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The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence.

ABSTRACT: SignificanceTuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis, and its close pathogenic relative Mycobacterium marinum, initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane-bound compartments called phagosomes. Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed to the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.

SUBMITTER: Osman MM

PROVIDER: S-EPMC8931374 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence.

Osman Morwan M MM Shanahan Jonathan K JK Chu Frances F Takaki Kevin K KK Pinckert Malte L ML Pagán Antonio J AJ Brosch Roland R Conrad William H WH Ramakrishnan Lalita L

Proceedings of the National Academy of Sciences of the United States of America 20220310 11

SignificanceTuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, <i>Mycobacterium tuberculosis</i>, and its close pathogenic relative <i>Mycobacterium marinum</i>, initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane-bound compartments called phagosomes. Mycobacterium-induced damage of the phagosomal membranes is int ...[more]

PMID: 35271388

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Project description:CpnT, a NAD+ glycohydrolase, is the only known toxin that is secreted by Mycobacterium tuberculosis CpnT is composed of two domains; the C-terminal domain is the toxin, whereas the N-terminal domain is required for secretion. CpnT shows characteristics of type VII secretion (T7S) substrates, including a predicted helix-turn-helix domain followed by a secretion motif (YxxxE). Disruption of this motif indeed abolished CpnT secretion. By analyzing different mutants, we established that CpnT is specifically secreted by the ESX-5 system in Mycobacterium marinum under axenic conditions and during macrophage infection. Surprisingly, intracellular secretion of CpnT was also dependent on both ESX-1 and ESX-4. These secretion defects could be partially rescued by coinfection with wild-type bacteria, indicating that secreted effectors are involved in this process. In summary, our data reveal that three different type VII secretion systems have to be functional in order to observe intracellular secretion of the toxin CpnT.IMPORTANCE For decades, it was believed that the intracellular pathogen M. tuberculosis does not possess toxins. Only fairly recently it was discovered that CpnT is a potent secreted toxin of M. tuberculosis, causing necrotic cell death in host cells. However, until now the secretion pathway remained unknown. In our study, we were able to identify CpnT as a substrate of the mycobacterial type VII secretion system. Pathogenic mycobacteria have up to five different type VII secretion systems, called ESX-1 to ESX-5, which play distinct roles for the pathogen during growth or infection. We were able to elucidate that CpnT is exclusively secreted by the ESX-5 system in bacterial culture. However, to our surprise we discovered that, during infection studies, CpnT secretion relies on intact ESX-1, ESX-4, and ESX-5 systems. We elucidate for the first time the intertwined interplay of three different and independent secretion systems to secrete one substrate during infection.

Project description:N-terminal protein acetylation is a ubiquitous post-translational modification that impacts diverse cellular processes in higher organisms. Bacterial proteins are also N-terminally acetylated, but the mechanisms and consequences of this modification in bacteria are poorly understood. The major virulence factor EsxA (ESAT-6, early secreted antigen, 6 kDa) was one of the first N-terminally acetylated proteins identified in bacteria. EsxA is conserved in mycobacterial pathogens, including Mycobacterium tuberculosis and Mycobacterium marinum, a non-tubercular mycobacterial species that causes tuberculosis-like disease in ectotherms. However, the enzyme responsible for EsxA N-terminal acetylation has been elusive. Here, we used genetics, molecular biology, and mass-spectroscopy-based proteomics to demonstrate that MMAR_1839 (renamed Emp1, ESX-1 modifying protein, 1) is the putative N-acetyltransferase (NAT) solely responsible for EsxA acetylation in M. marinum. We demonstrated that ERD_3144, the orthologous gene in M. tuberculosis Erdman, is functionally equivalent to Emp1. We previously quantified widespread N-terminal protein acetylation in pathogenic mycobacteria (C. R. Thompson, M. M. Champion, and P. A. Champion, J Proteome Res 17:3246-3258, 2018, https:// doi: 10.1021/acs.jproteome.8b00373). We identified at least 22 additional proteins that require Emp1 for acetylation, demonstrating that this putative NAT is not dedicated to EsxA. Finally, we showed that loss of emp1 did not prevent phagosomal escape but resulted in a significant reduction in macrophage cytolysis by and cell-to-cell spread of M. marinum during infection. Collectively, this study identified a NAT required for N-terminal acetylation and pathogenesis in Mycobacterium. IMPORTANCE N-terminal acetylation is a protein modification that broadly impacts basic cellular function and disease in higher organisms. Although bacterial proteins are N-terminally acetylated, little is understood how N-terminal acetylation impacts bacterial physiology and pathogenesis. Mycobacterial pathogens cause acute and chronic disease in humans and in animals. Approximately 15% of mycobacterial proteins are N-terminally acetylated, but the responsible enzymes are largely unknown. We identified a conserved mycobacterial protein required for the N-terminal acetylation of 23 mycobacterial proteins including the EsxA virulence factor. Loss of this enzyme from M. marinum reduced macrophage killing and spread of M. marinum to new host cells. Defining the acetyltransferases responsible for the N-terminal protein acetylation of essential virulence factors could lead to new targets for therapeutics against mycobacteria.

Dataset Information

The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence.

Publications

The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence.

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