ORCID Profile
0000-0002-3944-988X
Current Organisations
University of Sydney
,
Macquarie University
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Publisher: Oxford University Press (OUP)
Date: 11-01-2018
Abstract: N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from C ylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the C ylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of in idual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system.
Publisher: Cold Spring Harbor Laboratory
Date: 02-12-2019
DOI: 10.1101/862151
Abstract: Biofilms serve as a protective mechanism for bacteria to cope with environmental stress. Whilst ordinarily a fastidious organism, it has been long suggested that C. jejuni is able to utilise this mode of growth as a way to transmit infection from the avian host to humans. Herein, we undertook a combinatorial approach to examine differential expression of C. jejuni genes and protein abundance during biofilm formation. RNA sequencing and proteomics via quantitative liquid chromatography – tandem mass spectrometry (LC-MS/MS) revealed biofilm growth induced a substantial rearrangement of the C. jejuni transcriptome and proteome, with ∼600 genes differentially expressed in biofilms compared to planktonic cells. Biofilm-induced genes / proteins were those involved in iron metabolism and acquisition, cell ision, glycan production and attachment, while those repressed were associated with metabolism, amino acid uptake and utilisation, and large tracts of the chemotaxis pathway. We further examined the role of chemotaxis in C. jejuni biofilm formation by assessing the behaviour of isogenic strains with deletions of the cheV and cheW genes. Both Δ cheV and Δ cheW exhibited a significant decrease in directed motility when compared to wild-type C. jejuni . Both mutants also demonstrated an increase in autoagglutination ability and increased biofilm formation. A subtle difference was also observed between the phenotypes of Δ cheV and Δ cheW mutants, both in motility and biofilm formation. This suggests roles for the CheV and CheW signal transduction proteins and may present signal transduction as a potential method for modulating C. jejuni biofilm formation. C ylobacter jejuni is a gastroenteric bacterium that is responsible for most cases of bacterial food poisoning in the developed world. The organism commonly resides in avian reservoirs and is passed to humans through contaminated poultry and animal products. Ordinarily, C. jejuni requires a strict set of conditions in order to survive and cause infections in humans. Biofilms are a method of bacterial growth that may provide shielding from harsh environments and provide an important link between reservoirs and human hosts. In this study, we have utilised a multi-platform approach to compare gene expression and protein abundance in planktonic C. jejuni cells and those growing as a biofilm. We subsequently focused on the chemosensory system of C. jejuni and demonstrated that signal transduction proteins play a role in biofilm formation. Our work has provided a broad profile of which genes are important to C. jejuni biofilms and that the chemosensory pathway has an influence on biofilm formation.
Publisher: Portland Press Ltd.
Date: 10-08-2021
DOI: 10.1042/BST20191088
Abstract: Despite being considered the simplest form of life, bacteria remain enigmatic, particularly in light of pathogenesis and evolving antimicrobial resistance. After three decades of genomics, we remain some way from understanding these organisms, and a substantial proportion of genes remain functionally unknown. Methodological advances, principally mass spectrometry (MS), are paving the way for parallel analysis of the proteome, metabolome and lipidome. Each provides a global, complementary assay, in addition to genomics, and the ability to better comprehend how pathogens respond to changes in their internal (e.g. mutation) and external environments consistent with infection-like conditions. Such responses include accessing necessary nutrients for survival in a hostile environment where co-colonizing bacteria and normal flora are acclimated to the prevailing conditions. Multi-omics can be harnessed across temporal and spatial (sub-cellular) dimensions to understand adaptation at the molecular level. Gene deletion libraries, in conjunction with large-scale approaches and evolving bioinformatics integration, will greatly facilitate next-generation vaccines and antimicrobial interventions by highlighting novel targets and pathogen-specific pathways. MS is also central in phenotypic characterization of surface biomolecules such as lipid A, as well as aiding in the determination of protein interactions and complexes. There is increasing evidence that bacteria are capable of widespread post-translational modification, including phosphorylation, glycosylation and acetylation with each contributing to virulence. This review focuses on the bacterial genotype to phenotype transition and surveys the recent literature showing how the genome can be validated at the proteome, metabolome and lipidome levels to provide an integrated view of organism response to host conditions.
Publisher: Informa UK Limited
Date: 17-11-2018
Publisher: Elsevier BV
Date: 2014
DOI: 10.1016/J.JPROT.2013.08.012
Abstract: The post-translational modification (PTM) of proteins plays a critical role in the regulation of a broad range of cellular processes in eukaryotes. Yet their role in governing similar systems in the conventionally presumed 'simpler' forms of life has been largely neglected and, until recently, was thought to occur only rarely, with some modifications assumed to be limited to higher organisms alone. Recent developments in mass spectrometry-based proteomics have provided an unparalleled power to enrich, identify and quantify peptides with PTMs. Additional modifications to biological molecules such as lipids and carbohydrates that are essential for bacterial pathophysiology have only recently been detected on proteins. Here we review bacterial protein PTMs, focusing on phosphorylation, acetylation, proteolytic degradation, methylation and lipidation and the roles they play in bacterial adaptation - thus highlighting the importance of proteomic techniques in a field that is only just in its infancy. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.
Publisher: Elsevier BV
Date: 2016
DOI: 10.1016/J.JPROT.2015.09.011
Abstract: Staphylococcus epidermidis is an opportunistic pathogen that is an emerging risk factor in hospitals worldwide and is often difficult to eradicate as virulent strains produce a protective biofilm matrix. We utilized cell shaving proteomics to profile surface-exposed proteins from two fully genome sequenced S. epidermidis strains: the avirulent, non-biofilm forming ATCC12228 and the virulent, strongly adherent biofilm forming ATCC35984 (RP62A). A false positive control strategy was employed to calculate the probabilities of proteins being truly surface-exposed. A total of 78 surface-exposed proteins were identified, of which only 19 proteins were common to ATCC12228 and RP62A, and which thus represents the core surfaceome. S. epidermidis RP62A displayed additional proteins involved in biofilm formation (cell wall-associated Bhp and intercellular adhesion protein IcaB), surface antigenicity, peptidoglycan biosynthesis and antibiotic resistance. We concurrently profiled whole cell proteomes of the two strains using iTRAQ quantitation and LC-MS/MS. A total of 1610 proteins were confidently identified (representing 64% of the theoretical S. epidermidis proteome). One hundred and ninety one proteins were differentially abundant between strains. Proteins associated with RP62A were clustered into functions including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated defense, sulfate assimilation, antibiotic resistance and biofilm formation. Validation of the sulfate assimilation and cysteine/methionine biosynthesis pathways showed RP62A contained elevated levels (~25% increase) of methionine that are likely linked to biofilm formation. Cell shaving and quantitative proteomics identified proteins associated with a biofilm-forming, virulent strain of S. epidermidis (RP62A). These proteins show RP62A maintains an active CRISPR-mediated defense, as well as heightened antibiotic resistance in comparison to a non-virulent, non-biofilm forming strain. Increased abundances of sulfate assimilation proteins lead to elevated intracellular methionine. Proteins and their exposed peptides identified on the surface of S. epidermidis RP62A may be useful vaccine antigens in clinical settings if administered in at-risk patients prior to surgical implantations.
Publisher: Springer Science and Business Media LLC
Date: 22-04-2020
DOI: 10.1038/S41598-020-63569-5
Abstract: Biofilms of the gastroenteric pathogen C. jejuni may serve an important role in the transmission of infection from reservoirs of infection to humans. Herein, we undertook a combinatorial approach examining differential gene expression and protein abundance during biofilm formation in C. jejuni . Biofilms induced a substantial rearrangement of the C. jejuni transcriptome and proteome, with ~600 genes differentially expressed when compared to planktonic cells. Genes and proteins induced in biofilms were involved in iron metabolism and acquisition, cell ision, glycan production and attachment, while those repressed were associated with metabolism, amino acid usage, and large tracts of the chemotaxis pathway. We further examined the role of chemotaxis in C. jejuni biofilm formation by examining isogenic strains with deletions of the cheV and cheW signal transduction genes. Both ∆ cheV and ∆ cheW exhibited a significant decrease in directed motility when compared to wild-type C. jejuni as well as demonstrating an increase in autoagglutination ability and biofilm formation. A subtle difference was also observed between the phenotypes of ∆ cheV and ∆ cheW mutants, both in motility and biofilm formation. This suggests roles for CheV and CheW and may present signal transduction as a potential method for modulating C. jejuni biofilm formation.
Publisher: American Chemical Society (ACS)
Date: 25-08-2014
DOI: 10.1021/PR5005554
Abstract: C ylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the -2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA Gln at the -2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.
Publisher: Elsevier BV
Date: 08-2020
Publisher: American Society for Microbiology
Date: 02-01-2020
DOI: 10.1128/MRA.00982-19
Abstract: C ylobacter jejuni is a foodborne pathogen and an important contributor to gastroenteritis in humans. C. jejuni readily forms biofilms which may play a role in the transmission of the pathogen from animals to humans. Herein, we present RNA sequencing data investigating differential gene expression in biofilm and planktonic C. jejuni . These data provide insight into pathways which may be important to biofilm formation in this organism.
Publisher: Elsevier BV
Date: 04-2019
Publisher: Royal Society of Chemistry (RSC)
Date: 2020
DOI: 10.1039/D0MO00032A
Abstract: Virulence of C ylobacter jejuni is dependent on the ability to glycosylate membrane-associated proteins.
No related grants have been discovered for Joel Cain.