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The Campylobacter jejuni bacterium, which causes foodborne enteritis in humans, expresses the uncharacterized protein CJ0600. Based on sequence analysis, CJ0600 has been proposed to function as a 1-aminocyclopropane-1-carboxylate (ACC) deaminase (AccDA) or cysteine desulfhydrase (CysDS). However, it has never been investigated whether CJ0600 exerts AccDA or CysDS activity or how CJ0600 mediates its enzymatic activity. To reveal the structural features necessary for the function of CJ0600, we determined the crystal structure of CJ0600 and characterized its enzymatic activity. CJ0600 contains two domains and features an interdomain pocket, which accommodates a pyridoxal 5'-phosphate (PLP) molecule as a Schiff base with its lysine residue (K35), as observed in its structural homologs, including AccDA, CysDS, and serine deaminase (SerDA). However, unlike its structural homologs, CJ0600 exists as a monomer and exhibits unique structural features throughout its structure. Moreover, CJ0600 contains unique active site residues that are not observed in AccDA, CysDS, or SerDA. Consistently, phylogenetic analysis indicates that CJ0600 and its orthologs are evolutionarily distinct from AccDA, CysDS, and SerDA. Indeed, CJ0600 showed no CysDS or SerDA activity and extremely weak AccDA activity. These observations suggest that CJ0600 functions as a unique PLP-dependent enzyme that has not been reported., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bacteria power rotation of an extracellular flagellar filament for swimming motility. Thousands of flagellin subunits compose the flagellar filament, which extends several microns from the bacterial surface. It is unclear whether bacteria actively control filament length. Many polarly flagellated bacteria produce shorter flagellar filaments than peritrichous bacteria, and FlaG has been reported to limit flagellar filament length in polar flagellates. However, a mechanism for how FlaG may function is unknown. We observed that deletion of flaG in the polarly flagellated pathogens Vibrio cholerae, Pseudomonas aeruginosa , and Campylobacter jejuni caused extension of flagellar filaments to lengths comparable to peritrichous bacteria. Using C. jejuni as a model to understand how FlaG controls flagellar filament length, we found that FlaG and FliS chaperone-flagellin complexes antagonize each other for interactions with FlhA in the flagellar type III secretion system (fT3SS) export gate. FlaG interacted with an understudied region of FlhA, and this interaction appeared to be enhanced in Δ fliS and FlhA FliS-binding mutants. Our data support that FlaG evolved in polarly flagellated bacteria as an antagonist to interfere with the ability of FliS to interact with and deliver flagellins to FlhA in the fT3SS export gate to control flagellar filament length so that these bacteria produce relatively shorter flagella than peritrichous counterparts. This mechanism is similar to how some gatekeepers in injectisome T3SSs prevent chaperones from delivering effector proteins until completion of the T3SS and host contact occurs. Thus, flagellar and injectisome T3SSs have convergently evolved protein antagonists to negatively impact respective T3SSs to secrete their major terminal substrates., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Cytolethal distending toxin (CDT) belongs to the genotoxin family and is closely related to Campylobacter jejuni-associated gastroenteritis. We recently reported that CDT triggers the danger-associated molecular pattern (DAMP) signaling to exert deleterious effects on host cells. However, how CDT traffics in cells and the mechanism of CDT intoxication remain to be elucidated., Methods: Recombinant CDT subunits (CdtA, CdtB, and CdtC) were purified, and their activity was characterized in gastrointestinal cells. Molecular approaches and image tracking were employed to analyze the delivery of CDT in host cells., Results: In this study, we found that CDT interacts with the receptor of advanced glycation end products (RAGE) and high mobility group box 1 (HMGB1) to enter the cells. Our results further showed that CdtB transport in cells through the dynamin-dependent endocytic pathway and lysosome is involved in this process. Conversely, blockage of RAGE signaling resulted in a reduction in CDT-arrested cell cycles, indicating that RAGE is involved in CDT intracellular transport and its subsequent pathogenesis., Conclusion: Our results demonstrate that RAGE is important for CDT trafficking in the cells. These findings expand our understanding of important issues related to host cell intoxication by C. jejuni CDT., Competing Interests: Declaration of competing interest The authors report no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Species within the Campylobacter genus are recognized as emerging human pathogens. Common to all known members of the genus is the presence of an asparagine-linked glycosylation pathway encoded by the pgl operon. Campylobacter species are divided into two major groups, Group I and Group II. To date, most biochemical studies have focused on the Group I species including Campylobacter jejuni . We recently reported that the Group II Campylobacter concisus pathway deviates from that of Group I by the inclusion of a C-6″-oxidized GalNAc (GalNAcA) at the third position installed by PglJ. Herein, we investigate the diversification of the PglH enzymes that act subsequent to installation of GalNAcA. The majority of pgl operons from Group II species, including C. concisus , encode two GT-B fold glycosyltransferases (GTs), PglH1 and PglH2. As the functions of these GTs were not clear by simple comparison of their sequences to that of C. jejuni PglH, further analyses were required. We show that subsequent to the action of PglJ, PglH2 installs the next HexNAc followed by PglH1 adding a single sugar. These steps diverge from the C. jejuni pathway not only in the identity of the sugar donors (UDP-GlcNAc) but also in installing single sugars rather than acting processively. These biochemical studies were extended via bioinformatics to identify sequence signatures that provide predictive capabilities for unraveling the prokaryotic glycan landscape. Phylogenetic analysis showed early divergence between the C. jejuni PglH orthologs and C. concisus PglH1/PglH2 orthologs, leading to diversification of the final glycan.

Infection with Campylobacter jejuni is the major cause of human gastroenteritis in the United States and Europe, leading to debilitating autoimmune sequelae in many cases. While considerable progress has been made in detailing the infectious cycle of C. jejuni , a full understanding of the molecular mechanisms responsible for virulence remains to be elucidated. Here, we apply a novel approach by modulating protein expression on the pathogen's ribosomes by inactivating a highly conserved rRNA methyltransferase. Loss of the RsmA methyltransferase results in a more motile strain with greater adhesive and cell-invasive properties. These phenotypical effects correlate with enhanced expression of specific proteins related to flagellar formation and function, together with enzymes involved in cell wall/membrane and amino acid synthesis. Despite the enhancement of certain virulent traits, the null strain grows poorly on minimal media and is rapidly out-competed by the wild-type strain. Complementation with an active copy of the rsmA gene rescues most of the traits changed in the mutant. However, the complemented strain overexpresses rsmA and displays new flaws, including loss of the spiral cell shape, which is distinctive for C. jejuni . Proteins linked with altered virulence and morphology are identified here by mass spectrometry proteomic analyses of the strains., Competing Interests: The authors declare no conflicts of interest.

CorA is a Mg 2+ channel that plays a key role in the homeostasis of intracellular Mg 2+ in bacteria and archaea. CorA consists of a cytoplasmic domain and a transmembrane domain and generates a Mg 2+ pathway by forming a pentamer in the cell membrane. CorA gating is regulated via negative feedback by Mg 2+ , which is accommodated by the pentamerization interface of the CorA cytoplasmic domain (CorA CD ). The Mg 2+ -binding sites of CorA CD differ depending on the species, suggesting that the Mg 2+ -binding modes and Mg 2+ -mediated gating mechanisms of CorA vary across prokaryotes. To define the Mg 2+ -binding mechanism of CorA in the Campylobacter jejuni pathogen, we structurally and biochemically characterized C. jejuni CorA CD (cjCorA CD ). cjCorA CD adopts a three-layered α/β/α structure as observed in other CorA orthologs. Interestingly, cjCorA CD exhibited enhanced thermostability in the presence of Ca 2+ , Ni 2+ , Zn 2+ , or Mn 2+ in addition to Mg 2+ , indicating that cjCorA CD interacts with diverse divalent cations. This cjCorA CD stabilization is mediated by divalent cation accommodation by negatively charged residues located at the bottom of the cjCorA CD structure away from the pentamerization interface. Consistently, cjCorA CD exists as a monomer irrespective of the presence of divalent cations. We concluded that cjCorA CD binds divalent cations in a unique pentamerization-independent manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Campylobacter was considered asaccharolytic, but is now known to carry saccharide metabolization pathways for L-fucose and d-glucose. We hypothesized that these clusters are beneficial for Campylobacter niche adaptation and may help establish human infection. We investigated the distribution of d-glucose and L-fucose clusters among ∼9600 C. jejuni and C. coli genomes of different isolation sources in the Netherlands, the United Kingdom, the United States of America and Finland. The L-fucose utilization cluster was integrated at the same location in all C. jejuni and C. coli genomes, and was flanked by the genes rpoB, rpoC, rspL, repsG and fusA, which are associated with functions in transcription as well as translation and in acquired drug resistance. In contrast, the flanking regions of the d-glucose utilization cluster were variable among the isolates, and integration sites were located within one of the three different 16S23S ribosomal RNA areas of the C. jejuni and C. coli genomes. In addition, we investigated whether acquisition of the L-fucose utilization cluster could be due to horizontal gene transfer between the two species and found three isolates for which this was the case: one C. jejuni isolate carrying a C. coli L-fucose cluster, and two C. coli isolates which carried a C. jejuni L-fucose cluster. Furthermore, L-fucose utilization cluster alignments revealed multiple frameshift mutations, most of which were commonly found in the non-essential genes for L-fucose metabolism, namely, Cj0484 and Cj0489. These findings support our hypothesis that the L-fucose cluster was integrated multiple times across the C. coli/C. jejuni phylogeny. Notably, association analysis using the C. jejuni isolates from the Netherlands showed a significant correlation between human C. jejuni isolates and C. jejuni isolates carrying the L-fucose utilization cluster. This correlation was even stronger when the Dutch isolates were combined with the isolates from the UK, the USA and Finland. No such correlations were observed for C. coli or for the d-glucose cluster for both species. This research provides insight into the spread and host associations of the L-fucose and d-glucose utilization clusters in C. jejuni and C. coli, and the potential benefits in human infection and/or proliferation in humans, conceivably after transmission from any reservoir., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Campylobacter jejuni ( C. jejuni ) is one of the most common causes of foodborne infections worldwide and a major contributor to diarrheal diseases. This study aimed to explore the ability of commensal gut bacteria to control C. jejuni infection. Bacterial strains from the intestinal mucosa of broilers were screened in vitro against C. jejuni ATCC BAA1153. The cell-free supernatant (CFS) of Ligilactobacillus salivarius UO.C249 showed potent dose-dependent antimicrobial activity against the pathogen, likely due to the presence of bacteriocin-like moieties, as confirmed by protease treatment. Genome and exoproteome analyses revealed the presence of known bacteriocins, including Abp118. The genome of Lg. salivarius UO.C249 harbors a 1.8-Mb chromosome and a 203-kb megaplasmid. The strain was susceptible to several antibiotics and had a high survival rate in the simulated chicken gastrointestinal tract (GIT). Post-protease treatment revealed residual inhibitory activity, suggesting alternative antimicrobial mechanisms. Short-chain fatty acid (SCFA) quantification confirmed non-inhibitory levels of acetic (24.4 ± 1.2 mM), isovaleric (34 ± 1.0 µM), and butyric (32 ± 2.5 µM) acids. Interestingly, extracellular vesicles (EVs) isolated from the CFS of Lg. salivarius UO.C249 were found to inhibit C. jejuni ATCC BAA-1153. Proteome profiling of these EVs revealed the presence of unique proteins distinct from bacteriocins identified in CFS. The majority of the identified proteins in EVs are located in the membrane and play roles in transmembrane transport and peptidoglycan degradation, peptidase, proteolysis, and hydrolysis. These findings suggest that although bacteriocins are a primary antimicrobial mechanism, EV production also contributes to the inhibitory activity of Lg. salivarius UO.C249 against C. jejuni ., Importance: Campylobacter jejuni ( C. jejuni ) is a major cause of gastroenteritis and a global public health concern. The increasing antibiotic resistance and lack of effective alternatives in livestock production pose serious challenges for controlling C. jejuni infections. Therefore, alternative strategies are needed to control this pathogen, especially in the poultry industry where it is prevalent and can be transmitted to humans through contaminated food products. In this study, Ligilactobacillus salivarius UO.C249 isolated from broiler intestinal mucosa inhibited C. jejuni and exhibited important probiotic features. Beyond bacteriocins, Lg. salivarius UO.C249 secretes antimicrobial extracellular vesicles (EVs) with a unique protein set distinct from bacteriocins that are involved in transmembrane transport and peptidoglycan degradation. Our findings suggest that beyond bacteriocins, EV production is also a distinct inhibitory signaling mechanism used by Lg. salivarius UO.C249 to control C. jejuni . These findings hold promise for the application of probiotic EVs for pathogen control., Competing Interests: R.H., S.M., and W.M. are co-inventors and co-applicants with the University of Ottawa on a patent application (US Patent Application Number 63/607,682, filed 08 December 2023) entitled "LIGILACTOBACILLUS PROBIOTICS, LIGILACTOBACILLUS EXTRACELLULAR VESICLES AND METHODS OF USING SAME" that covers the inhibitory activity of probiotic extracellular vesicles. The remaining authors declare no competing interests.

Campylobacter jejuni and Arcobacter butzleri are microaerobic food-borne human gastrointestinal pathogens that mainly cause diarrheal disease. These related species of the Campylobacteria class face variable atmospheric environments during infection and transmission, ranging from nearly anaerobic to aerobic conditions. Consequently, their lifestyles require that both pathogens need to adjust their metabolism and respiration to the changing oxygen concentrations of the colonization sites. Our transcriptomic and proteomic studies revealed that C. jejuni and A. butzleri, lacking a Campylobacteria -specific regulatory protein, C. jejuni Cj1608, or a homolog, A. butzleri Abu0127, are unable to reprogram tricarboxylic acid cycle or respiration pathways, respectively, to produce ATP efficiently and, in consequence, adjust growth to changing oxygen supply. We propose that these Campylobacteria energy and metabolism regulators (CemRs) are long-sought transcription factors controlling the metabolic shift related to oxygen availability, essential for these bacteria's survival and adaptation to the niches they inhabit. Besides their significant universal role in Campylobacteria , CemRs, as pleiotropic regulators, control the transcription of many genes, often specific to the species, under microaerophilic conditions and in response to oxidative stress., Importance: C. jejuni and A. butzleri are closely related pathogens that infect the human gastrointestinal tract. In order to infect humans successfully, they need to change their metabolism as nutrient and respiratory conditions change. A regulator called CemR has been identified, which helps them adapt their metabolism to changing conditions, particularly oxygen availability in the gastrointestinal tract so that they can produce enough energy for survival and spread. Without CemR, these bacteria, as well as a related species, Helicobacter pylori , produce less energy, grow more slowly, or, in the case of C. jejuni , do not grow at all. Furthermore, CemR is a global regulator that controls the synthesis of many genes in each species, potentially allowing them to adapt to their ecological niches as well as establish infection. Therefore, the identification of CemR opens new possibilities for studying the pathogenicity of C. jejuni and A. butzleri ., Competing Interests: The authors declare no conflict of interest.

Background: Deoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly found in cereals and grains worldwide. The presence of this fungal secondary-metabolite raises public-health concerns at both the agriculture and food industry level. Recently, we have shown that DON has a negative impact on gut integrity, a feature also noticed for Campylobacter (C.) jejuni. We further demonstrated that DON increased the load of C. jejuni in the gut and inner organs. In contrast, feeding the less toxic DON metabolite deepoxy-deoxynivalenol (DOM-1) to broilers reduced the Campylobacter load in vivo. Consequently, it can be hypothesized that DON and DOM-1 have a direct effect on the growth profile of C. jejuni. The aim of the present study was to further resolve the nature of this interaction in vitro by co-incubation and RNA-sequencing., Results: The co-incubation of C. jejuni with DON resulted in significantly higher bacterial growth rates from 30 h of incubation onwards. On the contrary, the co-incubation of C. jejuni with DOM-1 reduced the CFU counts, indicating that this DON metabolite might contribute to reduce the burden of C. jejuni in birds, altogether confirming in vivo data. Furthermore, the transcriptomic profile of C. jejuni following incubation with either DON or DOM-1 differed. Co-incubation of C. jejuni with DON significantly increased the expression of multiple genes which are critical for Campylobacter growth, particularly members of the Flagella gene family, frr (ribosome-recycling factor), PBP2 futA-like (Fe 3+ periplasmic binding family) and PotA (ATP-binding subunit). Flagella are responsible for motility, biofilm formation and host colonization, which may explain the high Campylobacter load in the gut of DON-fed broiler chickens. On the contrary, DOM-1 downregulated the Flagella gene family and upregulated ribosomal proteins., Conclusion: The results highlight the adaptive mechanisms involved in the transcriptional response of C. jejuni to DON and its metabolite DOM-1, based on the following effects: (a) ribosomal proteins; (b) flagellar proteins; (c) engagement of different metabolic pathways. The results provide insight into the response of an important intestinal microbial pathogen against DON and lead to a better understanding of the luminal or environmental acclimation mechanisms in chickens., (© 2024. The Author(s).)

Heme trafficking is essential for cellular function, yet mechanisms of transport and/or heme interaction are not well defined. The System I and System II bacterial cytochrome c biogenesis pathways are developing into model systems for heme trafficking due to their functions in heme transport, heme stereospecific positioning, and mediation of heme attachment to apocytochrome c. Here we focus on the System II pathway, CcsBA, that is proposed to be a bi-functional heme transporter and holocytochrome c synthase. An extensive structure-function analysis of recombinantly expressed Helicobacter pylori and Campylobacter jejuni CcsBAs revealed key residues required for heme interaction and holocytochrome c synthase activity. Homologous residues were previously identified to be required for heme interaction in Helicobacter hepaticus CcsBA. This study provides direct, biochemical evidence that mechanisms of heme interaction are conserved, leading to the proposal that the CcsBA WWD heme-handling domain represents a novel target for therapeutics., (© 2024. The Author(s).)

Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 10 5  CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates., Competing Interests: Declaration of competing interest Our manuscript titled “Phenotypic, Genotypic, and Proteomic Variations of Poor and Robust Colonizing Campylobacter jejuni Strains” reflects the views of the authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 (PglB N311V ) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglB N311V 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.

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., (© 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

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., (© 2024. The Author(s).)

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. log 2 FC ≥ 0.585 or log 2 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., (© 2024. The Author(s).)

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., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Olson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jug, Šikić Pogačar, Sterniša, Tumpej, Karničar, Turk, Langerholc, Sabotič and Klančnik.)

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 (JlpAΔ 1 : S258-T295; JlpAΔ 2 : 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 JlpAΔ 2 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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 H 2 O 2 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 H 2 O 2 . 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 H 2 O 2 . 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., (© 2024. The Author(s).)

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 β-d-ido-heptopyranoside linkage via glycosylation with a β-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., Competing Interests: Declaration of competing interest Todd L. Lowary reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Todd Lowary reports financial support was provided by Canadian Glycomics Network. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 .

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 G 2 /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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Chen, Ang, Crowder, Brieher and Blanke.)

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.

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 lytic transglycosylases (LTs) contribute to peptidoglycan cell wall metabolism and are potential drug targets to potentiate β-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 -NH 2 , and C6 position, the addition of a -CH 3 , 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., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

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 ( d ouble Ca lcium channels and che motaxis 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., Competing Interests: The authors declare no conflict of interest.

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., (© 2023. The Author(s).)

C. jejuni is an important food-borne pathogen displaying high genetic diversity, substantially based on natural transformation. The mechanism of DNA uptake from the environment depends on a type II secretion/type IV pilus system, whose components are partially known. Here, we quantified DNA uptake in C. jejuni at the single cell level and observed median transport capacities of approximately 30 kb per uptake location. The process appeared to be limited by the initialization of DNA uptake, was finite, and, finalized within 30 min of contact to DNA. Mutants lacking either the outer membrane pore PilQ or the inner membrane channel ComEC were deficient in natural transformation. The periplasmic DNA binding protein ComE was negligible for DNA uptake, which is in contrast to its proposed function. Intriguingly, a mutant lacking the unique periplasmic protein Cj0683 displayed rare but fully functional DNA uptake events. We conclude that Cj0683 was essential for the efficient initialization of DNA uptake, consistent with the putative function as a competence pilus protein. Unravelling features important in natural transformation might lead to target identification, reducing the adaptive potential of pathogens.

In Gram-negative bacteria, lipopolysaccharide (LPS) is an essential component of the asymmetric outer membrane (OM). LptE is an OM lipoprotein that forms a complex with the β-barrel OM protein, LptD. Incorporation of LPS into the OM outer leaflet is essential for bacterial viability, and mediated by the LptD/E complex. The genome of Campylobacter jejuni, a major foodborne pathogen, contains over 20 putative lipoproteins including Cj1090c. Here, we report the crystal structure of Cj1090c at 2.4 Å resolution, revealing structural evidence for LptE in C. jejuni. The analysis of this crystal structure, along with the genomic context, allows us to propose the C. jejuni LPS transport system for the first time, and permits for discussion of the features of the LptD/E complex of C. jejuni., (© 2022 Wiley Periodicals LLC.)

Background: Campylobacter jejuni ( C. jejuni ) is one of the most common causes of bacterial gastroenteritis worldwide. One sequela of this infection is the development of post-infectious irritable bowel syndrome (PI-IBS). It has been suggested that a dysfunctional intestinal barrier may promote IBS development. We aimed to test this hypothesis against the background of the leaky gut concept for low-grade inflammation in PI-IBS., Methods: We identified patients with persistent PI-IBS symptoms after C. jejuni infection. During sigmoidoscopy, forceps biopsies were obtained for electrophysiological measurements of epithelial transport and barrier function in miniaturized Ussing devices. C. jejuni absence was checked by PCR and cytokine production with immunohistochemistry., Results: In PI-IBS, the epithelial resistance of the colon epithelium was unaltered, reflecting an intact paracellular pathway. In contrast, temperature-dependent horseradish peroxidase (HRP, 44 kDa) permeation increased. Short-circuit current (Isc) reflecting active anion secretion and ENaC-dependent electrogenic sodium absorption was unaffected. Early endosome antigen-1 (EEA1) and IL-4 levels increased. C. jejuni is not incorporated into the resident microbiota of the colon mucosa in PI-IBS., Conclusions: In PI-IBS after C. jejuni infection, macromolecule uptake via endocytosis was enhanced, leading to low-grade inflammation with pro-inflammatory cytokine release. The findings will allow C. jejuni -induced pathomechanisms to be targeted during infection and, thereafter to reduce sequelae such as PI-IBS.

Infection with the main human food-borne pathogen Campylobacter jejuni causes campylobacteriosis that accounts for a substantial percentage of gastrointestinal infections. The disease usually manifests as diarrhea that lasts for up to two weeks. C. jejuni possesses an array of peptidases and proteases that are critical for its lifestyle and pathogenesis. These include serine proteases Cj1365c, Cj0511 and HtrA; AAA+ group proteases ClpP, Lon and FtsH; and zinc-dependent protease PqqE, proline aminopeptidase PepP, oligopeptidase PepF and peptidase C26. Here, we review the numerous critical roles of these peptide bond-dissolving enzymes in cellular processes of C. jejuni that include protein quality control; protein transport across the inner and outer membranes into the periplasm, cell surface or extracellular space; acquisition of amino acids and biofilm formation and dispersal. In addition, we highlight their role as virulence factors that inflict intestinal tissue damage by promoting cell invasion and mediating cleavage of crucial host cell factors such as epithelial cell junction proteins. Furthermore, we reconstruct the evolution of these proteases in 34 species of the Campylobacter genus. Finally, we discuss to what extent C. jejuni proteases have initiated the search for inhibitor compounds as prospective novel anti-bacterial therapies.

Various CRISPR‒Cas9 orthologs are used in genome engineering. One of the smallest Cas9 orthologs is cjCas9 derived from Campylobacter jejuni, which is a highly specific genome editing tool. Here, we developed cjCas9-based base editors including a cytosine base editor (cjCBEmax) and an adenine base editor (cjABE8e) that can successfully induce endogenous base substitutions by up to 91.2% at the HPD gene in HEK293T cells. Analysis of the base editing efficiency of 13 endogenous target sites showed that the active windows of cjCBEmax and cjABE8e are wider than those of spCas9-based base editors and that their specificities are slightly lower than that of cjCas9. Importantly, engineered cjCas9 and gRNA scaffolds can improve the base editing efficiency of cjABE8e by up to 6.4-fold at the HIF1A gene in HEK293T cells. Due to its small size, cjABE8e can be packaged in a single adeno-associated virus vector with two tandem arrays of gRNAs, and the delivery of the resulting AAV could introduce base substitutions at endogenous ANGPT2 and HPD target sites. Overall, our findings have expanded the potential of the use of base editors for in vivo or ex vivo therapeutic approaches., (© 2023. The Author(s).)

Campylobacter jejuni is likely the most common bacterial cause of gastroenteritis worldwide, responsible for millions of cases of inflammatory diarrhea characterized by severe abdominal cramps and blood in the stool. Further, C. jejuni infections are associated with post-infection sequelae in developed countries and malnutrition and growth-stunting in low- and middle-income countries. Despite the increasing prevalence of the disease, campylobacteriosis, and the recognition that this pathogen is a serious health threat, our understanding of C. jejuni pathogenesis remains incomplete. In this review, we focus on the Campylobacter secretion systems proposed to contribute to host-cell interactions and survival in the host. Moreover, we have applied a genomics approach to defining the structural and mechanistic features of C. jejuni type III, IV, and VI secretion systems. Special attention is focused on the flagellar type III secretion system and the prediction of putative effectors, given that the proteins exported via this system are essential for host cell invasion and the inflammatory response. We conclude that C. jejuni does not possess a type IV secretion system and relies on the type III and type VI secretion systems to establish a niche and potentiate disease.

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 log 10 colony forming units (CFU)/mL after 24 h. Loss of C. jejuni viability was greater ( P  < 0.05) when incubated under 100% CO 2 compared to 50% H 2 :50% CO 2 , 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% CO 2 or 50% H 2 :50% CO 2 . 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 H 2 :CO 2 versus 100% CO 2 . Apparently, C. jejuni can compete for H 2 with methanogens but has limited survivability under rumen conditions.

Campylobacter jejuni is a highly successful enteric pathogen with a small, host-adapted genome (1.64 Mbp, ~1650 coding genes). As a result, C. jejuni has limited capacity in numerous metabolic pathways, including sulfur metabolism. Unable to utilise ionic sulfur, C. jejuni relies on the uptake of exogenous cysteine and its derivatives for its supply of this essential amino acid. Cysteine can also be synthesized de novo by the sole cysteine synthase, CysM. In this study, we explored the substrate specificity of purified C. jejuni CysM and define it as an O-acetyl-L-serine sulfhydrylase with an almost absolute preference for sulfide as sulfur donor. Sulfide is produced in abundance in the intestinal niche C. jejuni colonises, yet sulfide is generally viewed as highly toxic to bacteria. We conducted a series of growth experiments in sulfur-limited media and demonstrate that sulfide is an excellent sulfur source for C. jejuni at physiologically relevant concentrations, combating the view of sulfide as a purely deleterious compound to bacteria. Nonetheless, C. jejuni is indeed inhibited by elevated concentrations of sulfide and we sought to understand the targets involved. Surprisingly, we found that inactivation of the sulfide-sensitive primary terminal oxidase, the cbb 3 -type cytochrome c oxidase CcoNOPQ, did not explain the majority of growth inhibition by sulfide. Therefore, further work is required to reveal the cellular targets responsible for sulfide toxicity in C. jejuni .

Cytolethal distending toxin (CDT) is produced by a range of Gram-negative pathogenic bacteria such as Campylobacter jejuni . CDT represents an important virulence factor that is a heterotrimeric complex composed of CdtA, CdtB, and CdtC. CdtA and CdtC constitute regulatory subunits whilst CdtB acts as the catalytic subunit exhibiting phosphatase and DNase activities, resulting in cell cycle arrest and cell death. Extracellular vesicle (EV) secretion is an evolutionarily conserved process that is present throughout all kingdoms. Mammalian EVs play important roles in regular cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune responses. Here, we demonstrate that CDT targets the endo-lysosomal compartment, partially evading lysosomal degradation and exploiting unconventional secretion (EV release), which is largely involved in bacterial infections. CDT-like effects are transferred by Caco-2 cells to uninfected heterologous U937 and homologous Caco-2 cells. The journey of EVs derived from CDT-treated Caco-2 cells is associated with both intestinal and myeloid tumour cells. EV release represents the primary route of CDT dissemination, revealing an active toxin as part of the cargo. We demonstrated that bacterial toxins could represent suitable tools in cancer therapy, highlighting both the benefits and limitations. The global cell response involves a moderate induction of apoptosis and autophagic features may play a protective role against toxin-induced cell death. EVs from CDT-treated Caco-2 cells represent reliable CDT carriers, potentially suitable in colorectal cancer treatments. Our data present a potential bacterial-related biotherapeutic supporting a multidrug anticancer protocol.

Background: Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive ("dead") cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176., Results: The CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM)., Conclusions: This is the first report of a CRISPRi-based tool in C. jejuni, which will provide a valuable resource to the Campylobacter community., (© 2022. The Author(s).)

Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. A capsular polysaccharide that coats the exterior of the bacterium helps evade the host immune system. At least 33 different strains of C. jejuni have been identified, and the chemical structures of 12 different capsular polysaccharides (CPSs) have been characterized from various serotypes. Thus far, 10 different heptose sugars have been found in the chemically characterized CPSs, and each of these are currently thought to originate from the modification of GDP-d- glycero -d- manno -heptose by the successive action of 4,6-dehydratase (or C4-dehydrogenase), C3- or C3/C5-epimerase, and C4-reductase. Within the sequenced strains of C. jejuni , we have identified 25 different C4-reductases that cluster into nine groups at a sequence identity of >90%. Eight of the proteins from seven different clusters were purified, and their product profiles were determined with GDP-6-deoxy-4-keto-heptose substrates using NMR and ESI mass spectrometry. The isolated products included GDP-6-deoxy-l- gluco -heptose (serotype HS:2), GDP-6-deoxy-l- galacto -heptose (serotype HS:42), GDP-6-deoxy-l- gulo -heptose (serotype HS:15), GDP-6-deoxy-d- ido -heptose (serotypes HS:3, HS:4, and HS:33), GDP-6-deoxy-d- manno -heptose (serotype HS:53), and GDP-6-deoxy-d- altro -heptose (serotype HS:23/36). Based on these observations, the product specificity can be reliably predicted for 14 additional C4-reductases from C. jejuni . The remaining three C4-reductases are highly likely to be required for the biosynthesis of 3,6-dideoxy-heptose products.

The foodborne pathogen Campylobacter jejuni is typically found in an agricultural environment; in animals, such as birds, as an intestinal commensal; and also in food products, especially fresh poultry meat. Campylobacter interactions within mixed species biofilms are poorly understood, especially at the microscale. We have recently shown that the beneficial bacterium Bacillus subtilis reduces C. jejuni survival and biofilm formation in coculture by secreting the antibiotic bacillaene. We extend these studies here by providing evidence that besides bacillaene, the antagonistic effect of B. subtilis involves a nonribosomal peptide bacilysin and that the fully functional antagonism depends on the quorum-sensing transcriptional regulator ComA. Using confocal laser scanning microscopy, we also show that secreted antibiotics influence the distribution of C. jejuni and B. subtilis cells in the submerged biofilm and decrease the thickness of the pathogen's biofilm. Furthermore, we demonstrate that genes encoding structural or regulatory proteins of the efflux apparatus system ( cmeF and cmeR ), respectively, contribute to the survival of C. jejuni during interaction with B. subtilis PS-216. In conclusion, this study demonstrates a strong potential of B. subtilis PS-216 to reduce C. jejuni biofilm growth, which supports the application of the PS-216 strain to pathogen biofilm control. IMPORTANCE Campylobacter jejuni is a prevalent cause of foodborne infections worldwide, while Bacillus subtilis as a potential probiotic represents an alternative strategy to control this alimentary infection. However, only limited literature exists on the specific mechanisms that shape interactions between B. subtilis and C. jejuni in biofilms. This study shows that in the two species biofilms, B. subtilis produces two antibiotics, bacillaene and bacilysin, that inhibit C. jejuni growth. In addition, we provide the first evidence that specific pathogen efflux pumps contribute to the defense against B. subtilis attack. Specifically, the CmeDEF pump acts during the defense against bacilysin, while CmeR-dependent overexpression of CmeABC nullifies the bacillaene attack. The role of specific B. subtilis antibiotics and these polyspecific pumps, known for providing resistance against medically relevant antibiotics, has not been studied during bacterial competition in biofilms before. Hence, this work broadens our understanding of mechanisms that shape antagonisms and defense during probiotic-pathogen interactions.

Homologous recombination is involved in repairing DNA damage, contributing to maintaining the integrity and stability of viral and cellular genomes. In bacteria, the recombination mediator proteins RecO and RecR are required to load the RecA recombinase on ssDNA for homologous recombination. To structurally and functionally characterize RecO, we determined the crystal structure of RecO from Campylobacter jejuni (cjRecO) at a 1.8 Å resolution and biochemically assessed its capacity to interact with DNA and a metal ion. cjRecO folds into a curved rod-like structure that consists of an N-terminal domain (NTD), C-terminal domain (CTD), and Zn 2+ -binding domain (ZnD). The ZnD at the end of the rod-like structure coordinates three cysteine residues and one histidine residue to accommodate a Zn 2+ ion. Based on an extensive comparative analysis of RecO structures and sequences, we propose that the Zn 2+ -binding consensus sequence of RecO is CxxC…C/HxxC/H/D. The interaction with Zn 2+ is indispensable for the protein stability of cjRecO but does not seem to be required for the recombination mediator function. cjRecO also interacts with ssDNA as part of its biological function, potentially using the positively charged patch in the NTD and CTD. However, cjRecO displays a low ssDNA-binding affinity, suggesting that cjRecO requires RecR to efficiently recognize ssDNA for homologous recombination.

The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica. Here, we used electron cryo-tomography (cryo-ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Shewanella oneidensis. Our results reveal the in situ structures of flagellar intermediates, beginning with the earliest flagellar type III secretion system core complex (fT3SScc) and MS-ring. In high-torque motors of Beta-, Gamma-, and Epsilon-proteobacteria, we discovered novel cytoplasmic rings that interact with the cytoplasmic torque ring formed by FliG. These rings, associated with the MS-ring, assemble very early and persist until the stators are recruited into their periplasmic ring; in their absence the stator ring does not assemble. By imaging mutants in Helicobacter pylori, we found that the fT3SScc proteins FliO and FliQ are required for the assembly of these novel cytoplasmic rings. Our results show that rather than a simple accretion of components, flagellar motor assembly is a dynamic process in which accessory components interact transiently to assist in building the complex nanomachine., (© 2022 The Authors.)

The spiral shape of intestinal pathogen Campylobacter jejuni is critical for invasion of intestinal mucosa epithelial cells. Insofar as this cell morphology plays a role in the pathology of C. jejuni infection, its restructuring by pharmacological intervention could be an unexplored means to prevention of infection. We recently described that peptidoglycan hydrolase 3 (Pgp3) is involved in the spiral-shape formation of C. jejuni. We report herein the design and synthesis of the hydroxamate-based inhibitors targeting Pgp3. C. jejuni cells exposed to these inhibitors changed from the helical- to rod-shaped morphology, comparable to the case of the pgp3-deletion mutant. Evidence for the mechanism of action was provided by crystal structures of Pgp3 in complex with inhibitors, shedding light into the binding modes of inhibitors within the active site, supported by kinetics and molecular-dynamics simulations. C. jejuni exposed to these inhibitors underwent the morphological change from helical- to rod-shaped bacteria, an event that reduce the ability for invasion of the host cells. This proof of concept suggests that alteration of morphology affects the interference with the bacterial infection., (© 2022. The Author(s).)

Background: Cytolethal distending toxin (CDT) is a critical virulence factor of Campylobacter jejuni , and it induces cell death and regulates inflammation response in human epithelial cells. Pyroptosis is an inflammatory form of programmed cell death (PCD), but whether it is involved in CDT-mediated cytotoxicity remains elusive., Aims: This study explores the role and mechanism of pyroptosis in CDT-mediated cytotoxicity., Methods: HCT116 and FHC cell lines were treated with CDT. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability. Western blotting was used to measure the expression of related proteins in the pathway, and cell morphology observation, annexin V/propidium iodide (PI) staining and lactate dehydrogenase (LDH) release assay were performed to evaluate the occurrence of pyroptosis., Result: Our results show that C. jejuni CDT effectively induces pyroptosis in a dose- and time- dependent manner in human colonic epithelial cells owing to its DNase activity. Specific pyroptotic features including large bubbles emerging from plasma membrane and LDH release were observed upon CDT treatment. Moreover, CDT-induced pyroptosis involves the caspase-9/caspase-3 axis, which is followed by gasdermin E (GSDME) cleavage rather than gasdermin D (GSDMD). N-acetyl cysteine (NAC), a reactive oxygen species (ROS) inhibitor, attenuates the activation of caspase-9/3, the cleavage of GSDME and pyroptotic characteristic, therefore demonstrating ROS initiates pyroptotic signaling., Conclusions: We first clarify a molecular mechanism that CDT induces pyroptosis via ROS/caspase-9/caspase-3/GSDME signaling. These findings provide a new insight on understanding of CDT-induced pathogenesis at the molecular level., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gu, Lin, Wang, Pan, Cai, He, Xu and Cai.)

Campylobacter jejuni represents an eminent zoonotic germ responsible for foodborne infections causing campylobacteriosis. In addition, infections with C. jejuni constitute a risk factor for the occurrence of inflammatory bowel disease (IBD). In the latter case, patients show inflammatory reactions not only against C. jejuni, but also against the non-infectious microbiota. However, the involved mechanisms and molecular basis are still largely unclear. We recently reported that C. jejuni breaches the intestinal epithelial barrier by secretion of serine protease HtrA (high temperature requirement A), which cleaves several major tight and adherens junction proteins. In the present study, we aimed to study if HtrA-expressing C. jejuni may also trigger the transepithelial migration of non-pathogenic gastrointestinal microbiota. Using confocal immunofluorescence and scanning electron microscopy, we demonstrate that C. jejuni wild-type (wt) as well as the isogenic ∆ htrA mutant bind to the surface of polarized intestinal Caco-2 epithelial cells, but do not invade them at the apical side. Instead, C. jejuni wt, but not ∆ htrA mutant, disrupt the cellular junctions and transmigrate using the paracellular route between neighboring cells. Using transwell filter systems, we then co-incubated the cells with C. jejuni and non-invasive microbiota strains, either Escherichia coli or Lactococcus lactis . Interestingly, C. jejuni wt, but not ∆ htrA mutant, induced the efficient transmigration of these microbiota bacteria into the basal compartment. Thus, infection of the intestinal epithelium with C. jejuni causes local opening of cellular junctions and paracellular translocation in an HtrA-dependent manner, which paves the way for transmigration of microbiota that is otherwise non-invasive. Taken together, these findings may have impacts on various Campylobacter -associated diseases such as IBD, which are discussed here.

Campylobacter jejuni is a leading cause of food-borne disease worldwide. The pathogenicity of C. jejuni is closely associated with the internalization process in host epithelial cells, which is related to a host immune response. Autophagy indicates a key role in the innate immune system of the host to exclude invasive pathogens. Most bacteria are captured by autophagosomes and degraded by autophagosome-lysosome fusion in host cells. However, several pathogens, such as Salmonella and Shigella , avoid and/or escape autophagic degradation to establish infection. But autophagy involvement as a host immune response to C. jejuni infection has not been clarified. This study revealed autophagy association in C. jejuni infection. During infection, C. jejuni activated the Rho family small GTPase Rac1 signaling pathway, which modulates actin remodeling and promotes the internalization of this pathogen. In this study, we found the LC3 contribution to C. jejuni invasion signaling via the Rac1 signaling pathway. Interestingly, during C. jejuni invasion, LC3 was recruited to bacterial entry site depending on Rac1 GTPase activation just at the early step of the infection. C. jejuni infection induced LC3-II conversion, and autophagy induction facilitated C. jejuni internalization. Also, autophagy inhibition attenuated C. jejuni invasion step. Moreover, Rac1 recruited LC3 to the cellular membrane, activating the invasion of C. jejuni . Altogether, our findings provide insights into the new function of LC3 in bacterial invasion. We found the interaction between the Rho family small GTPase, Rac1, and autophagy-associated protein, LC3., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fukushima, Shimohata, Inoue, Kido, Uebanso, Mawatari and Takahashi.)

FlhF protein is critical for intact flagellar assembly in Campylobacter jejuni. It is a putative GTPase with B-, N- and G-domains. However, the role of the B- and N-domains in flagella biosynthesis remains unclear in C. jejuni. This study demonstrated that both the B- and N-domains are essential for flagellar synthesis, with the absence of B- and/or N-domains showing truncated variants of FlhF by TEM. Point mutations in the B- and N-domains (T13A, K159A, G231A) also induced flagella abnormalities. Furthermore, significant defects in GTPase activity and polar targeting of FlhF were triggered by point mutations of B- and N-domains. Flagella gene expression and transcription were also significantly disrupted in flhF(T13A), flhF(K159A) and flhF(G231A) strains. This study initially explored the effects of B- and N-domains on flagella synthesis. We speculated that B- and N-domains may directly or indirectly cause flagella abnormalities by affecting flagellar gene expression or GTPase activity, which helps us better understand the function of FlhF in flagella synthesis., (Crown Copyright © 2021. Published by Elsevier GmbH. All rights reserved.)

The nickel (Ni)-chelator dimethylglyoxime (DMG) was found to be bacteriostatic towards Campylobacter jejuni. Supplementation of nickel to DMG-containing media restored bacterial growth, whereas supplementation of cobalt or zinc had no effect on the growth inhibition. Unexpectedly, the combination of millimolar levels of DMG with micromolar levels of copper (Cu) was bactericidal, an effect not seen in select Gram-negative pathogenic bacteria. Both the cytoplasmic Ni-binding chaperone SlyD and the twin arginine translocation (Tat)-dependent periplasmic copper oxidase CueO were found to play a central role in the Cu-DMG hypersensitivity phenotype. Ni-replete SlyD is needed for Tat-dependent CueO translocation to the periplasm, whereas Ni-depleted (DMG-treated) SlyD is unable to interact with the CueO Tat signal peptide, leading to mislocalization of CueO and increased copper sensitivity. In support of this model, C. jejuni ΔslyD and ΔcueO mutants were more sensitive to copper than the wild-type (WT); CueO was less abundant in the periplasmic fraction of ΔslyD or DMG-grown WT cells, compared to WT cells grown on plain medium; SlyD binds the CueO signal sequence peptide, with DMG inhibiting and nickel enhancing the binding, respectively. Injection of Cu-DMG into Galleria mellonella before C. jejuni inoculation significantly increased the insect survival rate compared to the control group. In chickens, oral administration of DMG or Cu-DMG decreased and even abolished C. jejuni colonization in some cases, compared to both water-only and Cu-only control groups. The latter finding is important, since campylobacteriosis is the leading bacterial foodborne infection, and chicken meat constitutes the major foodborne source., (© The Author(s) 2021. Published by Oxford University Press.)

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that is commensal in poultry. Infection of C. jejuni leads to campylobacteriosis, the leading cause of gastroenteritis worldwide. Coating the surface of C. jejuni is a thick layer of sugar molecules known as the capsular polysaccharide (CPS). The CPS of C. jejuni NCTC 11168 (HS:2) is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronate, d- N -acetyl-galactosamine, and d-ribose. The glucuronate is further amidated with either ethanolamine or serinol, but it is unknown how this new amide bond is formed. Sequence similarity networks were used to identify a candidate enzyme for amide bond formation during the biosynthesis of the CPS of C. jejuni . The C-terminal domain of Cj1438 was shown to catalyze amide bond formation using MgATP and d-glucuronate in the presence of either ethanolamine phosphate or ( S )-serinol phosphate. Product formation was verified using 31 P NMR spectroscopy and ESI mass spectrometry, and the kinetic constants determined using a coupled enzyme assay by measuring the rate of ADP formation. This work represents the first functional characterization of an ATP-dependent amidoligase in the formation of amide bonds in the biosynthetic pathway for the assembly of the CPS in C. jejuni .