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

Campylobacter jejuni is the leading cause of foodborne human gastroenteritis in the developed world. Infections are largely acquired from poultry produced for human consumption and poor food handling is thus a major risk factor. Chicken exudate (CE) is a liquid produced from defrosted commercial chicken products that facilitates C. jejuni growth. We examined the response of C. jejuni to growth in CE using a multi-omics approach. Changes in the C. jejuni proteome were assessed by label-based liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We quantified 1328 and 1304 proteins, respectively, in experiments comparing 5% CE in Mueller-Hinton (MH) medium and 100% CE with MH-only controls. These proteins represent 81.8% and 80.3% of the predicted C. jejuni NCTC11168 proteome. Growth in CE induced profound remodelling of the proteome. These changes were typically conserved between 5% and 100% CE, with a greater magnitude of change observed in 100% CE. We confirmed that CE induced C. jejuni biofilm formation, as well as increasing motility and resistance against oxidative stress, consistent with changes to proteins representing those functions. Assessment of the C. jejuni metabolome showed CE also led to increased intracellular abundances of serine, proline, and lactate that were correlated with the elevated abundances of their respective transporters. Analysis of carbon source uptake showed prolonged culture supernatant retention of proline and succinate in CE-supplemented medium. Metabolomics data provided preliminary evidence for the uptake of chicken-meat-associated dipeptides. C. jejuni exposed to CE showed increased resistance to several antibiotics, including polymyxin B, consistent with changes to tripartite efflux system proteins and those involved in the synthesis of lipid A. The C. jejuni CE proteome was also characterised by very large increases in proteins associated with iron acquisition, while a decrease in proteins containing iron-sulphur clusters was also observed. Our data suggest CE is both oxygen- and iron-limiting and provide evidence of factors required for phenotypic remodelling to enable C. jejuni survival on poultry products.

Exploiting pglB Oligosaccharyltransferase-Positive and -Negative Campylobacter jejuni and a Multiprotease Digestion Strategy to Identify Novel Sites Modified by N-Linked Protein Glycosylation.

Campylobacter jejuni is a bacterial pathogen encoding a unique N-linked glycosylation (pgl) system that mediates attachment of a heptasaccharide to N-sequon-containing membrane proteins by the PglB oligosaccharyltransferase (OST). Many targets of PglB are known, yet only a fraction of sequons are experimentally confirmed, and site occupancy remains elusive. We exploited pglB-positive (wild-type; WT) and -negative (ΔpglB) proteomes to identify potential glycosites. The nonglycosylated forms of known glycopeptides were typically increased in protein normalized abundance in ΔpglB relative to WT and restored by pglB reintroduction (ΔpglB::pglB). Sequon-containing peptide abundances were thus consistent with significant site occupancy in the presence of the OST. Peptides with novel sequons were either unaltered (likely not glycosylated) or showed abundance consistent with known glycopeptides. Topology analysis revealed that unaltered sequons often displayed cytoplasmic localization, despite originating from membrane proteins. Novel glycosites were confirmed using parallel multiprotease digestion, LC-MS/MS, and FAIMS-MS to define the glycoproteomes of WT and ΔpglB::pglBC. jejuni. We identified 142 glycosites, of which 32 were novel, and 83% of sites predicted by proteomics were validated. There are now 166 experimentally verified C. jejuni glycosites and evidence for occupancy or nonoccupancy of 31 additional sites. This study serves as a model for the use of OST-negative cells and proteomics for highlighting novel glycosites and determining occupancy in a range of organisms.

Integrated mass spectrometry-based multi-omics for elucidating mechanisms of bacterial virulence.

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.

Proteomics of Campylobacter jejuni Growth in Deoxycholate Reveals Cj0025c as a Cystine Transport Protein Required for Wild-type Human Infection Phenotypes.

Campylobacter jejuni is a major cause of food-borne gastroenteritis. Proteomics by label-based two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) identified proteins associated with growth in 0.1% sodium deoxycholate (DOC, a component of gut bile salts), and system-wide validation was performed by data-independent acquisition (DIA-SWATH-MS). LC-MS/MS quantified 1326 proteins (∼82% of the predicted C. jejuni proteome), of which 1104 were validated in additional biological replicates by DIA-SWATH-MS. DOC resulted in a profound proteome shift with 512 proteins showing significantly altered abundance. Induced proteins were associated with flagellar motility and antibiotic resistance; and these correlated with increased DOC motility and resistance to polymyxin B and ciprofloxacin. DOC also increased human Caco-2 cell adherence and invasion. Abundances of proteins involved in nutrient transport were altered by DOC and aligned with intracellular changes to their respective carbon sources. DOC increased intracellular levels of sulfur-containing amino acids (cysteine and methionine) and the dipeptide cystine (Cys-Cys), which also correlated with reduced resistance to oxidative stress. A DOC induced transport protein was Cj0025c, which has sequence similarity to bacterial Cys-Cys transporters. Deletion of cj0025c (Δcj0025c) resulted in proteome changes consistent with sulfur starvation, as well as attenuated invasion, reduced motility, atypical morphology, increased antimicrobial susceptibility and poor biofilm formation. Targeted metabolomics showed Δcj0025c could use known C. jejuni amino and organic acid substrates commensurate with wild-type. Medium Cys-Cys levels however, were maintained in Δcj0025c relative to wild-type. A toxic Cys-Cys mimic (selenocystine) inhibited wild-type growth, but not Δcj0025c Provision of an alternate sulfur source (2 mm thiosulfate) restored Δcj0025c motility. Our data confirm that Cj0025c is a Cys-Cys transporter that we have named TcyP consistent with the nomenclature of homologous proteins in other species.

Identifying the targets and functions of N-linked protein glycosylation in Campylobacter jejuni.

Campylobacter jejuni is a major cause of bacterial gastroenteritis in humans that is primarily associated with the consumption of inadequately prepared poultry products, since the organism is generally thought to be asymptomatic in avian species. Unlike many other microorganisms, C. jejuni is capable of performing extensive post-translational modification (PTM) of proteins by N- and O-linked glycosylation, both of which are required for optimal chicken colonization and human virulence. The biosynthesis and attachment of N-glycans to C. jejuni proteins is encoded by the pgl (protein glycosylation) locus, with the PglB oligosaccharyltransferase (OST) enabling en bloc transfer of a heptasaccharide N-glycan from a lipid carrier in the inner membrane to proteins exposed within the periplasm. Seventy-eight C. jejuni glycoproteins (represented by 134 sites of experimentally verified N-glycosylation) have now been identified, and include inner and outer membrane proteins, periplasmic proteins and lipoproteins, which are generally of poorly defined or unknown function. Despite our extensive knowledge of the targets of this apparently widespread process, we still do not fully understand the role N-glycosylation plays biologically, although several phenotypes, including wild-type stress resistance, biofilm formation, motility and chemotaxis have been related to a functional pgl system. Recent work has described enzymatic processes (nitrate reductase NapAB) and antibiotic efflux (CmeABC) as major targets requiring N-glycan attachment for optimal function, and experimental evidence also points to roles in cell binding via glycan-glycan interactions, protein complex formation and protein stability by conferring protection against host and bacterial proteolytic activity. Here we examine the biochemistry of the N-linked glycosylation system, define its currently known protein targets and discuss evidence for the structural and functional roles of this PTM in individual proteins and globally in C. jejuni pathogenesis.

Assigning a role for chemosensory signal transduction in Campylobacter jejuni biofilms using a combined omics approach.

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 division, 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.

RNA Sequencing Data Sets Identifying Differentially Expressed Transcripts during Campylobacter jejuni Biofilm Formation.

Campylobacter 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.

Proteomics Reveals Multiple Phenotypes Associated with N-linked Glycosylation in Campylobacter jejuni.

Campylobacter jejuni is a major gastrointestinal pathogen generally acquired via consumption of poorly prepared poultry. N-linked protein glycosylation encoded by the pgl gene cluster targets >80 membrane proteins and is required for both nonsymptomatic chicken colonization and full human virulence. Despite this, the biological functions of N-glycosylation remain unknown. We examined the effects of pgl gene deletion on the C. jejuni proteome using label-based liquid chromatography/tandem mass spectrometry (LC-MS/MS) and validation using data independent acquisition (DIA-SWATH-MS). We quantified 1359 proteins corresponding to ∼84% of the C. jejuni NCTC 11168 genome, and 1080 of these were validated by DIA-SWATH-MS. Deletion of the pglB oligosaccharyltransferase (ΔpglB) resulted in a significant change in abundance of 185 proteins, 137 of which were restored to their wild-type levels by reintroduction of pglB (Δaaz.batpglB::ΔpglB). Deletion of pglB was associated with significantly reduced abundances of pgl targets and increased stress-related proteins, including ClpB, GroEL, GroES, GrpE and DnaK. pglB mutants demonstrated reduced survival following temperature (4 °C and 46 °C) and osmotic (150 mm NaCl) shock and altered biofilm phenotypes compared with wild-type C. jejuni Targeted metabolomics established that pgl negative C. jejuni switched from aspartate (Asp) to proline (Pro) uptake and accumulated intracellular succinate related to proteome changes including elevated PutP/PutA (proline transport and utilization), and reduced DctA/DcuB (aspartate import and succinate export, respectively). ΔpglB chemotaxis to some substrates (Asp, glutamate, succinate and α-ketoglutarate) was reduced and associated with altered abundance of transducer-like (Tlp) proteins. Glycosylation negative C. jejuni were depleted of all respiration-associated proteins that allow the use of alternative electron acceptors under low oxygen. We demonstrate for the first time that N-glycosylation is required for a specific enzyme activity (Nap nitrate reductase) that is associated with reduced abundance of the NapAB glycoproteins. These data indicate a multifactorial role for N-glycosylation in C. jejuni physiology.

Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system.

N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter 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 Campylobacter 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 individual 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.

Comparative analysis of Staphylococcus epidermidis strains utilizing quantitative and cell surface shaving proteomics.

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. BIOLOGICAL SIGNIFICANCE: 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.

Comparative proteomics and glycoproteomics reveal increased N-linked glycosylation and relaxed sequon specificity in Campylobacter jejuni NCTC11168 O.

Campylobacter 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.

Beyond gene expression: the impact of protein post-translational modifications in bacteria.

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.