An N-acetyltransferase required for ESAT-6 N-terminal acetylation and virulence in Mycobacterium marinum

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.