Anticytolytic Screen Identifies Inhibitors of Mycobacterial Virulence Protein Secretion

Mycobacterium tuberculosis (Mtb) requires protein secretion systems like ESX-1 for intracellular survival and virulence. The major virulence determinant and ESX-1 substrate, EsxA, arrests phagosome maturation and lyses cell membranes, resulting in tissue damage and necrosis that promotes pathogen spread. To identify inhibitors of Mtb protein secretion, we developed a fibroblast survival assay exploiting this phenotype and selected molecules that protect host cells from Mtb-induced lysis without being bactericidal in vitro. Hit compounds blocked EsxA secretion and promoted phagosome maturation in macrophages, thus reducing bacterial loads. Target identification studies led to the discovery of BTP15, a benzothiophene inhibitor of the histidine kinase MprB that indirectly regulates ESX-1, and BBH7, a benzyloxybenzylidene-hydrazine compound. BBH7 affects Mtb metal-ion homeostasis and revealed zinc stress as an activating signal for EsxA secretion. This screening approach extends the target spectrum of small molecule libraries and will help tackle the mounting problem of antibiotic-resistant mycobacteria.

Structural and functional characterization of the mycobacterial ESX-1 secretion system

Ye Lou

Human tuberculosis (TB) is a frequently fatal lung disease mostly caused by Mycobacterium tuberculosis (M. tb), a slow-growing intracellular pathogen. M. tb utilizes the ESX-1 secretion system, classified as type VII, to export the virulence factors needed for host infection and pathogenesis. A functional ESX-1 pathway largely involves the proteins encoded by two gene clusters, esx-1 and espACD. These proteins are diverse in functions, such as cytolysis, immunogenicity, pore forming, stabilization and energization. Although many of the ESX-1 associated proteins were characterized individually, the overall mechanism of ESX-1 secretion is still far from clear. The main objective of my thesis is to better understand the structure and function of the ESX-1 secretion system in M. tb. To achieve this, an integrated approach which combines biochemistry, biophysics and genetics was applied. The experimental results are organized into three parts, in which I investigated EspC, EccCb1 and the effect of calcium on ESX-1 secretion. Firstly, the focus of this thesis is EspC, a secreted protein encoded in the espACD operon which is not linked to the esx-1 locus, but mediates ESX-1-associated virulence. Based on what I observed from a series of experiments, we propose a novel working model of the ESX-1 apparatus, in which EspC interacts with EspA in the cytosol and then translocates across the membrane to assemble into a channel in the outer membrane and spans the capsular layer of M. tb. This model could explain the mechanism by which the major virulence factors EsxA and EsxB (also named ESAT-6 and CFP-10) rely on EspC for secretion. Moreover, EspC is a potential outer membrane pore protein which has not been previously identified in M. tb. Secondly, the putative ATPase EccCb1 is another protein that was studied. EccCb1 is predicted as an ATPase that targets the EsxA/EsxB heterodimer during translocation. EccCb1 was overexpressed and purified in the form of a NusA- EccCb1 fusion protein due to its low stability. The protein was characterized as a hexamer with an ATPase activity. This result confirms that EccCb1 belongs to the FtsK/SpoIIIE ATPase family. Thirdly, we observed that ESX-1 secretion can be inhibited by a high concentration of calcium (~500 ÎŒM) in the culture medium. Moreover, bacterial persistence, rather than growth rate, decreased with the elevated calcium. RNA- seq was performed to evaluate the changes of global gene expression of M. tb in the presence of high calcium levels. Surprisingly, calcium dramatically repressed hypoxia response genes in the DevRS regulon, leading to reduced persistence. I also include a section that describes the additional work I was involved with, in which we investigated the activation of cGAS-dependent host response by ESX-1. Taken together, these findings provide new insights into the structural and functional aspects of ESX-1 secretion system, and will contribute to discovery of ESX-1 inhibitors with potential applications in TB therapy.

EPFL2016

The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis

Andrej Benjak, Stewart Cole, Jacques Rougemont, Claudia Sala, Swapna Uplekar

The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host.

Public Library of Science2014

Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence

Stewart Cole

The dedicated secretion system ESX-1 of Mycobacterium tuberculosis encoded by the extended RD1 region (extRD1) assures export of the ESAT-6 protein and its partner, the 10-kDa culture filtrate protein CFP-10, and is missing from the vaccine strains M. bovis BCG and M. microti. Here, we systematically investigated the involvement of each individual ESX-1 gene in the secretion of both antigens, specific immunogenicity, and virulence. ESX-1-complemented BCG and M. microti strains were more efficiently engulfed by bone-marrow-derived macrophages than controls, and this may account for the enhanced in vivo growth of ESX-1-carrying strains. Inactivation of gene pe35 (Rv3872) impaired expression of CFP-10 and ESAT-6, suggesting a role in regulation. Genes Rv3868, Rv3869, Rv3870, Rv3871, and Rv3877 encoding an ATP-dependent chaperone and translocon were essential for secretion of ESAT-6 and CFP-10 in contrast to ppe68 Rv3873 and Rv3876, whose inactivation did not impair secretion of ESAT-6. A strict correlation was found between ESAT-6 export and the generation of ESAT-6 specific T-cell responses in mice. Furthermore, ESAT-6 secretion and specific immunogenicity were almost always correlated with enhanced virulence in the SCID mouse model. Only loss of Rv3865 and part of Rv3866 did not affect ESAT-6 secretion or immunogenicity but led to attenuation. This suggests that Rv3865/66 represent a new virulence factor that is independent from ESAT-6 secretion. The present study has allowed us to identify new aspects of the extRD1 region of M. tuberculosis and to explore its role in the pathogenesis of tuberculosis.