ABSTRACT
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: EccB1, EccCa1, EccCb1, EccD1, and EccE1 In this study, we have characterized the membrane component EccE1 and found that deletion of eccE1 lowers the levels of EccB1, EccCa1, and EccD1, thereby abolishing ESX-1 secretion and attenuating M. tuberculosisex vivo Surprisingly, secretion of EspB was not affected by loss of EccE1 Furthermore, EccE1 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 EccE1 and its localization in M. tuberculosisIMPORTANCE 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 EccE1, 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 EccE1 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.
Subject(s)
Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Mycobacterium tuberculosis/physiology , Tuberculosis/microbiology , Gene-Environment Interaction , Genome, Bacterial , Host-Pathogen Interactions , Humans , Membrane Proteins , Metals/metabolism , Mutation , Protein Stability , Protein Transport , Proteomics , Stress, Physiological , VirulenceABSTRACT
The ESX-1, type VII, secretion system represents the major virulence determinant of Mycobacterium tuberculosis, one of the most successful intracellular pathogens. Here, by combining genetic and high-throughput approaches, we show that EspL, a protein of 115 amino acids, is essential for mediating ESX-1-dependent virulence and for stabilization of EspE, EspF and EspH protein levels. Indeed, an espL knock-out mutant was unable to replicate intracellularly, secrete ESX-1 substrates or stimulate innate cytokine production. Moreover, proteomic studies detected greatly reduced amounts of EspE, EspF and EspH in the espL mutant as compared to the wild type strain, suggesting a role for EspL as a chaperone. The latter conclusion was further supported by discovering that EspL interacts with EspD, which was previously demonstrated to stabilize the ESX-1 substrates and effector proteins, EspA and EspC. Loss of EspL also leads to downregulation in M. tuberculosis of WhiB6, a redox-sensitive transcriptional activator of ESX-1 genes. Overall, our data highlight the importance of a so-far overlooked, though conserved, component of the ESX-1 secretion system and begin to delineate the role played by EspE, EspF and EspH in virulence and host-pathogen interaction.
