An ancestral mycobacterial effector promotes dissemination of infection

Saelens, Joseph W.; Sweeney, Mollie I; Viswanathan, Gopinath; Xet-Mull, Ana M.; Smith, Kristen L. Jurcic; Sisk, Dana M.; Hu, Daniel D.; Cronin, Rachel M.; Hughes, Erika J.; Brewer, W. Jared; Coers, Jörn; Champion, Matthew M.; Champion, Patricia A.; Lowe, Craig B.; Smith, Clare M.; Lee, Sunhee; Stout, Jason E.; Tobin, David M.

doi:10.1016/j.cell.2022.10.019

Cited by 31 publications

(22 citation statements)

References 129 publications

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“…Using the zebrafish larval model of mycobacterial infection, we monitored infection burden over five days of infection as measured by a validated Fluorescence Pixel Count (FPC) assay (Fig. 5C and 5D) (37, 38). Bacterial burden was substantially reduced at five days post-infection.…”

Section: Resultsmentioning

confidence: 99%

The EspN transcription factor is an infection-dependent regulator of the ESX-1 system inM. marinum

Nicholson

Cronin

Menon³

et al. 2023

Preprint

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Bacterial pathogens use protein secretion systems to translocate virulence factors into the host and to control bacterial gene expression. The ESX-1 (ESAT-6 system 1) secretion system facilitates disruption of the macrophage phagosome during infection, enabling access to the cytoplasm, and regulates widespread gene expression in the mycobacterial cell. The transcription factors contributing to the ESX-1 transcriptional network during mycobacterial infection are not known. We showed that the EspM and WhiB6 transcription factors regulate the ESX-1 transcriptional networkin vitrobut are dispensable for macrophage infection byMycobacterium marinum. In this study, we used our understanding of the ESX-1 system to identify EspN, a critical transcription factor that controls expression of the ESX-1 genes during infection, but whose effect is not detectable under standard laboratory growth conditions. Under laboratory conditions, EspN activity is masked by the EspM repressor. In the absence of EspM, we found that EspN is required for ESX-1 function because it activates expression of thewhiB6transcription factor gene, and specific ESX-1 substrate and secretory component genes. Unlike the other transcription factors that regulate ESX-1, EspN is required forM. marinumgrowth within and cytolysis of macrophages, and for disease burden in a zebrafish larval model of infection. These findings demonstrate that EspN is an infection-dependent regulator of the ESX-1 transcriptional network, which is essential for mycobacterial pathogenesis. Moreover, our findings suggest that ESX-1 expression is controlled by a genetic switch that responds to host specific signals.

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Section: Resultsmentioning

confidence: 99%

The EspN transcription factor is an infection-dependent regulator of the ESX-1 system inM. marinum

Nicholson

Cronin

Menon³

et al. 2023

Preprint

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“…In this study, we used strain H37Rv for the sake of consistency with other prior studies on antigen presentation on MHC-I and phagosome membrane damage in Mtb -infection, but antigen presentation may vary among Mtb isolates. Mtb isolates have been shown to differ in their level of ESX-1 activity ( Solans et al, 2014 ), as well as carrying mutations in Esx-family proteins and other T7SS substrates ( Saelens et al, 2022 ; Uplekar et al, 2011 ). Further studies across multiple isolates may therefore reveal differences in antigen presentation that could be relevant for the design of broadly protective vaccines.…”

Section: Discussionmentioning

confidence: 99%

Immunopeptidomics reveals determinants of Mycobacterium tuberculosis antigen presentation on MHC class I

Leddy

White

Bryson

2023

eLife

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CD8+ T cell recognition of Mycobacterium tuberculosis (Mtb)-specific peptides presented on major histocompatibility complex class I (MHC-I) contributes to immunity to tuberculosis (TB), but the principles that govern presentation of Mtb antigens on MHC-I are incompletely understood. In this study, mass spectrometry (MS) analysis of the MHC-I repertoire of Mtb-infected primary human macrophages reveals that substrates of Mtb's type VII secretion systems (T7SS) are overrepresented among Mtb-derived peptides presented on MHC-I. Quantitative, targeted MS shows that ESX-1 activity is required for presentation of Mtb peptides derived from both ESX-1 substrates and ESX-5 substrates on MHC-I, consistent with a model in which proteins secreted by multiple T7SSs access a cytosolic antigen processing pathway via ESX-1-mediated phagosome permeabilization. Chemical inhibition of proteasome activity, lysosomal acidification, or cysteine cathepsin activity did not block presentation of Mtb antigens on MHC-I, suggesting involvement of other proteolytic pathways or redundancy among multiple pathways. Our study identifies Mtb antigens presented on MHC-I that could serve as targets for TB vaccines, and reveals how the activity of multiple T7SSs interacts to contribute to presentation of Mtb antigens on MHC-I.

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“…Whole colony MALDI mass spectrometry to measure surface associated EsxA, Nt-EsxA and EsxB was performed as in(58). Label free Quantitative Mass Spectrometry was used to measure Nt-acetylation from M. marinum whole cell lysates, similar to (68, 75). Hemolytic activity of M. marinum was measured against sheep Red Blood Cells (sRBCs) as previously described (14).…”

Section: Methodsmentioning

confidence: 99%

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

Collars

Jones

et al. 2023

Preprint

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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.

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An ancestral mycobacterial effector promotes dissemination of infection

Cited by 31 publications

References 129 publications

The EspN transcription factor is an infection-dependent regulator of the ESX-1 system inM. marinum

The EspN transcription factor is an infection-dependent regulator of the ESX-1 system inM. marinum

Immunopeptidomics reveals determinants of Mycobacterium tuberculosis antigen presentation on MHC class I

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

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