An N-acetyltransferase required for EsxA N-terminal protein acetylation and virulence in Mycobacterium marinum.

Abstract

N-terminal protein acetylation is a ubiquitous post-translational modification that broadly 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. 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(9): 3246-3258, 2018, https://doi:10.1021/acs.jproteome.8b00373). The major virulence factor EsxA (ESAT-6, Early secreted antigen, 6kDa) 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, 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-acetyl transferase (NAT) solely responsible for EsxA acetylation in Mycobacterium marinum. We demonstrated that ERD_3144, the orthologous gene in M. tuberculosis Erdman, is functionally equivalent to Emp1. 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 resulted in a significant reduction in the ability of M. marinum to cause macrophage cytolysis. Collectively, this study identified a NAT required for N-terminal acetylation in Mycobacterium and provided insight into the requirement of N-terminal acetylation of EsxA and other proteins in mycobacterial virulence in the macrophage. Significance StatementN-terminal acetylation is a protein modification that broadly impacts basic cellular function, protein turnover and disease in higher organisms. In bacteria, very little is understood how N-terminal acetylation impacts bacterial physiology and pathogenesis. Mycobacterial pathogens cause acute and chronic diseases in humans and in animals. [~]15% of mycobacterial proteins are N-terminally acetylated, but the enzymes responsible for this protein modification are largely unknown. We identified a conserved mycobacterial protein, MMAR_1839, that is required for the N-terminal acetylation of 23 mycobacterial proteins including EsxA, a protein essential for mycobacteria to cause disease. Loss of this enzyme from Mycobacterium marinum reduced macrophage killing, which is required for bacterial spread in the host. Defining the acetyltransferases responsible for the N-terminal protein acetylation of essential virulence factors could lead to new targets for therapeutics against mycobacterial pathogens.