of RshA, but not SigH, interferes with the interaction of these 2 proteins in vitro. Consistent with this finding, negative regulation of SigH activity by RshA in viva is partially relieved in strains in which pknB is over-expressed, resulting in increased resistance to oxidative stress. These findings demonstrate an interaction between the signaling pathways mediated by PknB and the stress response regulon controlled by SigH. The intersection of these apparently discrete regulatory systems provides a mechanism by which limited activation of the SigH-dependent stress response in M. tuberculosis can be achieved. Coordination of the PknB and SigH regulatory pathways through phosphorylation of RshA may lead to adaptive responses that are important in the pathogenesis of M. tuberculosis infection. [ABSTRACT FROM AUTHOR]

mRNA interferases are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems in bacterial genomes. Previously, we demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7) and determined cleavage specificities for MazF-mt1 and MazF-mt6. Here we have developed a new general method for the determination of recognition sequences longer than three bases for mRNA interferases with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. Using this method, we determined that MazF-mt3 cleaves RNA at UU˘CCU or CU˘CCU and MazF-mt7 at U˘CGCU (˘indicates the cleavage site). As pentad sequence recognition is more specific than those of previously characterized mRNA interferases, bioinformatics analysis was carried out to identify M. tuberculosis mRNAs that may be resistant to MazF-mt3 and MazF-mt7 cleavage. The pentad sequence was found to be significantly underrepresented in several genes, including members of the PE and PPE families, large families of proteins that play a role in tuberculosis immunity and pathogenesis. These data suggest that MazF-mt3 and MazF-mt7 or other mRNA interferases that target longer RNA sequences may alter protein expression through differential mRNA degradation, a regulatory mechanism that may allow adaptation to environmental conditions, including those encountered by pathogens such as M. tuberculosis during infection. [ABSTRACT FROM AUTHOR]

Protective immunity to human immunodeficiency virus (HIV) was examined in 228 serial sera from 58 HIV-infected children before and during antiretroviral therapy. Binding antibodies to putative protective V3 epitopes of HIV-IIIB and HIV-1MN were investigated by a peptide ELISA, and neutralizing antibodies by inhibition of HIV-IM N cell-free viral infection. No difference in binding of total IgG or IgG subclasses was observed between patients with mild (group A) or advanced disease (group B). However, group A patients were more likely to possess neutralizing antibody titers ⩾225 (P = .040 after adjustment for CD4). This threshold titer also predicted clinical outcome in patients with age-adjusted CD4 count >10%of normal median. Patients with titers <225 more frequently encountered major clinical events during follow-up than did patients with titers ⩾225 (P = .0028). Epitopes other than the linear V3100p contribute to this protective immune response. Identification of these epitopes should assist immune therapy of AIDS and HIV vaccine development. [ABSTRACT FROM PUBLISHER]

Background: A recently licensed pneumococcal conjugate vaccine has been shown to be highly effective in the prevention of bacteremia in immunized children but the degree of protection against pneumonia has been difficult to determine. Methods: We sought to develop a model of Streptococcus pneumoniae pneumonia in Sprague-Dawley rats. We challenged three-week old Sprague-Dawley pups via intrapulmonary injection of S. pneumoniae serotypes 3 and 6B. Outcomes included bacteremia, mortality as well histologic sections of the lungs. Results: Pneumonia was reliably produced in animals receiving either 10 or 100 cfu of type 3 pneumococci, with 30% and 50% mortality respectively. Similarly, with type 6B, the likelihood of pneumonia increased with the inoculum, as did the mortality rate. Prophylactic administration of a preparation of high-titered anticapsular antibody prevented the development of type 3 pneumonia and death. Conclusion: We propose that this model may be useful for the evaluation of vaccines for the prevention of pneumococcal pneumonia.

Examines the effects of mycobacterium avium complex (MAC) related strains on children with AIDS and lymphadenitis in the United States. Development of disseminated MAC following a T cell depletion; Isolation of MAC patients from pediatric patients with AIDS and lymphadenitis.

Background: A recently licensed pneumococcal conjugate vaccine has been shown to be highly effective in the prevention of bacteremia in immunized children but the degree of protection against pneumonia has been difficult to determine. Methods: We sought to develop a model of Streptococcus pneumoniae pneumonia in Sprague-Dawley rats. We challenged three-week old Sprague-Dawley pups via intrapulmonary injection of S. pneumoniae serotypes 3 and 6B. Outcomes included bacteremia, mortality as well histologic sections of the lungs. Results: Pneumonia was reliably produced in animals receiving either 10 or 100 cfu of type 3 pneumococci, with 30% and 50% mortality respectively. Similarly, with type 6B, the likelihood of pneumonia increased with the inoculum, as did the mortality rate. Prophylactic administration of a preparation of high-titered anticapsular antibody prevented the development of type 3 pneumonia and death. Conclusion: We propose that this model may be useful for the evaluation of vaccines for the prevention of pneumococcal pneumonia. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis (Mtb) VapBC4 toxin-antitoxin system is essential for the establishment of Mtb infection. Using a multitier, systems-level approach, we uncovered the sequential molecular events triggered by the VapC4 toxin that activate a circumscribed set of critical stress survival pathways which undoubtedly underlie Mtb virulence. VapC4 exclusively inactivated the sole transfer RNACys (tRNACys) through cleavage at a single site within the anticodon sequence. Depletion of the pool of tRNACys led to ribosome stalling at Cys codons within actively translating messenger RNAs. Genome mapping of these Cys-stalled ribosomes unexpectedly uncovered several unannotated Cys-containing open reading frames (ORFs). Four of these are small ORFs (sORFs) encoding Cys-rich proteins of fewer than 50 amino acids that function as Cys-responsive attenuators that engage ribosome stalling at tracts of Cys codons to control translation of downstream genes. Thus, VapC4 mimics a state of Cys starvation, which then activates Cys attenuation at sORFs to globally redirect metabolism toward the synthesis of free Cys. The resulting newly enriched pool of Cys feeds into the synthesis of mycothiol, the glutathione counterpart in this pathogen that is responsible for maintaining cellular redox homeostasis during oxidative stress, as well as into a circumscribed subset of cellular pathways that enable cells to defend against oxidative and copper stresses characteristically endured by Mtb within macrophages. Our ability to pinpoint activation or down-regulation of pathways that collectively align with Mtb virulence-associated stress responses and the nonreplicating persistent state brings to light a direct and vital role for the VapC4 toxin in mediating these critical pathways. [ABSTRACT FROM AUTHOR]

The ability of Mycobacterium tuberculosis (Mtb) to adapt to diverse stresses in its host environment is crucial for pathogenesis. Two essential Mtb serine/threonine protein kinases, PknA and PknB, regulate cell growth in response to environmental stimuli, but little is known about their downstream effects. By combining RNA-Seq data, following treatment with either an inhibitor of both PknA and PknB or an inactive control, with publicly available ChIP-Seq and protein–protein interaction data for transcription factors, we show that the Mtb transcription factor (TF) regulatory network propagates the effects of kinase inhibition and leads to widespread changes in regulatory programs involved in cell wall integrity, stress response, and energy production, among others. We also observe that changes in TF regulatory activity correlate with kinase-specific phosphorylation of those TFs. In addition to characterizing the downstream regulatory effects of PknA/PknB inhibition, this demonstrates the need for regulatory network approaches that can incorporate signal-driven transcription factor modifications. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis genome contains an unusually high number of toxin-antitoxin modules, some of which have been suggested to play a role in the establishment and maintenance of latent tuberculosis. Nine of these toxin-antitoxin loci belong to the mazEF family, encoding the intracellular toxin MazF and its antitoxin inhibitor MazE. Nearly every MazF ortholog recognizes a unique three- or five-base RNA sequence and cleaves mRNA. As a result, these toxins selectively target a subset of the transcriptome for degradation and are known as "mRNA interferases." Here we demonstrate that a MazF family member from M. tuberculosis, MazF-mt6, has an additional role—inhibiting translation through targeted cleavage of 23S rRNA in the evolutionarily conserved helix/loop 70. We first determined that MazF-mt6 cleaves mRNA at5′UU↓CCU3′ sequences. We then discovered that MazF-mt6 also cleaves M. tuberculosis 23S rRNA at a single UUCCU in the ribosomal A site that contacts tRNA and ribosome recycling factor. To gain further mechanistic insight, we demonstrated that MazF-mt6-mediated cleavage of rRNA can inhibit protein synthesis in the absence of mRNA cleavage. Finally, consistent with the position of 23S rRNA cleavage, MazF-mt6 destabilized 50S-30S ribosomal subunit association. Collectively, these results show that MazF toxins do not universally act as mRNA interferases, because MazF-mt6 inhibits protein synthesis by cleaving 23S rRNA in the ribosome active center. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis genome harbors an unusually large number of toxin-antitoxin (TA) modules. Curiously, over half of these are VapBC (virulence-associated protein) family members. Nonetheless, the cellular target, precise mode of action, and physiological role of the VapC toxins in this important pathogen remain unclear. To better understand the function of this toxin family, we studied the features and biochemical properties of a prototype M. tuberculosis VapBC TA system, vapBC-mt4 (Rv0596c-Rv0595c). VapC-mt4 expression resulted in growth arrest, a hallmark of all TA toxins, in Escherichia coli, Mycobacterium smegmatis, and M. tuberculosis. Its expression led to translation inhibition accompanied by a gradual decrease in the steady-state levels of several mRNAs. VapC-mt4 exhibited sequence-specific endoribonuclease activity on mRNA templates at ACGC and AC(A/U)GC sequences. However, the cleavage activity of VapC-mt4 was comparatively weak relative to the TA toxin MazF-mt1 (Rv2801c). Unlike other TA toxins, translation inhibition and growth arrest preceded mRNA cleavage, suggesting that the RNA binding property of VapC-mt4, not RNA cleavage, initiates toxicity. In support of this hypothesis, expression of VapC-mt4 led to an increase in the recovery of total RNA with time in contrast to TA toxins that inhibit translation via direct mRNA cleavage. Additionally, VapC-mt4 exhibited stable, sequence-specific RNA binding in an electrophoretic mobility shift assay. Finally, VapC-mt4 inhibited protein synthesis in a cell-free system without cleaving the corresponding mRNA. Therefore, the activity of VapC-mt4 is mechanistically distinct from other TA toxins because it appears to primarily inhibit translation through selective, stable binding to RNA. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis. We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred, by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins. [ABSTRACT FROM AUTHOR]

The article examines the localization and function of Wag31 in mycobacteria and the role of phosphorylation in modulation of its function. The researchers found that Wag31, a homologue of the cell division protein DivIVA, regulates growth, morphology and polar cell wall synthesis in mycobacteria. Wag31 is also highly localized to the cell poles and that it is essential in mycobacteria and that depletion of wag31 causes a striking morphological change in which one end of the cell becomes round rather than rod-shaped apparently because of a defect in polar peptidoglycan synthesis.

Mycobacterium tuberculosis sigL encodes an extracytoplasmic function (ECF) sigma factor and is adjacent to a gene for a membrane protein (Rv0736) that contains a conserved HXXXCXXC sequence. This motif is found in anti-sigma factors that regulate several ECF sigma factors, including those that control oxidative stress responses. In this work, SigL and Rv0736 were found to be cotranscribed, and the intracellular domain of Rv0736 was shown to interact specifically with SigL, suggesting that Rv0736 may encode an anti-sigma factor of SigL. An M. tuberculosis sigL mutant was not more susceptible than the parental strain to several oxidative and nitrosative stresses, and sigL expression was not increased in response to these stresses. In vivo, sigL is expressed from a weak SigL-independent promoter and also from a second SigL-dependent promoter. To identify SigL-regulated genes, sigL was overexpressed and microarray analysis of global transcription was performed. Four small operons, sigL (Rv0735)-Rv0736, mpt53 (Rv2878c)-Rv2877c, pks10 (Rv1660)-pks7 (Rv1661), and Rv1139c-Rv1138c, were among the most highly upregulated genes in the sigL-overexpressing strain. SigL-dependent transcription start sites of these operons were mapped, and the consensus promoter sequences TGAACC in the -35 region and CGTgtc in the -10 region were identified. In vitro, purified SigL specifically initiated transcription from the promoters of sigL, mpt53, and pks10. Additional genes, including four PE̱PGRS genes, appear to be regulated indirectly by SigL. In an in vivo murine infection model, the sigL mutant strain showed marked attenuation, indicating that the sigL regulon is important in M. tuberculosis pathogenesis. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis genome contains 11 serine/threonine kinase genes including two, pknA and pknB, that are part of an operon encoding genes involved in cell shape control and cell wall synthesis. Here we demonstrate that pknA and pknB are predominantly expressed during exponential growth, and that overexpression of these kinases slows growth and alters cell morphology. We determined the preferred substrate motifs of PknA and PknB, and identified three in vivo substrates of these kinases: PknB; Wag31, an ortholog of the cell division protein DivIVA; and Rv1422, a conserved protein of unknown function. Expression of different alleles of wag31 in vivo alters cell shape, in a manner dependent on the phosphoacceptor residue in the protein produced. Partial depletion of pknA or pknB results in narrow, elongated cells. These data indicate that signal transduction mediated by these kinases is a novel mechanism for the regulation of cell shape in mycobacteria, one that may be conserved among gram-positive bacteria. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis, an obligate mammalian pathogen, adapts to its host during the course of infection via the regulation of gene expression. Of the regulators of transcription that play a role in this response, several alternative sigma factors of M. tuberculosis have been shown to control gene expression in response to stresses, and some of these are required for virulence or persistence in vivo. For this study, we examined the role of the alternative sigma factor SigD in M. tuberculosis gene expression and virulence. Using microarray analysis, we identified several genes whose expression was altered in a strain with a sigD deletion. A small number of these genes, including sigD itself, the gene encoding the autocrine growth factor RpfC, and a gene of unknown function, Rv1815, appear to be directly regulated by this sigma factor. By identifying the in vivo promoters of these genes, we have determined a consensus promoter sequence that is putatively recognized by SigD. The expression of several genes encoding PE-PGRS proteins, part of a large family of related genes of unknown function, was significantly increased in the sigD mutant. We found that the expression of sigD is stable throughout log phase and stationary phase but that it declines rapidly with oxygen depletion. In a mouse infection model, the sigD mutant strain was attenuated, with differences in survival and the inflammatory response in the lung between mice infected with the mutant and those infected with the wild type. [ABSTRACT FROM AUTHOR]

Interviews John Dorman, a staff physician at the Stanford University Health Service, on tuberculosis. Information on the Detection and Treatment of Latent Tuberculosis (TB) Infection in Massachusetts College and University Students operational guide; TB skin test for students implemented by the university; Benefits of the Bacillus Calmette-Guérin vaccine.

Examines a mycobacterial extracytoplasmic function sigma factor involved in survival following stress. Reconstitution of specific RNA polymerase activity on Mycobacterium smegmatis; Comparison between wild-type Mycobacterium smegmatis and the mutant strain; Absence of an inducible protective response to oxidative stress in the wild type.

The Mycobacterium tuberculosis genome harbors an unusually high number of toxin-antitoxin (TA) systems. These TA systems have been implicated in establishing the nonreplicating persistent state of this pathogen during latent tuberculosis infection. More than half of the M. tuberculosis TA systems belong to the VapBC (virulence associated protein) family. In this work, we first identified the RNA targets for the M. tuberculosis VapC-mt11 (VapC11, Rv1561) toxin in vitro to learn more about the general function of this family of toxins. Recombinant VapC-mt11 cleaved 15 of the 45 M. tuberculosis tRNAs at a single site within their anticodon stem loop (ASL) to generate tRNA halves. Cleavage was dependent on the presence of a GG consensus sequence immediately before the cut site and a structurally intact ASL. However, in striking contrast to the broad enzyme activity exhibited in vitro, we used a specialized RNA-seq method to demonstrate that tRNA cleavage was highly specific in vivo. Expression of VapC-mt11 in M. tuberculosis resulted in cleavage of only two tRNA isoacceptors containing the GG consensus sequence, tRNAGln32-CUG and tRNALeu3-CAG. Therefore, our results indicate that although in vitro studies are useful for identification of the class of RNA cleaved and consensus sequences required for accurate substrate recognition by endoribonuclease toxins, definitive RNA target identification requires toxin expression in their native host. The restricted in vivo specificity of VapC-mt11 suggests that it may be enlisted to surgically manipulate pathogen physiology in response to stress. [ABSTRACT FROM AUTHOR]

Mycobacterial SigE and SigH both initiate transcription from the sigB promoter, suggesting that they recognize similar sequences. Through mutational and primer extension analyses, we determined that SigE and SigH recognize nearly identical promoters, with differences at the 3' end of the -35 element distinguishing between SigE- and SigH-dependent promoters. [ABSTRACT FROM AUTHOR]

Mycobacterium leprae, a major human pathogen, grows poorly at 37°C. The basis for its inability to survive at elevated temperatures was investigated. We determined that M. leprae lacks a protective heat shock response as a result of the lack of transcriptional induction of the alternative sigma factor genes sigE and sigB and the major heat shock operons, HSP70 and HSP60, even though heat shock promoters and regulatory circuits for these genes appear to be intact. M. leprae sigE was found to be capable of complementing the defective heat shock response of mycobacterial sigE knockout mutants only in the presence of a functional mycobacterial sigH, which orchestrates the mycobacterial heat shock response. Since the sigH of M. leprae is a pseudogene, these data support the conclusion that a key aspect of the defective heat shock response in M. leprae is the absence of a functional sigH. In addition, 68% of the genes induced during heat shock in M. tuberculosis were shown to be either absent from the M. leprae genome or were present as pseudogenes. Among these is the hsp/acr2 gene, whose product is essential for M. tuberculosis survival during heat shock. Taken together, these results suggest that the reduced ability of M. leprae to survive at elevated temperatures results from the lack of a functional transcriptional response to heat shock and the absence of a full repertoire of heat stress response genes, including sigH. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis genome encodes 12 alternative sigma factors, several of which regulate stress responses and are required for virulence in animal models of acute infection. In this work we investigated M. tuberculosis SigM, a member of the extracytoplasmic function subfamily of alternative sigma factors. This sigma factor is expressed at low levels in vitro and does not appear to function in stress response regulation. Instead, SigM positively regulates genes required for the synthesis of surface or secreted molecules. Among these are genes encoding two pairs of Esx secreted proteins, a multisubunit nonribosomal peptide synthetase operon, and genes encoding two members of the proline-proline-glutamate (PPE) family of proteins. Genes up regulated in a sigM mutant strain include a different PPE gene, as well as several genes involved in surface lipid synthesis. Among these are genes involved in synthesis of phthiocerol dimycocerosate (PDIM), a surface lipid critical for virulence during acute infection, and the kasA-kasB operon, which is required for mycolic acid synthesis. Analysis of surface lipids showed that PDIM synthesis is increased in a sigM-disrupted strain and is undetectable in a sigM overexpression strain. These findings demonstrate that SigM positively and negatively regulates cell surface and secreted molecules that are likely to function in host-pathogen interactions. [ABSTRACT FROM AUTHOR]

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins may also bind to promoter region sequences and repress the expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis , we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. In growth inhibition experiments, M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46 , whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation.IMPORTANCEIntracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here, we show that antitoxin interactions with its cognate toxin and with promoter DNA required for antitoxin and toxin expression can be altered by Ser/Thr phosphorylation of the antitoxin and, thus, affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection., Competing Interests: The authors declare no conflict of interest.