Biology subjects

Jones, B. S.

Publications and source records attributed to Jones, B. S..

7 recordsLinked to original sources

bioRxiv2026

pH-responsive substrate switching in mycobacterial Type VII ESX secretion.

During infection, pathogenic mycobacteria reside within phagosomes of varying acidity based on the macrophage activation state. The ESX-1 secretion system [early secreted antigen 6 kilodaltons (ESAT-6) system-1] delivers protein virulence factors essential for phagosome lysis, facilitating infection. The mechanisms underlying ESX-1 lytic activity in heterogeneous environments remain unknown. Here we show that the canonical Type VII secretion system, ESX-1, orchestrates substrate switching in response to different environments. Growing Mycobacterium marinum at acidic pH resulted in substrate switching in vitro. Substrate switching was accompanied by significant changes to the levels of ESX-1 substrate transcripts, and to the levels of both ESX-1 substrates and chaperones at the protein level. We showed that specific ESX-1 transcripts were significantly upregulated in vivo, and that distinct substrate sets are required in an acidic infection model. Significance StatementPathogenic mycobacteria cause chronic and acute disease. Mycobacterial pathogens promote infection by transporting bacterial proteins into the host using ESX/Type VII secretion systems. The ESX-1 system secretes proteins into the phagosome that release the bacteria into the cytoplasm and promote bacterial survival in the macrophage. We show that Mycobacterium marinum, an animal pathogen and model for studying ESX-1 and tuberculosis, switches which ESX-1 proteins are secreted in response to acidic pH, an infection relevant signal. We demonstrate that protein secretion reflects changes in substrate transcripts and in substrate and chaperone protein levels. Finally, we leveraged two infection models to support that ESX-1 substrate switching likely occurs during infection. Our findings support a model in which mycobacterial pathogens use different proteins to lyse macrophage phagosomes of different pH.

microbiology↗

bioRxiv2025

One-Pot Quantitation of Mycobacterial N-terminal Protein Acetylation Peptidoforms and Proteome

Isotopic labeling of proteins for quantitative proteomics is a popular technique to increase sample throughput and provide improved accuracy and precision for relative or absolute quantitation between samples. Derivatives of this technique are used to label protein and peptide N-termini for selective enrichment and analysis. We previously reported on a method to enrich and quantify protein N-terminal acetylation in model-system and pathogenic mycobacteria. Significant recent advancements in silica filter-based protein digestion have improved identification of proteins in bottom-up proteomics. However, these are not yet compatible with existing methods which detect and quantify protein termini and N-terminal modifications. Here, we present a one-pot method (OnePotNTA) that incorporates silica filter digestion with protein N-terminal labeling and subsequent quantitation. This technique achieves high-density coverage of the N-terminome and obviates the need to enrich N-terminal peptides prior to analysis. OnePotN TA identified 54% of the canonical proteome from whole-cell lysates of Mycobacterium marinum and eliminated biases in peptides identified by forgoing enrichment. This significantly reduced sample preparation time by [≥]5-fold and preserves protein-level abundance measurements (LFQ) from the same injections and analysis. Analysis of a mutant strain of M. marinum lacking Emp1 ({Delta}emp1)-an N-terminal Acetyltransferase required for efficient pathogenesis-identified 37 putative substrates of this enzyme. Additionally, analysis of the remaining peptides identified at least 34 proteins with alternate true N-termini, distinct from the canonical genome.

biochemistry↗

bioRxiv2024

N-acetyl-transferases required for iron uptake and aminoglycoside resistance promote virulence lipid production in M. marinum

Phagosomal lysis is a key aspect of mycobacterial infection of host macrophages. Acetylation is a protein modification mediated enzymatically by N-acetyltransferases (NATs) that impacts bacterial pathogenesis and physiology. To identify NATs required for lytic activity, we leveraged Mycobacterium marinum, a nontubercular pathogen and an established model for M. tuberculosis. M. marinum hemolysis is a proxy for phagolytic activity. We generated M. marinum strains with deletions in conserved NAT genes and screened for hemolytic activity. Several conserved lysine acetyltransferases (KATs) contributed to hemolysis. Hemolysis is mediated by the ESX-1 secretion system and by phthiocerol dimycocerosate (PDIM), a virulence lipid. For several strains, the hemolytic activity was restored by the addition of second copy of the ESX-1 locus. Using thin-layer chromatography (TLC), we found a single NAT required for PDIM and phenolic glycolipid (PGL) production. MbtK is a conserved KAT required for mycobactin siderophore synthesis and virulence. Mycobactin J exogenously complemented PDIM/PGL production in the {Delta}mbtK strain. The {Delta}mbtK M. marinum strain was attenuated in macrophage and Galleria mellonella infection models. Constitutive expression of either eis or papA5, which encode a KAT required for aminoglycoside resistance and a PDIM/PGL biosynthetic enzyme, rescued PDIM/PGL production and virulence of the {Delta}mbtK strain. Eis N-terminally acetylated PapA5 in vitro, supporting a mechanism for restored lipid production. Overall, our study establishes connections between the MbtK and Eis NATs, and between iron uptake and PDIM and PGL synthesis in M. marinum. Our findings underscore the multifunctional nature of mycobacterial NATs and their connection to key virulence pathways. Significance StatementAcetylation is a modification of protein N-termini, lysine residues, antibiotics and lipids. Many of the enzymes that promote acetylation belong to the GNAT family of proteins. M. marinum is a well-established as a model to understand how M. tuberculosis causes tuberculosis. In this study we sought to identify conserved GNAT proteins required for early stages of mycobacterial infection. Using M. marinum, we determined that several GNAT proteins are required for the lytic activity of M. marinum. We uncovered previously unknown connections between acetyl-transferases required for iron uptake and antimicrobial resistance, and the production of the unique mycobacterial lipids, PDIM and PGLOur data support that acetyl-transferases from the GNAT family are interconnected, and have activities beyond those previously reported.

microbiology↗

bioRxiv2024

Clonal determinants of organotropism and survival in metastatic uveal melanoma

Uveal melanoma (UM), the most common intraocular primary cancer in adults, demonstrates a unique proclivity for liver metastasis. To understand the molecular underpinnings of this organotropism, we analyzed the genomic features of liver and extrahepatic UM metastases, identifying distinct molecular signatures that mirror the clonal diversity in primary UM tumors. Liver metastases were enriched in BAP1 mutations and exhibited a higher prevalence of monosomy 3 compared to extrahepatic metastases. Analysis of the tumor-liver microenvironment crosstalk at the single-cell level underscored a significant role for hepatic stellate cells in facilitating UM growth and establishment in the liver. Notably, within the primary tumor, clones that demonstrated a high affinity for the liver, compared to those with low liver affinity, exhibited a distinct transcriptional profile characterized by the upregulation of pathways that activate hepatic stellate cells, specifically involving TGF-{beta} signaling, cytokine signaling, extracellular matrix remodeling, and angiogenesis. Liver-tropic clones displayed not only an increased affinity for liver colonization but were also associated with worse survival outcomes, underscoring the adverse prognostic significance of hepatic metastases in UM. Our findings demonstrate that trajectories of metastatic dissemination and patient survival in UM are established early in the primary tumors evolution, opening pathways for the development of targeted therapeutic interventions to improve patient outcomes.

cancer biology↗

bioRxiv2024

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

The mycobacterial cell envelope is a major virulence determinant in pathogenic mycobacteria. Specific outer lipids play roles in pathogenesis, modulating the immune system and promoting the secretion of virulence factors. ESX-1 (ESAT-6 system-1) is a conserved protein secretion system required for mycobacterial pathogenesis (1, 2). Previous studies revealed that mycobacterial strains lacking the outer lipid PDIM have impaired ESX-1 function during laboratory growth and infection (3-5). The mechanisms underlying changes in ESX-1 function are unknown. We used a proteo-genetic approach to measure PDIM and PGL-dependent protein secretion in M. marinum, a non-tubercular mycobacterial pathogen that causes tuberculosis-like disease in ectothermic animals (6, 7). Importantly, M. marinum is a well-established model for mycobacterial pathogenesis (8, 9). Our findings showed that M. marinum strains without PDIM and PGL showed specific, significant reductions in protein secretion compared to the WT and complemented strains. We recently established a hierarchy for the secretion of ESX-1 substrates in four (I-IV) groups (10). Loss of PDIM differentially impacted secretion of Groups III and IV ESX-1 substrates, which are likely the effectors of pathogenesis. Our data suggests that the altered secretion of specific ESX-1 substrates is responsible for the observed ESX-1-related effects in PDIM-deficient strains. ImportanceMycobacterium tuberculosis, the cause of human tuberculosis, killed an estimated 1.3 million people in 2022. Non-tubercular mycobacterial species are causing acute and chronic human infections. Understanding how these bacteria cause disease is critical. Lipids in the cell envelope are essential for mycobacteria to interact with the host and promote disease. Strains lacking outer lipids are attenuated for infection, but the reasons are unclear. Our research aims to identify a mechanism for attenuation of mycobacterial strains without the PDIM and PGL outer lipids in M. marinum. These findings will enhance our understanding of the importance of lipids in pathogenesis, and how these lipids contribute to other established virulence mechanisms.

microbiology↗

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↗

bioRxiv2021

Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

{beta}-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative {beta}-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 {degrees}C towards 4-Nitrophenyl-{beta}-galactofuranoside (pNP-{beta}-Galf), with a Km of 17.9 {+/-} 1.9 mM and Vmax of 70.6 {+/-} 5.3 mol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.

biochemistry↗