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

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.

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

Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation

Andrej Benjak, Stewart Cole, Gaëlle Séraphine Kolly, Andréanne Lupien, Nina Theres Odermatt, Claudia Sala, Anthony Vocat

A handful of nucleoid-associated proteins (NAPs) regulate the vast majority of genes in a bacterial cell. H-NS, the histone-like nucleoid-structuring protein, is one of these NAPs and protects Escherichia coli from foreign gene expression. Though lacking any sequence similarity with E. coli H-NS, Rv3852 was annotated as the H-NS ortholog in Mycobacterium tuberculosis, as it resembles human histone H1. The role of Rv3852 was thoroughly investigated by immunoblotting, subcellular localization, construction of an unmarked rv3852 deletion in the M. tuberculosis genome, and subsequent analysis of the resulting Delta rv3852 strain. We found that Rv3852 was predominantly present in the logarithmic growth phase with a decrease in protein abundance in stationary phase. Furthermore, it was strongly associated with the cell membrane and not detected in the cytosolic fraction, nor was it secreted. The Delta rv3852 strain displayed no growth defect or morphological abnormalities. Quantitative measurement of nucleoid localization in the Delta rv3852 mutant strain compared to that in the parental H37Rv strain showed no difference in nucleoid position or spread. Infection of macrophages as well as severe combined immunodeficient (SCID) mice demonstrated that loss of Rv3852 had no detected influence on the virulence of M. tuberculosis. We thus conclude that M. tuberculosis Rv3852 is not involved in pathogenesis and is not a typical NAP. The existence of an as yet undiscovered Rv3852 ortholog cannot be excluded, although this role is likely played by the well-characterized Lsr2 protein. IMPORTANCE Mycobacterium tuberculosis is the causative agent of the lung infection tuberculosis, claiming more than 1.5 million lives each year. To understand the mechanisms of latent infection, where M. tuberculosis can stay dormant inside the human host, we require deeper knowledge of the basic biology and of the regulatory networks. In our work, we show that Rv3852, previously annotated as H-NS, is not a typical nucleoid-associated protein (NAP) as expected from its initial annotation. Rv3852 from M. tuberculosis has neither influence on nucleoid shape or compaction nor a role in virulence. Our findings reduce the repertoire of identified nucleoid-associated proteins in M. tuberculosis to four transcription regulators and underline the importance of genetic studies to assign a function to bacterial genes.

Amer Soc Microbiology2017

Functional Characterization of Nucleoid Associated Proteins Acting as Global Transcription Factors in Mycobacterium tuberculosis

Nina Theres Odermatt

The fatal lung disease tuberculosis is caused by the airborne Mycobacterium tuberculosis, a versatile pathogen adapted to rapidly changing environments. Instead of being eradicated by phagocytic cells of its human host, bacilli tune macrophages to support their own growth and even mask their presence from the immune system for several decades. Rapid adjustment of gene expression is critical for bacterial survival and heavily relies on nucleoid-associated proteins (NAPs). NAPs contribute to active DNA management by altering the chromosomal topology through bending, bridging and looping the DNA. These conformational changes can bring distant genetic loci into close spatial proximity or influence DNA supercoiling and therefore accessibility of the transcription machinery. Apart from their architectural role, NAPs moreover act as global transcription factors by direct regulation of numerous genes. In M. tuberculosis, five proteins were assigned a role as NAP. Among these, EspR, HupB and Lsr2 are crucial not only for virulence but also for cellular metabolism. This thesis focuses on the NAPs mIHF and H-NS with the objective of determining their function and target regulon. Both proteins were investigated by means of genetic manipulation, phenotype assessment and structural studies, and the efficiency of a potential new anti-tuberculosis drug acting on Lsr2 was assessed. Chrysomycin, described as specific inhibitor of Lsr2-DNA complex formation, was found to intercalate into the DNA. The resulting toxic effect on both its target M. tuberculosis as well as on eukaryotic cells rendered further development of the compound as an anti-tuberculosis drug futile. We demonstrated that Rv3852, formerly annotated as H-NS, does not act as a NAP. Deletion of the rv3852 gene had no effect on the in vitro phenotype of M. tuberculosis, did not alter nucleoid spread nor position and had no influence on virulence in mice. The mIHF protein on the other hand is not only essential for active bacterial growth, but also indispensable for survival. Generation of a conditional knockdown mutant showed that depletion of mIHF led to elongated cells devoid of septa with abnormal DNA localization and finally to cell death. The target regulon of mIHF was thoroughly studied by mapping its binding sites on the bacterial genome and by identifying genes that were differentially expressed upon depletion of the protein. We found that mIHF has a strong effect on virulence gene expression and, similar to EspR, possesses a major binding site upstream of one of the main virulence factor operons espACD. Analysis of the transcriptional response revealed that mIHF is further involved in the bacterial response to the hostâs immune system, including control of nutrient pathways as well as global protein and nucleic acid synthesis. To define how mIHF interacts with DNA and influences its 3D organisation, the protein structure of mIHF was determined by nuclear magnetic resonance spectroscopy. Binding of mIHF introduced left-hand loops into linear as well as supercoiled DNA substrates, therefore unwinding condensed DNA. We identified two DNA binding domains in mIHF and showed that its stability increased substantially upon DNA binding. All together, the findings of this thesis contribute to a better understanding of the complex gene regulatory network of M. tuberculosis, advancing the knowledge necessary to eventually defeat tuberculosis, a disease that has plagued humanity for millennia.

EPFL2017