Structural studies suggest a peptidoglycan hydrolase function for the Mycobacterium tuberculosis Tat-secreted protein Rv2525c

Among the few proteins shown to be secreted by the Tat system in Mycobacterium tuberculosis, Rv2525c is of particular interest, since its gene is conserved in the minimal genome of Mycobacterium leprae. Previous evidence linked this protein to cell wall metabolism and sensitivity to beta-lactams. We describe here the crystal structure of Rv2525c that shows a TIM barrel-like fold characteristic of glycoside hydrolases of the GH25 family, which includes prokaryotic and phage-encoded peptidoglycan hydrolases. Structural comparison with other members of this family combined with substrate docking suggest that, although the 'neighbouring group' catalytic mechanism proposed for this family still appears as the most plausible, the identity of residues involved in catalysis in GH25 hydrolases might need to be revised. (C) 2014 Elsevier Inc. All rights reserved.

The crystal structure of Rv0813c from Mycobacterium tuberculosis reveals a new family of fatty acid-binding protein-like proteins in bacteria

Stewart Cole

The gene Rv0813c from Mycobacterium tuberculosis, which codes for a hypothetical protein of unknown function, is conserved within the order Actinomycetales but absent elsewhere. The crystal structure of Rv0813c reveals a new family of proteins that resemble the fatty acid-binding proteins (FABPs) found in eukaryotes. Rv0813c adopts the 10-stranded beta-barrel fold typical of FABPs but lacks the double-helix insert that covers the entry to the binding site in the eukaryotic proteins. The barrel encloses a deep cavity, at the bottom of which a small cyclic ligand was found to bind to the hydroxyl group of Tyr192. This residue is part of a conserved Arg-X-Tyr motif much like the triad that binds the carboxylate group of fatty acids in FABPs. Most of the residues forming the internal surface of the cavity are conserved in homologous protein sequences found in CG-rich prokaryotes, strongly suggesting that Rv0813c is a member of a new family of bacterial FABP-like proteins that may have roles in the recognition, transport, and/or storage of small molecules in the bacterial cytosol.

2006

A protein linkage map of the ESAT-6 secretion system 1 (ESX-1) of Mycobacterium tuberculosis

Stewart Cole

Tuberculosis is a chronic infectious disease caused by bacteria of the Mycobacterium tuberculosis complex. One of the major contributors to virulence and intercellular spread of M. tuberculosis is the ESAT-6 secretion system 1 (ESX-1) that has been lost by the live vaccines Mycobacterium bovis BCG (Bacille Calmette Guérin) and Mycobacterium microti as a result of independent deletions. ESX-1 consists of at least 10 genes (Rv3868-Rv3877) encoding the T-cell antigens ESAT-6 and CFP-10 as well as AAA-ATPases, chaperones, and membrane proteins which probably form a novel export system. To better understand the mode of action of the ESX-1 proteins, as a prelude to drug development, we examined systematically the interactions between the various proteins using the two-hybrid system in Saccharomyces cerevisiae. Interestingly, ESAT-6 and CFP-10 formed both hetero- and homodimers. Moreover, Rv3866, Rv3868, and CFP-10 interacted with Rv3873 which also homodimerized. The data were summarized in a protein linkage map that is consistent with the model for the secretion apparatus and can be used as a basis to identify inhibitors of specific interactions.

2007

Polarly Localized EccE(1) Is Required for ESX-1 Function and Stabilization of ESX-1 Membrane Proteins in Mycobacterium tuberculosis

Stewart Cole, Jérémie Piton, Claudia Sala, Paloma Julia Soler Arnedo, Ming Zhang

Mycobacterium tuberculosis is a slow-growing intracellular bacterium with the ability to induce host cell death and persist indefinitely in the human body. This pathogen uses the specialized ESX-1 secretion system to secrete virulence factors and potent immunogenic effectors required for disease progression. ESX-1 is a multisubunit apparatus with a membrane complex that is predicted to form a channel in the cytoplasmic membrane. In M. tuberculosis this complex is composed of five membrane proteins: EccB(1), EccCa(1), EccCb(1), EccD(1), and EccE(1). In this study, we have characterized the membrane component EccE(1) and found that deletion of eccE(1) lowers the levels of EccB(1), EccCa(1), and EccD(1), thereby abolishing ESX-1 secretion and attenuating M. tuberculosis ex vivo. Surprisingly, secretion of EspB was not affected by loss of EccE(1). Furthermore, EccE(1) was found to be a membrane- and cell wall-associated protein that needs the presence of other ESX-1 components to assemble into a stable complex at the poles of M. tuberculosis. Overall, this investigation provides new insights into the role of EccE(1) and its localization in M. tuberculosis. IMPORTANCE Tuberculosis (TB), the world's leading cause of death of humans from an infectious disease, is caused by the intracellular bacterium Mycobacterium tuberculosis. The development of successful strategies to control TB requires better understanding of the complex interactions between the pathogen and the human host. We investigated the contribution of EccE(1), a membrane protein, to the function of the ESX-1 secretion system, the major virulence determinant of M. tuberculosis. By combining genetic analysis of selected mutants with eukaryotic cell biology and proteomics, we demonstrate that EccE(1) is critical for ESX-1 function, secretion of effector proteins, and pathogenesis. Our research improves knowledge of the molecular basis of M. tuberculosis virulence and enhances our understanding of pathogenesis.

AMER SOC MICROBIOLOGY2020

Polarly Localized EccE(1) Is Required for ESX-1 Function and Stabilization of ESX-1 Membrane Proteins in Mycobacterium tuberculosis

Stewart Cole, Jérémie Piton, Claudia Sala, Paloma Julia Soler Arnedo, Ming Zhang

AMER SOC MICROBIOLOGY2020