EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis

The function of EspI, a 70kDa protein in Mycobacterium tuberculosis, has remained unclear. Although EspI is encoded by a gene within the esx-1 locus, in this study we clarify previous conflicting results and show that EspI is not essential for ESX-1-mediated secretion or virulence in M. tuberculosis. We also provide evidence that reduction of cellular ATP levels in wild-type M. tuberculosis using the drug bedaquiline completely blocks ESX-1-mediated secretion. Remarkably, M. tuberculosis lacking EspI fails to exhibit this phenotype. Furthermore, mutagenesis of a highly conserved ATP-binding motif in EspI renders M. tuberculosis incapable of shutting down ESX-1-mediated secretion during ATP depletion. Collectively these results show that M. tuberculosisEspI negatively regulates the ESX-1 secretion system in response to low cellular ATP levels and this function requires the ATP-binding motif. In light of our results the potential significance of EspI in ESX-1 function during latent tuberculosis infection and reactivation is also discussed.

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

Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis

Stewart Cole

Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis.

2008

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