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.

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6-specific nanobody restricts M. tuberculosis growth in macrophages

“…Previous studies have found major differences in function between recombinant ESAT-6 in general when compared with ‘native’ ESAT-6 purified from Mtb cultures, which are typically purified using much harsher techniques than recombinant protein due to the lack of affinity tags. One attractive theory for the observed difference between recombinant and native ESAT-6 which is gaining traction is N-terminal acetylation being required for ESAT-6 function, but this has not yet been proven in Mtb infection ( Aguilera et al, 2020 ; Collars et al, 2023 ; Mba Medie et al, 2014 ; Okkels et al, 2004 ). The most commonly given reason for the importance of N-acetylation is that acetylated ESAT-6 is more easily released from CFP-10 under acidic conditions.…”

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages

“…Previous studies have found major differences in function between recombinant ESAT-6 in general when compared with “native” ESAT-6 purified from Mtb cultures, which are typically purified using much harsher techniques than recombinant protein due to the lack of affinity tags. One attractive theory for the observed difference between recombinant and native ESAT-6 which is gaining traction is N-terminal acetylation being required for ESAT-6 function, but this has not yet been proven in Mtb infection (Aguilera et al, 2020; Collars et al, 2023; Mba Medie et al, 2014; Okkels et al, 2004). The most commonly given reason for the importance of N-acetylation is that acetylated ESAT-6 is more easily released from CFP-10 under acidic conditions.…”

The loss of the PDIM/PGL virulence lipids causes differential secretion of ESX-1 substrates inMycobacterium marinum

“…Cell-associated and secreted cell lysate samples were prepared for LC-MS analysis as described (72, 73). 50 μg of each sample was prepared in 100 mM triethylammonium bicarbonate (TEAB), 5% sodium dodecyl sulfate (SDS), and 100 mM tris(2-carboxyethyl)phosphine (TCEP).…”