Atypical DNA recognition mechanism used by the EspR virulence regulator of Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis requires the ESX-1 secretion system for full virulence. EspR plays a key role in ESX-1 regulation via direct binding and transcriptional activation of the espACD operon. Here, we describe the crystal structures of EspR, a C-terminally truncated form, EspR Delta 10, as well as an EspR-DNA complex. EspR forms a dimer with each monomer containing an N-terminal helix-turn-helix DNA binding motif and an atypical C-terminal dimerization domain. Structural studies combined with footprinting experiments, atomic force microscopy and molecular dynamic simulations allow us to propose a model in which a dimer of EspR dimers is the minimal functional unit with two subunits binding two consecutive major grooves. The other two DNA binding domains are thus free to form higher-order oligomers and to bridge distant DNA sites in a cooperative way. These features are reminiscent of nucleoid-associated proteins and suggest a more general regulatory role for EspR than was previously suspected.

Functional Dissection of Intersubunit Interactions in the EspR Virulence Regulator of Mycobacterium tuberculosis

Benjamin Blasco, Stewart Cole, Matteo Dal Peraro, Giovanni Dietler, Aleksandre Japaridze, Florence Pojer, Marco Stenta, Basile Isidore Martin Wicky

The nucleoid-associated protein EspR, a chromosome organizer, has pleiotropic effects on expression of genes associated with cell wall function and pathogenesis in Mycobacterium tuberculosis. In particular, EspR binds to several sites upstream of the espACD locus to promote its expression, thereby ensuring full function of the ESX-1 secretion system, a major virulence determinant. The N terminus of EspR contains the helix-turn-helix DNA-binding domain, whereas the C-terminal dimerization domain harbors residues involved in intersubunit interactions. While direct binding to DNA appears to be mediated by an EspR dimer-of-dimers, where two helix-turn-helix motifs remain free for long-range interactions, the mechanism of EspR higher-order organization and its impact on chromosome structure and gene expression are not understood. To investigate these processes, we identified seven amino acid residues using molecular dynamics and replaced them with Ala in order to probe interactions at either the dimer or the dimer-of-dimers interfaces. Arg70, Lys72, and Arg101 were important for protein stability and optimal DNA-binding activity. Moreover, the Arg70 mutant showed decreased dimerization in a mycobacterial two-hybrid system. To correlate these defects with higher-order organization and transcriptional activity, we used atomic force microscopy to observe different EspR mutant proteins in complex with the espACD promoter region. In addition, complementation of an M. tuberculosis espR knockout mutant was performed to measure their impact on EspA expression. Our results pinpoint key residues required for EspR function at the dimer (Arg70) and the dimer-of-dimers (Lys72) interface and demonstrate that EspR dimerization and higher-order oligomerization modulate espACD transcriptional activity and hence pathogenesis.

Amer Soc Microbiology2014

Structural and DNA binding properties of mycobacterial integration host factor mIHF

Luciano Andres Abriata, Stewart Cole, Giovanni Dietler, David Lyndon Emsley, Aleksandre Japaridze, Nina Theres Odermatt, Jérémie Piton, Rajkumar Singh

In bacteria, nucleoid associated proteins (NAPs) take part in active chromosome organization by supercoil management, three-dimensional DNA looping and direct transcriptional control. Mycobacterial integration host factor (mIHF, rv1388) is a NAP restricted to Actinobacteria and essential for survival of the human pathogen Mycobacterium tuberculosis. We show in vitro that DNA binding by mIHF strongly stabilizes the protein and increases its melting temperature. The structure obtained by Nuclear Magnetic Resonance (NMR) spectroscopy characterizes mIHF as a globular protein with a protruding alpha helix and a disordered N-terminus, similar to Streptomyces coelicolor IHF (sIHF). NMR revealed no residues of high flexibility, suggesting that mIHF is a rigid protein overall that does not undergo structural rearrangements. We show that mIHF only binds to double stranded DNA in solution, through two DNA binding sites (DBSs) similar to those identified in the X-ray structure of sIHF. According to Atomic Force Microscopy, mIHF is able to introduce left-handed loops of ca. 100 nm size (similar to 300 bp) in supercoiled cosmids, thereby unwinding and relaxing the DNA.

2020

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.