Biology subjects

Hu, D. D.

Publications and source records attributed to Hu, D. D..

5 recordsLinked to original sources

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

Gender- and Age-Based Characterization and Comparison of the Murine Primary Peritoneal Mesothelial Cell Proteome

Organs in the abdominal cavity are covered by a peritoneal membrane, which is comprised of a monolayer of mesothelial cells (MC). Diseases involving the peritoneal membrane include peritonitis, primary cancer (mesothelioma), and metastatic cancers (ovarian, pancreatic, colorectal). These diseases have gender- and/or age-related pathologies; however, the impact of gender and age on the peritoneal MC is not well evaluated. To address this, we identified and characterized gender- and age-related differences in the proteomes of murine primary peritoneal MC. Primary peritoneal MC were isolated from young female (FY) or male (MY) mice (3-6 months) and aged female (FA) or male (MA) mice (20-23 months), lysed, trypsin digested using S-Traps, then subjected to bottom-up proteomics using an LC-Orbitrap mass spectrometer. In each cohort, we identified >1000 protein groups. Proteins were categorized using Gene Ontology and pairwise comparisons between gender and age cohorts were conducted. This study establishes baseline information for studies on peritoneal MC in health and disease at two physiologic age/gender points. Segregation of the data by gender and age could reveal novel factors to specific disease states involving the peritoneum. [This in vitro primary cell model has utility for future studies on the interaction between the mesothelium and foreign materials.] SUMMARY STATEMENTMany diseases initiate from or involve peritoneal mesothelial cells including peritonitis, primary cancer (mesothelioma) and metastatic cancers. Progression of these diseases is influenced by many host factors including gender and age; however, the influence of these factors on the peritoneal mesothelial cell proteome has not been evaluated. This study provides novel information and identifies proteins exclusive to both male and female young and aged cohorts. Given the importance of the peritoneal mesothelial cell in abdominal homeostasis, and the impact of gender and age on disease progression, these data will be key for future studies examining mesothelium in both health and disease.

cancer biology↗

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

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↗