Interplay of two small RNAs fine-tunes hierarchical flagella gene expression in Campylobacter jejuni.

Like for many bacteria, flagella are crucial for Campylobacter jejuni motility and virulence. Biogenesis of the flagellar machinery requires hierarchical transcription of early, middle (RpoN-dependent), and late (FliA-dependent) genes. However, little is known about post-transcriptional regulation of flagellar biogenesis by small RNAs (sRNAs). Here, we characterized two sRNAs with opposing effects on C. jejuni filament assembly and motility. We demonstrate that CJnc230 sRNA (FlmE), encoded downstream of the flagellar hook protein, is processed from the RpoN-dependent flgE mRNA by RNase III, RNase Y, and PNPase. We identify mRNAs encoding a flagella-interaction regulator and the anti-sigma factor FlgM as direct targets of CJnc230 repression. CJnc230 overexpression upregulates late genes, including the flagellin flaA, culminating in longer flagella and increased motility. In contrast, overexpression of the FliA-dependent sRNA CJnc170 (FlmR) reduces flagellar length and motility. Overall, our study demonstrates how the interplay of two sRNAs post-transcriptionally fine-tunes flagellar biogenesis through balancing of the hierarchically-expressed components.

Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N-GlcNAcylation.

N-linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human N-linked glycans is the difficulty of installing a single N-acetylglucosamine (GlcNAc), the reducing end sugar of many human-type glycans, onto asparagine in a single step (N-GlcNAcylation). Here, we develop an in vitro method for N-GlcNAcylating proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. We use cell-free protein synthesis (CFPS) to test promiscuous PglB variants previously reported in the literature for the ability to produce N-GlcNAc and successfully determine that PglB with an N311V mutation (PglBN311V) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglBN311V and further optimize the reaction conditions, achieving a 98.6 ± 0.5% glycosylation efficiency. We anticipate this method will expand the glycoengineering toolbox for therapeutic research and biomanufacturing.

Campylobacter jejuni and casein hydrolysate addition: Impact on poultry in vitro cecal microbiota and metabolome.

This study investigates the impact of casein hydrolysates on the poultry ceca inoculated with Campylobacter focusing on microbial molecular preferences for different protein sources in the presence of Campylobacter jejuni. Three casein sources (intact casein (IN), casein enzyme hydrolysate (EH), and casein acid hydrolysate (AH)) were introduced to cecal contents in combination with inoculated C. jejuni in an in vitro model system incubated for 48 h at 42°C under microaerophilic conditions. Samples were collected at 0, 24, and 48 h. Genomic DNA was extracted and amplified using custom dual-indexed primers, followed by sequencing on an Illumina MiSeq platform. The obtained sequencing data were then analyzed via QIIME2-2021.11. Metabolite extracts were analyzed with ultra-high-performance liquid orbitrap chromatography-mass spectrometry (UHPLC-MS). Statistical analysis of metabolites was conducted using MetaboAnalyst 5.0, while functional analysis was performed using Mummichog 2.0 with a significance threshold set at P < 0.00001. DNA sequencing and metabolomic analyses revealed that C. jejuni was most abundant in the EH group. Microbial diversity and richness improved in casein supplemented groups, with core microbial differences observed, compared to non-supplemented groups. Vitamin B-associated metabolites significantly increased in the supplemented groups, displaying distinct patterns in vitamin B6 and B9 metabolism between EH and AH groups (P < 0.05). Faecalibacterium and Phascolarctobacterium were associated with AH and EH groups, respectively. These findings suggest microbial interactions in the presence of C. jejuni and casein supplementation are influenced by microbial community preferences for casein hydrolysates impacting B vitamin production and shaping competitive dynamics within the cecal microbial community. These findings underscore the potential of nutritional interventions to modulate the poultry GIT microbiota for improved health outcomes.

Characterization of Ssc, an N-acetylgalactosamine-containing Staphylococcus aureus surface polysaccharide.

Whole genome sequencing has revealed that the genome of Staphylococcus aureus possesses an uncharacterized 5-gene operon (SAOUHSC_00088-00092 in strain 8325 genome) that encodes factors with functions related to polysaccharide biosynthesis and export, indicating the existence of a new extracellular polysaccharide species. We designate this locus as ssc for staphylococcal surface carbohydrate. We found that the ssc genes were weakly expressed and highly repressed by the global regulator MgrA. To characterize Ssc, Ssc was heterologously expressed in Escherichia coli and extracted by heat treatment. Ssc was also conjugated to AcrA from Campylobacter jejuni in E. coli using protein glycan coupling technology (PGCT). Analysis of the heat-extracted Ssc and the purified Ssc-AcrA glycoconjugate by tandem mass spectrometry revealed that Ssc is likely a polymer consisting of N-acetylgalactosamine. We further demonstrated that the expression of the ssc genes in S. aureus affected phage adsorption and susceptibility, suggesting that Ssc is surface-exposed. IMPORTANCE: Surface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens. Staphylococcus aureus produces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide in S. aureus. Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide.

Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors.

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents.

Proteome profiling of Campylobacter jejuni 81-176 at 37 °C and 42 °C by label-free mass spectrometry.

BACKGROUND: The main natural reservoir for Campylobacter jejuni is the avian intestinal tract. There, C. jejuni multiplies optimally at 42 °C - the avian body temperature. After infecting humans through oral intake, the bacterium encounters the lower temperature of 37 °C in the human intestinal tract. Proteome profiling by label-free mass spectrometry (DIA-MS) was performed to examine the processes which enable C. jejuni 81-176 to thrive at 37 °C in comparison to 42 °C. In total, four states were compared with each other: incubation for 12 h at 37 °C, for 24 h at 37 °C, for 12 h at 42 °C and 24 h at 42 °C. RESULTS: It was shown that the proteomic changes not only according to the different incubation temperature but also to the length of the incubation period were evident when comparing 37 °C and 42 °C as well as 12 h and 24 h of incubation. Altogether, the expression of 957 proteins was quantifiable. 37.1 - 47.3% of the proteins analyzed showed significant differential regulation, with at least a 1.5-fold change in either direction (i.e. log2 FC ≥ 0.585 or log2 FC ≤ -0.585) and an FDR-adjusted p-value of less than 0.05. The significantly differentially expressed proteins could be arranged in 4 different clusters and 16 functional categories. CONCLUSIONS: The C. jejuni proteome at 42 °C is better adapted to high replication rates than that at 37 °C, which was in particular indicated by the up-regulation of proteins belonging to the functional categories "replication" (e.g. Obg, ParABS, and NapL), "DNA synthesis and repair factors" (e.g. DNA-polymerase III, DnaB, and DnaE), "lipid and carbohydrate biosynthesis" (e.g. capsular biosynthesis sugar kinase, PrsA, AccA, and AccP) and "vitamin synthesis, metabolism, cofactor biosynthesis" (e.g. MobB, BioA, and ThiE). The relative up-regulation of proteins with chaperone function (GroL, DnaK, ClpB, HslU, GroS, DnaJ, DnaJ-1, and NapD) at 37 °C in comparison to 42 °C after 12 h incubation indicates a temporary lower-temperature proteomic response. Additionally the up-regulation of factors for DNA uptake (ComEA and RecA) at 37 °C compared to 42 °C indicate a higher competence for the acquisition of extraneous DNA at human body temperature.

Structural and biochemical analysis of the unique interactions of the Campylobacter jejuni CorA channel protein with divalent cations.

CorA is a Mg2+ channel that plays a key role in the homeostasis of intracellular Mg2+ in bacteria and archaea. CorA consists of a cytoplasmic domain and a transmembrane domain and generates a Mg2+ pathway by forming a pentamer in the cell membrane. CorA gating is regulated via negative feedback by Mg2+, which is accommodated by the pentamerization interface of the CorA cytoplasmic domain (CorACD). The Mg2+-binding sites of CorACD differ depending on the species, suggesting that the Mg2+-binding modes and Mg2+-mediated gating mechanisms of CorA vary across prokaryotes. To define the Mg2+-binding mechanism of CorA in the Campylobacter jejuni pathogen, we structurally and biochemically characterized C. jejuni CorACD (cjCorACD). cjCorACD adopts a three-layered α/ß/α structure as observed in other CorA orthologs. Interestingly, cjCorACD exhibited enhanced thermostability in the presence of Ca2+, Ni2+, Zn2+, or Mn2+ in addition to Mg2+, indicating that cjCorACD interacts with diverse divalent cations. This cjCorACD stabilization is mediated by divalent cation accommodation by negatively charged residues located at the bottom of the cjCorACD structure away from the pentamerization interface. Consistently, cjCorACD exists as a monomer irrespective of the presence of divalent cations. We concluded that cjCorACD binds divalent cations in a unique pentamerization-independent manner.

Identification and functional characterization of putative ligand binding domain(s) of JlpA protein of Campylobacter jejuni.

Among the major Surface Exposed Colonization Proteins (SECPs) of Campylobacter jejuni (C. jejuni), Jejuni lipoprotein A (JlpA) plays a crucial role in host cell adhesion specifically by binding to the N-terminal domain of the human heat shock protein 90α (Hsp90α-NTD). Although the JlpA binding to Hsp90α activates NF-κB and p38 MAP kinase pathways, the underlying mechanism of JlpA association with the cellular receptor remains unclear. To this end, we predicted two potential receptor binding sites within the C-terminal domain of JlpA: one spanning from amino acid residues Q332-A354 and the other from S258-T295; however, the latter exhibited weaker binding. To assess the functional attributes of these predicted sequences, we generated two JlpA mutants (JlpAΔ1: S258-T295; JlpAΔ2: Q332-A354) and assessed the Hsp90α-binding affinity-kinetics by in vitro and ex vivo experiments. Our findings confirmed that the residues Q332-A354 are of greater importance in host cell adhesion with a measurable impact on cellular responses. Further, thermal denaturation by circular dichroism (CD) confirmed that the reduced binding affinity of the JlpAΔ2 to Hsp90α is not associated with protein folding or stability. Together, this study provides a possible framework for determining the molecular function of designing rational inhibitors efficiently targeting JlpA.

Campylobacter jejuni Surface-Bound Protease HtrA, but Not the Secreted Protease nor Protease in Shed Membrane Vesicles, Disrupts Epithelial Cell-to-Cell Junctions.

Fundamental functions of the intestinal epithelium include the digestion of food, absorption of nutrients, and its ability to act as the first barrier against intruding microbes. Campylobacter jejuni is a major zoonotic pathogen accounting for a substantial portion of bacterial foodborne illnesses. The germ colonizes the intestines of birds and is mainly transmitted to humans through the consumption of contaminated poultry meat. In the human gastrointestinal tract, the bacterium triggers campylobacteriosis that can progress to serious secondary disorders, including reactive arthritis, inflammatory bowel disease and Guillain-Barré syndrome. We recently discovered that C. jejuni serine protease HtrA disrupts intestinal epithelial barrier functions via cleavage of the tight and adherens junction components occludin, claudin-8 and E-cadherin. However, it is unknown whether epithelial damage is mediated by the secreted soluble enzyme, by HtrA contained in shed outer-membrane vesicles (OMVs) or by another mechanism that has yet to be identified. In the present study, we investigated whether soluble recombinant HtrA and/or purified OMVs induce junctional damage to polarized intestinal epithelial cells compared to live C. jejuni bacteria. By using electron and confocal immunofluorescence microscopy, we show that HtrA-expressing C. jejuni bacteria trigger efficient junctional cell damage, but not soluble purified HtrA or HtrA-containing OMVs, not even at high concentrations far exceeding physiological levels. Instead, we found that only bacteria with active protein biosynthesis effectively cleave junctional proteins, which is followed by paracellular transmigration of C. jejuni through the epithelial cell layer. These findings shed new light on the pathogenic activities of HtrA and virulence strategies of C. jejuni.

Synthesis of 6-deoxy-d-ido-heptopyranose-containing fragments of the Campylobacter jejuni strain CG8486 capsular polysaccharide.

Campylobacters are important causes of gastrointestinal illness and the capsular polysaccharides (CPS) they produce are key virulence factors and targets for vaccine development. We report here the synthesis of two fragments of the Campylobacter jejuni CG8486 strain CPS that contain a rare 6-deoxy-d-ido-heptopyranose residue and, in one target, two O-methyl phosphoramidate (MeOPN) motifs. The synthetic approach features the stereoselective construction of the ß-d-ido-heptopyranoside linkage via glycosylation with a ß-d-galacto-heptopyranoside donor followed by a one-pot sequential C-2 and C-3 inversion. During the syntheses, we uncovered a number of interesting conformational effects with regard to the 6-deoxy-ido-heptopyranose ring, the glycosidic linkage connecting the two monosaccharides, and the MeOPN groups.

Identification of Campylobacter jejuni and Campylobacter coli genes contributing to oxidative stress response using TraDIS analysis.

BACKGROUND: Campylobacter jejuni and Campylobacter coli are the major causative agents of bacterial gastroenteritis worldwide and are known obligate microaerophiles. Despite being sensitive to oxygen and its reduction products, both species are readily isolated from animal food products kept under atmospheric conditions where they face high oxygen tension levels. RESULTS: In this study, Transposon Directed Insertion-site Sequencing (TraDIS) was used to investigate the ability of one C. jejuni strain and two C. coli strains to overcome oxidative stress, using H2O2 to mimic oxidative stress. Genes were identified that were required for oxidative stress resistance for each individual strain but also allowed a comparison across the three strains. Mutations in the perR and ahpC genes were found to increase Campylobacter tolerance to H2O2. The roles of these proteins in oxidative stress were previously known in C. jejuni, but this data indicates that they most likely play a similar role in C. coli. Mutation of czcD decreased Campylobacter tolerance to H2O2. The role of CzcD, which functions as a zinc exporter, has not previously been linked to oxidative stress. The TraDIS data was confirmed using defined deletions of perR and czcD in C. coli 15-537360. CONCLUSIONS: This is the first study to investigate gene fitness in both C. jejuni and C. coli under oxidative stress conditions and highlights both similar roles for certain genes for both species and highlights other genes that have a role under oxidative stress.

Supplementation of lactobacillus-fermented rapeseed meal in broiler diet reduces Campylobacter jejuni cecal colonization and limits the l-tryptophan and l-histidine biosynthesis pathways.

BACKGROUND: Campylobacter jejuni (C. jejuni), a widely distributed global foodborne pathogen, primarily linked with contaminated chicken meat, poses a significant health risk. Reducing the abundance of this pathogen in poultry meat is challenging but essential. This study assessed the impact of Lactobacillus-fermented rapeseed meal (LFRM) on broilers exposed to C. jejuni-contaminated litter, evaluating growth performance, Campylobacter levels, and metagenomic profile. RESULTS: By day 35, the litter contamination successfully colonized broilers with Campylobacter spp., particularly C. jejuni. In the grower phase, LFRM improved (P < 0.05) body weight and daily weight gain, resulting in a 9.2% better feed conversion ratio during the pre-challenge period (the period before artificial infection; days 13-20). The LFRM also reduced the C. jejuni concentration in the ceca (P < 0.05), without altering alpha and beta diversity. However, metagenomic data analysis revealed LFRM targeted a reduction in the abundance of C. jejuni biosynthetic pathways of l-tryptophan and l-histidine and gene families associated with transcription and virulence factors while also possibly leading to selected stress-induced resistance mechanisms. CONCLUSION: The study demonstrated that LFRM inclusion improved growth and decreased cecal Campylobacter spp. concentration and the relative abundance of pivotal C. jejuni genes. Performance benefits likely resulted from LFRM metabolites. At the molecular level, LFRM may have reduced C. jejuni colonization, likely by decreasing the abundance of energy transduction and l-histidine and l-tryptophan biosynthesis genes otherwise required for bacterial survival and increased virulence. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Revisiting bacterial cytolethal distending toxin structure and function.

Cytolethal distending toxins (CDTs) are intracellular-acting bacterial genotoxins generated by a diverse group of mucocutaneous human pathogens. CDTs must successfully bind to the plasma membrane of host cells in order to exert their modulatory effects. Maximal toxin activity requires all three toxin subunits, CdtA, CdtB, and CdtC, which, based primarily on high-resolution structural data, are believed to preassemble into a tripartite complex necessary for toxin activity. However, biologically active toxin has not been experimentally demonstrated to require assembly of the three subunits into a heterotrimer. Here, we experimentally compared concentration-dependent subunit interactions and toxin cellular activity of the Campylobacter jejuni CDT (Cj-CDT). Co-immunoprecipitation and dialysis retention experiments provided evidence for the presence of heterotrimeric toxin complexes, but only at concentrations of Cj-CdtA, Cj-CdtB, and Cj-CdtC several logs higher than required for Cj-CDT-mediated arrest of the host cell cycle at the G2/M interface, which is triggered by the endonuclease activity associated with the catalytic Cj-CdtB subunit. Microscale thermophoresis confirmed that Cj-CDT subunit interactions occur with low affinity. Collectively, our data suggest that at the lowest concentrations of toxin sufficient for arrest of cell cycle progression, mixtures of Cj-CdtA, Cj-CdtB, and Cj-CdtC consist primarily of non-interacting, subunit monomers. The lack of congruence between toxin tripartite structure and cellular activity suggests that the widely accepted model that CDTs principally intoxicate host cells as preassembled heterotrimeric structures should be revisited.

Molecular structures mediating adhesion of Campylobacter jejuni to abiotic and biotic surfaces.

Microaerophilic, Gram-negative Campylobacter jejuni is the causative agent of campylobacteriosis, the most common bacterial gastrointestinal infection worldwide. Adhesion is the crucial first step in both infection or interaction with the host and biofilm formation, and is a critical factor for bacterial persistence. Here we describe the proteins and other surface structures that promote adhesion to various surfaces, including abiotic surfaces, microorganisms, and animal and human hosts. In addition, we provide insight into the distribution of adhesion proteins among strains from different ecological niches and highlight unexplored proteins involved in C. jejuni adhesion. Protein-protein, protein-glycan, and glycan-glycan interactions are involved in C. jejuni adhesion, with different factors contributing to adhesion to varying degrees under different circumstances. As adhesion is essential for survival and persistence, it represents an interesting target for C. jejuni control. Knowledge of the adhesion process is incomplete, as different molecular and functional aspects have been studied for different structures involved in adhesion. Therefore, it is important to strive for an integration of different approaches to obtain a clearer picture of the adhesion process on different surfaces and to consider the involvement of proteins, glycoconjugates, and polysaccharides and their cooperation.

Survival of Campylobacter jejuni during in vitro culture with mixed bovine ruminal microorganisms in the presence of methanogen inhibitors.

Foodborne pathogen Campylobacter jejuni has been associated with ruminants. The objectives of this experiment were to determine C. jejuni survivability in mixed in vitro rumen microbial populations and the impact on methane production with or without methane inhibitors 2-bromosulfonate (BES) and/or sodium nitrate. When inoculated into rumen microbial populations without or with 0.5 mM BES, 5.0 mM nitrate or their combination, C. jejuni viability decreased from 4.7 ± 0.1 log10 colony forming units (CFU)/mL after 24 h. Loss of C. jejuni viability was greater (P < 0.05) when incubated under 100% CO2 compared to 50% H2:50% CO2, decreasing 1.46 versus 1.15 log units, respectively. C. jejuni viability was also decreased (P < 0.05) by more than 0.43 log units by the anti-methanogen treatments. Rumen microbial populations produced less methane (P = 0.05) when incubated with than without C. jejuni regardless of whether under 100% CO2 or 50% H2:50% CO2. For either gas phase, nitrate was decreased (13.2 versus 37.9%) by the anti-methanogen treatments versus controls although not always significant. C. jejuni-inoculated populations metabolized 16.4% more (P < 0.05) nitrate under H2:CO2 versus 100% CO2. Apparently, C. jejuni can compete for H2 with methanogens but has limited survivability under rumen conditions.

Heterogeneity and Compositional Diversities of Campylobacter jejuni Outer Membrane Vesicles (OMVs) Drive Multiple Cellular Uptake Processes.

Naturally secreted outer membrane vesicles (OMVs) from gut microbes carry diverse cargo, including proteins, nucleic acids, toxins, and many unidentified secretory factors. Bacterial OMVs can shuttle molecules across different cell types as a generalized secretion system, facilitating bacterial pathogenicity and self-survival. Numerous mucosal pathogens, including Campylobacter jejuni (C. jejuni), share a mechanism of harmonized secretion of major virulence factors. Intriguingly, as a common gut pathogen, C. jejuni lacks some classical virulence-associated secretion systems; alternatively, it often employs nanosized lipid-bound OMVs as an intensive strategy to deliver toxins, including secretory proteins, into the target cells. To better understand how the biophysical and compositional attributes of natural OMVs of C. jejuni regulate their cellular interactions to induce a biologically relevant host response, we conducted an in-depth morphological and compositional analysis of naturally secreted OMVs of C. jejuni. Next, we focused on understanding the mechanism of host cell-specific OMVs uptake from the extracellular milieu. We showed that intracellular perfusion of OMVs is mediated by cytosolic as well as multiple endocytic uptake processes due to the heterogenic nature, abundance of surface proteins, and membrane phospholipids acquired from the source bacteria. Furthermore, we used human and avian cells as two different host targets to provide evidence of target cell-specific preferential uptake of OMVs. Together, the present study provides insight into the unique functionality of natural OMVs of C. jejuni at the cellular interface, upholding their potential for multimodal use as prophylactic and therapeutic carriers.

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

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.

Exploring the inhibition of the soluble lytic transglycosylase Cj0843c of Campylobacter jejuni via targeting different sites with different scaffolds.

Bacterial lytic transglycosylases (LTs) contribute to peptidoglycan cell wall metabolism and are potential drug targets to potentiate ß-lactam antibiotics to overcome antibiotic resistance. Since LT inhibitor development is underexplored, we probed 15 N-acetyl-containing heterocycles in a structure-guided fashion for their ability to inhibit and bind to the Campylobacter jejuni LT Cj0843c. Ten GlcNAc analogs were synthesized with substitutions at the C1 position, with two having an additional modification at the C4 or C6 position. Most of the compounds showed weak inhibition of Cj0843c activity. Compounds with alterations at the C4 position, replacing the -OH with a -NH2 , and C6 position, the addition of a -CH3 , yielded improved inhibitory efficacy. All 10 GlcNAc analogs were crystallographically analyzed via soaking experiments using Cj0843c crystals and found to bind to the +1 +2 saccharide subsites with one of them additionally binding to the -2 -1 subsite region. We also probed other N-acetyl-containing heterocycles and found that sialidase inhibitors N-acetyl-2,3-dehydro-2-deoxyneuraminic acid and siastatin B inhibited Cj0843c weakly and crystallographically bound to the -2 -1 subsites. Analogs of the former also showed inhibition and crystallographic binding and included zanamivir amine. This latter set of heterocycles positioned their N-acetyl group in the -2 subsite with additional moieties interacting in the -1 subsite. Overall, these results could provide novel opportunities for LT inhibition via exploring different subsites and novel scaffolds. The results also increased our mechanistic understanding of Cj0843c regarding peptidoglycan GlcNAc subsite binding preferences and ligand-dependent modulation of the protonation state of the catalytic E390.

The dCache Domain of the Chemoreceptor Tlp1 in Campylobacter jejuni Binds and Triggers Chemotaxis toward Formate.

Chemotaxis is an important virulence factor in some enteric pathogens, and it is involved in the pathogenesis and colonization of the host. However, there is limited knowledge regarding the environmental signals that promote chemotactic behavior and the sensing of these signals by chemoreceptors. To date, there is no information on the ligand molecule that directly binds to and is sensed by Campylobacter jejuni Tlp1, which is a chemoreceptor with a dCache-type ligand-binding domain (LBD). dCache (double Calcium channels and chemotaxis receptor) is the largest group of sensory domains in bacteria, but the dCache-type chemoreceptor that directly binds to formate has not yet been discovered. In this study, formate was identified as a direct-binding ligand of C. jejuni Tlp1 with high sensing specificity. We used the strategy of constructing a functional hybrid receptor of C. jejuni Tlp1 and the Escherichia coli chemoreceptor Tar to screen for the potential ligand of Tlp1, with the binding of formate to Tlp1-LBD being verified using isothermal titration calorimetry. Molecular docking and experimental analyses indicated that formate binds to the membrane-proximal pocket of the dCache subdomain. Chemotaxis assays demonstrated that formate elicits robust attractant responses of the C. jejuni strain NCTC 11168, specifically via Tlp1. The chemoattraction effect of formate via Tlp1 promoted the growth of C. jejuni, especially when competing with Tlp1- or CheY-knockout strains. Our study reveals the molecular mechanisms by which C. jejuni mediates chemotaxis toward formate, and, to our knowledge, is the first report on the high-specificity binding of the dCache-type chemoreceptor to formate as well as the physiological role of chemotaxis toward formate. IMPORTANCE Chemotaxis is important for Campylobacter jejuni to colonize favorable niches in the gastrointestinal tract of its host. However, there is still a lack of knowledge about the ligand molecules for C. jejuni chemoreceptors. The dCache-type chemoreceptor, namely, Tlp1, is the most conserved chemoreceptor in C. jejuni strains; however, the direct-binding ligand(s) triggering chemotaxis has not yet been discovered. In the present study, we found that the ligand that binds directly to Tlp1-LBD with high specificity is formate. C. jejuni exhibits robust chemoattraction toward formate, primarily via Tlp1. Tlp1 is the first reported dCache-type chemoreceptor that specifically binds formate and triggers strong chemotaxis. We further demonstrated that the formate-mediated promotion of C. jejuni growth is correlated with Tlp1-mediated chemotaxis toward formate. Our work provides important insights into the mechanism and physiological function of chemotaxis toward formate and will facilitate further investigations into the involvement of microbial chemotaxis in pathogen-host interactions.

Campylobacter jejuni: targeting host cells, adhesion, invasion, and survival.

Campylobacter jejuni, causing strong enteritis, is an unusual bacterium with numerous peculiarities. Chemotactically controlled motility in viscous milieu allows targeted navigation to intestinal mucus and colonization. By phase variation, quorum sensing, extensive O-and N-glycosylation and use of the flagellum as type-3-secretion system C. jejuni adapts effectively to environmental conditions. C. jejuni utilizes proteases to open cell-cell junctions and subsequently transmigrates paracellularly. Fibronectin at the basolateral side of polarized epithelial cells serves as binding site for adhesins CadF and FlpA, leading to intracellular signaling, which again triggers membrane ruffling and reduced host cell migration by focal adhesion. Cell contacts of C. jejuni results in its secretion of invasion antigens, which induce membrane ruffling by paxillin-independent pathway. In addition to fibronectin-binding proteins, other adhesins with other target structures and lectins and their corresponding sugar structures are involved in host-pathogen interaction. Invasion into the intestinal epithelial cell depends on host cell structures. Fibronectin, clathrin, and dynein influence cytoskeletal restructuring, endocytosis, and vesicular transport, through different mechanisms. C. jejuni can persist over a 72-h period in the cell. Campylobacter-containing vacuoles, avoid fusion with lysosomes and enter the perinuclear space via dynein, inducing signaling pathways. Secretion of cytolethal distending toxin directs the cell into programmed cell death, including the pyroptotic release of proinflammatory substances from the destroyed cell compartments. The immune system reacts with an inflammatory cascade by participation of numerous immune cells. The development of autoantibodies, directed not only against lipooligosaccharides, but also against endogenous gangliosides, triggers autoimmune diseases. Lesions of the epithelium result in loss of electrolytes, water, and blood, leading to diarrhea, which flushes out mucus containing C. jejuni. Together with the response of the immune system, this limits infection time. Based on the structural interactions between host cell and bacterium, the numerous virulence mechanisms, signaling, and effects that characterize the infection process of C. jejuni, a wide variety of targets for attenuation of the pathogen can be characterized. The review summarizes strategies of C. jejuni for host-pathogen interaction and should stimulate innovative research towards improved definition of targets for future drug development. KEY POINTS: • Bacterial adhesion of Campylobacter to host cells and invasion into host cells are strictly coordinated processes, which can serve as targets to prevent infection. • Reaction and signalling of host cell depend on the cell type. • Campylobacter virulence factors can be used as targets for development of antivirulence drug compounds.