Global Acetylomics of Campylobacter jejuni Shows Lysine Acetylation Regulates CadF Adhesin Processing and Human Fibronectin Binding

Abstract

Lysine acetylation (KAc) is a reversible post-translational modification (PTM) that can alter protein structure and function; however, specific roles for KAc are largely undefined in bacteria. Acetyl-lysine immunoprecipitation and LC–MS/MS identified 5567 acetylated lysines on 1026 proteins from the gastrointestinal pathogen Campylobacter jejuni (∼63% of the predicted proteome). KAc was identified on proteins from all subcellular locations, including the outer membrane (OM) and extracellular proteins. Label-based LC–MS/MS identified proteins and KAc sites during growth in 0.1% sodium deoxycholate (DOC, a component of gut bile salts). 3410 acetylated peptides were quantified, and 784 (from 409 proteins) were differentially abundant in DOC growth. Changes in KAc involved multiple pathways, suggesting a dynamic role for this PTM in bile resistance. As observed elsewhere, we show KAc is primarily nonenzymatically mediated via acetyl-phosphate; however, the deacetylase CobB also contributes to a global elevation of this modification in DOC. We observed several multiply acetylated OM proteins and altered DOC abundance of acetylated peptides in the fibronectin (Fn)-binding adhesin CadF. We show KAc reduces CadF Fn binding and prevalence of lower mass variants. This study provides the first system-wide lysine acetylome of C. jejuni and contributes to our understanding of KAc as an emerging PTM in bacteria.

Bacterial protein acetylation: mechanisms, functions, and methods for study

“…As a first step in the investigation, detection, and semi-quantification of the targeted acetylated protein can be made by western blot analysis using specific antibodies against acetylated proteins ( Yu et al., 2008 ; Hirano, 2012 ; Diallo et al., 2019 ). This type of analysis gives a global view of the acetylation profiles under different conditions (temperature, growth phase, stressors, among others) and in different strains to determine the differences in acetylation levels qualitatively ( Kosono et al., 2015 ; Schilling et al., 2015 ; Christensen et al., 2018 ; Xu et al., 2018 ; Dale et al., 2023 ). However, this approach usually presents an insensitive and non-specific signal, so it is necessary to introduce negative controls to demonstrate the specificity of the anti-acetyl lysine antibody.…”

Multi-Omics of Campylobacter jejuni Growth in Chicken Exudate Reveals Molecular Remodelling Associated with Altered Virulence and Survival Phenotypes

“…Peptide spectral matches (PSMs) corresponding to a 1% FDR were exported, and reporter intensities normalised to total reporter ion signals across all channels. For relative quantitation and statistical analysis, data were imported into Perseus (v. 1.6.1.1) and analysed as per [25].…”

“…Despite a limited genome of ~1620 genes (in the NCTC11168 strain [18]), C. jejuni has an unusual capacity for post-translationally modifying proteins. Such modifications include O-glycosylation of the flagellin subunits of the flagellar apparatus, a general N-linked glycosylation system that modifies ~100 membrane-associated proteins and is required for host cell binding, nitrate reductase activity, and antimicrobial resistance, along with widespread lysine acetylation on >1000 proteins that plays a role in metabolism and appears to regulate CadF binding to host fibronectin [19][20][21][22][23][24][25].…”