Biology subjects

Champion, P. A.

Publications and source records attributed to Champion, P. A..

3 recordsLinked to original sources

bioRxiv2023

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

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.

microbiology↗

bioRxiv2023

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

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 network in vitro but are dispensable for macrophage infection by Mycobacterium 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 the whiB6 transcription factor gene, and specific ESX-1 substrate and secretory component genes. Unlike the other transcription factors that regulate ESX-1, EspN is required for M. marinum growth 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. ImportancePathogenic mycobacteria cause acute and long-term diseases, including human tuberculosis. The ESX-1 system transports proteins that control the host response to infection and promotes bacterial survival. Although ESX-1 transports proteins, it also controls gene expression in the bacteria. In this study, we identify an undescribed transcription factor that controls the expression of ESX-1 genes, and is required for both macrophage and animal infection. However, this transcription factor is not the primary regulator of ESX-1 genes under standard laboratory conditions. These findings identify a critical transcription factor that controls expression of a major virulence pathway during infection, but whose effect is not detectable with standard laboratory strains and growth conditions.

microbiology↗

bioRxiv2023

Mycobacterium tuberculosis SecA2-dependent activation of host Rig-I/MAVs signaling is not conserved in Mycobacterium marinum

Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis, have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo. In M. tuberculosis, this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M. tuberculosis and M. marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-{beta} secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a {Delta}secA2 M. marinum strain along with complementation strains expressing either the M. marinum or M. tuberculosis secA2 genes. Our results suggest that the {Delta}secA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the {Delta}secA2 strain. Therefore, to better examine secreted IFN-{beta} levels when bacterial infection levels are equal across strains, we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-{beta} to a standard number of bacteria. Applying this approach to both WT and MAVS-/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-{beta} from macrophages infected with the {Delta}secA2 M. marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-{beta} response in a SecA2-dependent manner is not conserved in M. marinum under the conditions tested.

microbiology↗