EspR-dependent ESAT-6 Protein Secretion of Mycobacterium tuberculosis Requires the Presence of Virulence Regulator PhoP

Abstract

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...

Metabolic switching ofMycobacterium tuberculosisduring hypoxia is controlled by the virulence regulator PhoP

“…Considering the above results, recently-reported PhoP-DosR interaction (23) emerged as a possible explanation to account for the regulation of hypoxia-inducible genes. To examine this, whole cell lysate of ΔphoP expressing His-tagged PhoP, as described previously (35), was incubated with Ni-NTA, and proteins were eluted from Ni-NTA column using imidazole (Fig. 4B).…”

“…While these results suggest convergence of two different types of signalling modules for a common function, along the similar line DosR interacts with the house keeping sigma factor SigA for bacterial survival under dormancy (17). Recently, we have shown that PhoP interacts with nucleoid-associated protein EspR to control ESAT-6 secretion (35). In this study, we show that N-domains which get phosphorylated for activation of respective regulators, interact to each other (Fig.…”

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

“…PhoP is part of a two-component system that regulates several virulence pathways in M. tuberculosis, including biosynthetic genes of the M. tuberculosis-specific lipids, sulfolipid, DAT, and PAT (103). In addition to regulating lipid biosynthesis genes, PhoP also regulates whiB6 and works directly with EspR, and both regulate ESX-1 gene expression (96,104,105). Therefore, under many of the conditions in which cell envelope remodeling may be occurring or where cell wall genes are regulated, ESX genes are also regulated.…”

“…Alternatively, the ESX substrates may direct their own transit across the MOM, by either forming the apparatus spanning the MOM or by transiting the MOM directly (48,104,(157)(158)(159). Notably, the EspC substrate of the ESX-1 system in M. tuberculosis (160) was recently reported by Lou et al to self-assemble into filaments in vitro.…”

The role of two‐component systems in the physiology of Mycobacterium tuberculosis

“…phoP deletion mutants of M. tuberculosis are attenuated for growth in THP‐1 macrophages as well as in murine bone marrow‐derived macrophages . PhoP/PhoR regulates espB and espR which are essential for ESX‐1 mediated secretion of ESAT‐6. In a recent study, it has been demonstrated that PhoP and EspR interact directly with each other and are recruited to the espACD promoter.…”