The ability to sense acid stress and mount an appropriate adaptive response by Mycobacterium tuberculosis, which adapts a long-term residence in the macrophage phagosome, remains one of the critical features that defines mycobacterial physiology and its intracellular location. To understand the mechanistic basis of adaptation of the intracellular pathogen, we studied global regulation of M. tuberculosis gene expression in response to acid stress. Although recent studies indicate a role for the virulenceassociated phoP locus in pH-driven adaptation, in this study, we discovered a strikingly novel regulatory mechanism, which controls acid-stress homeostasis. Using mycobacterial protein fragment complementation and in vitro interaction analyses, we demonstrate that PhoP interacts with acidinducible extracytoplasmic SigE (one of the 13 M. tuberculosis sigma factors) to regulate a complex transcriptional program. Based on these results, we propose a model to suggest that PhoP-SigE interaction represents a major requirement for the global acid stress response, absence of which leads to strongly reduced survival of the bacilli under acidic pH conditions. These results account for the significant growth attenuation of the phoP mutant in both cellular and animal models, and unravel the underlying global mechanism of how PhoP induces an adaptive program in response to acid stress.Regulation of acid-stress response of M. tuberculosis 401 Additional supporting information may be found in the online version of this article at the publisher's web-site.Regulation of acid-stress response of M. tuberculosis 411

Attenuation of Mycobacterium bovis BCG strain is related to the loss of the RD1-encoded ESX-1 secretion system. The ESX-1 system secretes virulence factor ESAT-6 that plays a critical role in modulation of the host immune system, which is essential for establishment of a productive infection. Previous studies suggest that among the reasons for attenuation of Mycobacterium tuberculosis H37Ra is a mutation in the phoP gene that interferes with the ESX-1 secretion system and inhibits secretion of ESAT-6. Here, we identify a totally different and distinct regulatory mechanism involving PhoP and transcription regulator EspR on transcriptional control of the espACD operon, which is required for ESX-1-dependent ESAT-6 secretion. Although both of these regulators are capable of influencing espACD expression, we show that activation of espACD requires direct recruitment of both PhoP and EspR at the espACD promoter. The most fundamental insights are derived from the inhibition of EspR binding at the espACD regulatory region of the phoP mutant strain because of PhoP-EspR protein-protein interactions. Based on these results, a model is proposed suggesting how PhoP and EspR protein-protein interactions contribute to activation of espACD expression and, in turn, control ESAT-6 secretion, an essential pathogenic determinant of M. tuberculosis. Together, these results have significant implications on the mechanism of virulence regulation of M. tuberculosis.Mycobacterium tuberculosis uses the ESX-1 secretion system to transport virulence factors in host cells (1-5). ESX-1 secretion system is encoded by the genes of the esx-1 locus, which is highly conserved in members of the M. tuberculosis complex and in other pathogenic mycobacteria (5-9). The best known ESX-1 substrates, the secreted proteins EsxA (ESAT-6) and EsxB (CFP10), have been implicated in the majority of the ESX-1-dependent modulations of host cell defense (10 -19) and therefore are considered to be essential for virulence (12)(13)(14). Consistently, deletion of the esx-1 locus abrogates ESX-1-dependent secretion and strongly attenuates M. tuberculosis (15,20). Notably, the genes encoding esx-1, located within the M. tuberculosis RD1 locus, are absent in the attenuated Mycobacterium bovis BCG and the potential vaccine strain Mycobacterium microti (15,17). However, a chromosomally unlinked non-RD1 locus (Rv3616c-Rv3615c-Rv3614c, also designated as espA, espC, and espD) is essential for ESX-1 function (13, 21). In fact, EspA and EspC proteins themselves are substrates of the ESX-1 secretion system, thus constituting mutually dependent secretion of substrates, a notable feature of this secretion system. More recently, EspA of the tubercle bacilli has been implicated as a critical mediator of bacterial cell wall integrity and ESX-1-dependent virulence regulation (22).ESX-1 represents the first and the most characterized member of the ESX secretion family. Although intracellular concentrations of ESAT-6 are similar in both virulent M. tuberculosis H37Rv and the attenuated...

For centuries, hydrogen sulfide (H2S) was considered primarily as a poisonous gas and environmental hazard. However, with the discovery of prokaryotic and eukaryotic enzymes for H2S production, breakdown, and utilization, H2S has emerged as an important signaling molecule in a wide range of physiological and pathological processes. Hence, H2S is considered a gasotransmitter along with nitric oxide (•NO) and carbon monoxide (CO). Surprisingly, despite having overlapping functions with •NO and CO, the role of host H2S in microbial pathogenesis is understudied and represents a gap in our knowledge. Given the numerous reports that followed the discovery of •NO and CO and their respective roles in microbial pathogenesis, we anticipate a rapid increase in studies that further define the importance of H2S in microbial pathogenesis, which may lead to new virulence paradigms. Therefore, this review provides an overview of sulfide chemistry, enzymatic production of H2S, and the importance of H2S in metabolism and immunity in response to microbial pathogens. We then describe our current understanding of the role of host-derived H2S in tuberculosis (TB) disease, including its influences on host immunity and bioenergetics, and on Mycobacterium tuberculosis (Mtb) growth and survival. Finally, this review discusses the utility of H2S-donor compounds, inhibitors of H2S-producing enzymes, and their potential clinical significance.

A hallmark feature of Mycobacterium tuberculosis pathogenesis lies in the ability of the pathogen to survive within macrophages under a stressful environment. Thus, coordinated regulation of stress proteins is critically important for an effective adaptive response of M. tuberculosis, the failure of which results in elevated immune recognition of the tubercle bacilli with reduced survival during chronic infections. Here, we show that virulence regulator PhoP impacts the global regulation of heat shock proteins, which protect M. tuberculosis against stress generated by macrophages during infection. Our results identify that in addition to classical DNAprotein interactions, newly discovered protein-protein interactions control complex mechanisms of expression of heat shock proteins, an essential pathogenic determinant of M. tuberculosis. While the C-terminal domain of PhoP binds to its target promoters, the N-terminal domain of the regulator interacts with the C-terminal end of the heat shock repressors. Remarkably, our findings delineate a regulatory pathway which involves three major transcription factors, PhoP, HspR, and HrcA, that control in vivo recruitment of the regulators within the target genes and regulate stressspecific expression of heat shock proteins via protein-protein interactions. The results have implications on the mechanism of regulation of PhoP-dependent stress response in M. tuberculosis. IMPORTANCE The regulation of heat shock proteins which protect M. tuberculosis against stress generated by macrophages during infection is poorly understood. In this study, we show that PhoP, a virulence regulator of the tubercle bacilli, controls heat shock-responsive genes, an essential pathogenic determinant of M. tuberculosis. Our results unravel that in addition to classical DNA-protein interactions, complex mechanisms of regulation of heat shock-responsive genes occur through multiple protein-protein interactions. Together, these findings delineate a fundamental regulatory pathway where transcription factors PhoP, HspR, and HrcA interact with each other to control stress-specific expression of heat shock proteins.