MprA and DosR coregulate a Mycobacterium tuberculosis virulence operon encoding Rv1813c and Rv1812c.
ABSTRACT: Mycobacterium tuberculosis remains a significant global pathogen, causing extensive morbidity and mortality worldwide. This bacterium persists within granulomatous lesions in a poorly characterized, nonreplicating state. The two-component signal transduction systems MprAB and DosRS-DosT (DevRS-Rv2027c) are responsive to conditions likely to be present within granulomatous lesions and mediate aspects of M. tuberculosis persistence in vitro and in vivo. Here, we describe a previously uncharacterized locus, Rv1813c-Rv1812c, that is coregulated by both MprA and DosR. We demonstrate that MprA and DosR bind to adjacent and overlapping sequences within the promoter region of Rv1813c and direct transcription from an initiation site located several hundred base pairs upstream of the Rv1813 translation start site. We further show that Rv1813c and Rv1812c are cotranscribed, and that the genomic organization of this operon is specific to M. tuberculosis and Mycobacterium bovis. Although Rv1813c is not required for survival of M. tuberculosis in vitro, including under conditions in which MprAB and DosRST signaling are activated, an M. tuberculosis ?Rv1813c mutant is attenuated in the low-dose aerosol model of murine tuberculosis, where it exhibits a lower bacterial burden, delayed time to death, and decreased ability to stimulate proinflammatory cytokines interleukin-1? (IL-1?) and IL-12. Interestingly, overcomplementation of these phenotypes is observed in the M. tuberculosis ?Rv1813c mutant expressing both Rv1813c and Rv1812c, but not Rv1813c alone, in trans. Therefore, Rv1813c and Rv1812c may represent general stress-responsive elements that are necessary for aspects of M. tuberculosis virulence and the host immune response to infection.
Project description:Mycobacterium tuberculosis, the etiological agent of tuberculosis, remains a significant cause of morbidity and mortality throughout the world despite a vaccine and cost-effective antibiotics. The success of this organism can be attributed, in part, to its ability to adapt to potentially harmful stress within the host and establish, maintain, and reactivate from long-term persistent infection within granulomatous structures. The DosRS-DosT/DevRS-Rv2027c, and MprAB two-component signal transduction systems have previously been implicated in aspects of persistent infection by M. tuberculosis and are known to be responsive to conditions likely to be found within the granuloma. Here, we describe initial characterization of a locus (Rv0081-Rv0088) encoding components of a predicted formate hydrogenylase enzyme complex that is directly regulated by DosR/DevR and MprA, and the product of the first gene in this operon, Rv0081. In particular, we demonstrate that Rv0081 negatively regulates its own expression and that of downstream genes by binding an inverted repeat element in its upstream region. In contrast, DosR/DevR and MprA positively regulate Rv0081 expression by binding to recognition sequences that either partially or completely overlap that recognized by Rv0081, respectively. Expression of Rv0081 initiates from two promoter elements; one promoter located downstream of the DosR/DevR binding site but overlapping the sequence recognized by both Rv0081 and MprA and another promoter downstream of the DosR/DevR, Rv0081, and MprA binding sites. Interestingly, Rv0081 represses Rv0081 and downstream determinants following activation of DosRS-DosT/DevRS-Rv2027c by nitric oxide, suggesting that expression of this locus is complex and subject to multiple levels of regulation. Based on this and other published information, a model is proposed detailing Rv0081-Rv0088 expression by these transcription factors within particular growth environments.
Project description: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.
Project description:The mechanisms utilized by Mycobacterium tuberculosis to establish, maintain, or reactivate from latent infection in the host are largely unknown but likely include genes that mediate adaptation to conditions encountered during persistence. Previously, a two-component signal transduction system, mprAB, was found to be required in M. tuberculosis for establishment and maintenance of persistent infection in a tissue- and stage-specific fashion. To begin to characterize the role of this system in M. tuberculosis physiology and virulence, a functional analysis of the mprA and mprB gene products was initiated. Here, evidence is presented demonstrating that sensor kinase MprB and response regulator MprA function as an intact signal-transducing pair in vitro and in vivo. Sensor kinase MprB can be autophosphorylated, can donate phosphate to MprA, and can act as a phospho-MprA phosphatase in vitro. Correspondingly, response regulator MprA can accept phosphate from MprB or from small phosphodonors including acetyl phosphate. Mutagenesis of residues His249 in MprB and Asp48 in MprA abolished the ability of these proteins to be phosphorylated in vitro. Introduction of these alleles into Mycobacterium bovis BCG attenuated virulence in macrophages in vivo. Together, these results support a role for the mprAB two-component system in M. tuberculosis physiology and pathogenesis. Characterization of two-component signal transduction systems will enhance our understanding of processes regulated by M. tuberculosis during acute and/or persistent infection in the host.
Project description:The ?-propeller gene Rv1057 of Mycobacterium tuberculosis is activated by envelope stress and was first characterized as a regulatory target of the TrcRS two-component system (TCS). Rv1057 expression is repressed by TrcRS, and the Rv1057 proximal promoter contains a TrcR binding site. In this study, we determined that Rv1057 is also directly regulated by MprAB, a TCS associated with envelope stress. Multiple potential MprA binding sites (MprA boxes) were identified in the 1 kb intergenic region upstream of Rv1057, and four sites were shown to bind MprA. Although MprA boxes were found in the proximal promoter, analyses suggest that MprA and TrcR do not compete for binding in this region. An MprAB-dependent, detergent-inducible transcriptional start point for Rv1057 was identified downstream of the MprA boxes, and a second TrcR binding site and small ORF of the 13E12 family were discovered in the distal promoter. MprAB was required for activation of Rv1057 during growth in macrophages and under detergent stress, and lacZ promoter constructs suggest the entire intergenic region is utilized during MprAB-dependent activation of Rv1057. These findings indicate that Rv1057 has an extensive and complex promoter, and provide evidence for coordinated regulation of stress response genes by TCSs.
Project description:In Mycobacterium tuberculosis, the sensor kinases DosT and DosS activate the transcriptional regulator DosR, resulting in the induction of the DosR regulon, which is important for anaerobic survival and perhaps latent infection. The individual and collective roles of these sensors have been postulated biochemically, but their roles in vivo have remained unclear. This work demonstrates distinct and additive roles for each sensor during anaerobic dormancy. Both sensors are necessary for wild-type levels of DosR regulon induction, and concomitantly, full induction of the regulon is required for wild-type anaerobic survival. In the anaerobic model, DosT plays an early role, responding to hypoxia. DosT then induces the regulon and with it DosS, which sustains and further induces the regulon. DosT then loses its functionality as oxygen becomes limited, and DosS alone maintains induction of the genes from that point forward. Thus, M. tuberculosis has evolved a system whereby it responds to hypoxic conditions in a stepwise fashion as it enters an anaerobic state.
Project description:DosS/DosR is a two-component regulatory system in which DosS, a heme-containing sensor also known as DevS, under certain conditions undergoes autophosphorylation and then transfers the phosphate to DosR, a DNA-binding protein that controls the entry of Mycobacterium tuberculosis and other mycobacteria into a latent, dormant state. DosT, a second sensor closely related to DosS, is present in M. tuberculosis and participates in the control of the dormancy response mediated by DosR. The binding of phosphorylated DosR to DNA initiates the expression of approximately fifty dormancy-linked genes. DosT is accepted to be a gas sensor that is activated in the ferrous state by the absence of an oxygen ligand or by the binding of NO or CO. DosS functions in a similar fashion as a gas sensor, but contradictory evidence has led to the suggestion that it also functions as a redox state sensor. This review focuses on the structure, biophysical properties, and function of the DosS/DosT heme sensors.
Project description:As part of our ongoing efforts to uncover the phenotypic consequences of genetic variability among clinical Mycobacterium tuberculosis isolates, we previously reported that isolates of the "East Asian" or "W/Beijing" lineage constitutively overexpress the coordinately regulated transcriptional program known as the DosR regulon under standard in vitro conditions. This phenotype distinguishes the W/Beijing lineage from all other M. tuberculosis lineages, which normally induce expression of this regulon only once exposed to low oxygen or nitric oxide, both of which result in inhibition of bacterial respiration and replication. Transcription of the DosR regulon is controlled through a two-component regulatory system comprising the transcription factor DosR and two possible cognate histidine sensor kinases, DosS and DosT. Through sequence analysis of a carefully selected set of isolates representing each of the major M. tuberculosis lineages, we describe herein a naturally occurring frameshift mutation in the gene encoding the DosT sensor kinase for isolates of the most recently evolved W/Beijing sublineages. Intriguingly, the occurrence of the frameshift mutation correlates precisely with the appearance of the constitutive DosR regulon phenotype displayed by the same "modern" W/Beijing strains. However, complementation studies have revealed that the mutation in dosT alone is not directly responsible for the constitutive DosR regulon phenotype. Our data serve to highlight the evolutionary pressure that exists among distinct M. tuberculosis lineages to maintain tight control over DosR regulon expression.
Project description:The genetic mechanisms mediating the adaptation of Mycobacterium tuberculosis within the host are poorly understood. The best-characterized regulatory systems in this organism include sigma factors and two-component signal transduction systems. mprAB is a two-component system required by M. tuberculosis for growth in vivo during the persistent stage of infection. In this report, we demonstrate that MprAB is stress responsive and regulates the expression of numerous stress-responsive genes in M. tuberculosis. With DNA microarrays and quantitative real-time reverse transcription-PCR, genes regulated by MprA in M. tuberculosis that included two stress-responsive sigma factors were identified. Response regulator MprA bound to conserved motifs in the upstream regions of both sigB and sigE in vitro and regulated the in vivo expression of sigB and sigE in M. tuberculosis. In addition, mprA itself was induced following exposure to stress, establishing a direct role for this regulatory system in stress response pathways of M. tuberculosis. Induction of mprA and sigE by MprA in response to stress was mediated through the cognate sensor kinase MprB and required expression of the extracytoplasmic loop domain. These results provide the first evidence that recognition of and adaptation to specific stress in M. tuberculosis are mediated through activation of a two-component signal transduction system that directly regulates the expression of stress-responsive determinants.
Project description:The DosR regulon, a set of 48 genes normally expressed in Mycobacterium tuberculosis under conditions that inhibit aerobic respiration, is controlled via the DosR-DosS/DosT two-component system. While the regulon requires induction in most M. tuberculosis isolates, for members of the Beijing lineage, its expression is uncoupled from the need for signaling. In our attempts to understand the mechanistic basis for this uncoupling in the Beijing background, we previously reported the identification of two synonymous single-nucleotide polymorphisms (SNPs) within the adjacent Rv3134c gene. In the present study, we have interrogated the impact of these SNPs on dosR expression in wild-type strains, as well as a range of dosR-dosS-dosT mutants, for both Beijing and non-Beijing M. tuberculosis backgrounds. In this manner, we have unequivocally determined that the C601T dosR promoter SNP is the sole requirement for the dramatic shift in the pattern of DosR regulon expression seen in this globally important lineage. Interestingly, we also show that DosT is completely nonfunctional within these strains. Thus, a complex series of evolutionary steps has led to the present-day Beijing DosR phenotype that, in turn, potentially confers a fitness advantage in the face of some form of host-associated selective pressure. IMPORTANCE:Mycobacterium tuberculosis strains of the Beijing lineage have been described as being of enhanced virulence compared to other lineages, and in certain regions, they are associated with the dramatic spread of multidrug-resistant tuberculosis (TB). In terms of trying to understand the functional basis for these broad epidemiological phenomena, it is interesting that, in contrast to the other major lineages, the Beijing strains all constitutively overexpress members of the DosR regulon. Here, we identify the mutational events that led to the evolution of this unique phenotype. In addition, our work highlights the fact that important phenotypic differences exist between distinct M. tuberculosis lineages, with the potential to impact the efficacy of diagnosis, vaccination, and treatment programs.
Project description:Mycobacterium tuberculosis (Mtb) must counter hypoxia within granulomas to persist. DosR, in concert with sensor kinases DosS and DosT, regulates the response to hypoxia. Yet Mtb lacking functional DosR colonize the lungs of C57Bl/6 mice, presumably owing to the lack of organized lesions with sufficient hypoxia in that model. We compared the phenotype of the ?-dosR, ?-dosS, and ?-dosT mutants to Mtb using C3HeB/FeJ mice, an alternate mouse model where lesions develop hypoxia. C3HeB/FeJ mice were infected via aerosol. The progression of infection was analyzed by tissue bacterial burden and histopathology. A measure of the comparative global immune responses was also analyzed. Although ?-dosR and ?-dosT grew comparably to wild-type Mtb, ?-dosS exhibited a significant defect in bacterial burden and pathology in vivo, accompanied by ablated proinflammatory response. ?-dosS retained the ability to induce DosR. The ?-dosS mutant was also attenuated in murine macrophages ex vivo, with evidence of reduced expression of the proinflammatory signature. Our results show that DosS, but not DosR and DosT, is required by Mtb to survive in C3HeB/FeJ mice. The attenuation of ?-dosS is not due to its inability to induce the DosR regulon, nor is it a result of the accumulation of hypoxia. That the in vivo growth restriction of ?-dosS could be mimicked ex vivo suggested sensitivity to macrophage oxidative burst. Anoxic caseous centers within tuberculosis lesions eventually progress to cavities. Our results provide greater insight into the molecular mechanisms of Mtb persistence within host lungs.