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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.

The Campylobacter genus is the leading cause of human gastroenteritis, with the consumption of contaminated poultry meat as the main route of infection. Probiotic bacteria, such as Lactobacillus , Bacillus , Escherichia coli Nissle, and Bifidobacterium species, have a great immunomodulatory capacity and exhibit antipathogenic effects through various molecular mechanisms. Reducing Campylobacter levels in livestock animals, such as poultry, will have a substantial benefit to humans as it will reduce disease transmissibility through the food chain. Moreover, probiotic-based strategies might attenuate intestinal inflammatory processes, which consequently reduce the severity of Campylobacter disease progression. At a molecular level, probiotics can also negatively impact on the functionality of various Campylobacter virulence and survival factors (e.g., adhesion, invasion), and on the associated colonization proteins involved in epithelial translocation. The current review describes recent in vitro , in vivo , and preclinical findings on probiotic therapies, aiming to reduce Campylobacter counts in poultry and reduce the pathogen's virulence in the avian and human host. Moreover, we focused in particular on probiotics with known anti- Campylobacter activity seeking to understand the biological mechanisms involved in their mode of action.

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.)

Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis world wide and represents a major public health concern. Over the past two decades, significant progress in functional genomics, proteomics, enzymatic-based virulence profiling (EBVP), and the cellular biology of C. jejuni have improved our basic understanding of this important pathogen. We review key advances in our understanding of the multitude of emerging virulence factors that influence the outcome of C. jejuni -mediated infections. We highlight, the spatial and temporal dynamics of factors that promote C. jejuni to sense, adapt and survive in multiple hosts. Finally, we propose cohesive research directions to obtain a comprehensive understanding of C. jejuni virulence mechanisms., 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 © 2021 Elmi, Nasher, Dorrell, Wren and Gundogdu.)

The Gram-negative Campylobacter jejuni is a major cause of foodborne gastroenteritis in humans worldwide. The cytotoxic effects of Campylobacter have been mainly ascribed to the actions of the cytolethal distending toxin (CDT): it is mandatory to put in evidence risk factors for sequela development, such as reactive arthritis (ReA) and Guillain-Barré syndrome (GBS). Several researches are directed to managing symptom severity and the possible onset of sequelae. We found for the first time that rapamycin (RM) is able to largely inhibit the action of C. jejuni lysate CDT in U937 cells, and to partially avoid the activation of specific sub-lethal effects. In fact, we observed that the ability of this drug to redirect lysosomal compartment, stimulate ER-remodeling (highlighted by ER-lysosome and ER-mitochondria contacts), protect mitochondria network, and downregulate CD317/tetherin, is an important component of membrane microdomains. In particular, lysosomes are involved in the process of the reduction of intoxication, until the final step of lysosome exocytosis. Our results indicate that rapamycin confers protection against C. jejuni bacterial lysate insults to myeloid cells.

Campylobacter jejuni is considered to be the most common bacterial cause of human gastroenteritis worldwide. C. jejuni can cause bloody diarrhoea, fever and abdominal pain in humans along with post-infectious sequelae such as Guillain-Barré syndrome (a paralytic autoimmune complication). C. jejuni infections can be fatal, particularly among young children. C. jejuni are distributed in most warm-blooded animals, and therefore the main route of transmission is generally foodborne, via the consumption and handling of meat products (particularly poultry). C. jejuni is microaerophilic and oxygen-sensitive, although it appears to be omnipresent in the environment, one of the many contradictions of Campylobacter .

Campylobacter jejuni outer membrane vesicles (OMVs) contain numerous virulence-associated proteins including the cytolethal distending toxin and three serine proteases. As C. jejuni lacks the classical virulence-associated secretion systems of other enteric pathogens that deliver effectors directly into target cells, OMVs may have a particularly important role in virulence. C. jejuni OMV production is stimulated by the presence of physiological concentrations of the bile salt sodium taurocholate (ST) through an unknown mechanism. The maintenance of lipid asymmetry (MLA) pathway has been implicated in a novel mechanism for OMV biogenesis, open to regulation by host signals. In this study we investigated the role of the MLA pathway in C. jejuni OMV biogenesis with ST as a potential regulator. OMV production was quantified by analyzing protein and lipid concentrations of OMV preparations and OMV particle counts produced by nanoparticle tracking analysis. Mutation of mlaA which encodes the outer membrane component of the MLA pathway significantly increased OMV production compared to the wild-type strain. Detergent sensitivity and membrane permeability assays confirmed the increased OMV production was not due to changes in membrane stability. The presence of 0.2% (w/v) ST increased wild-type OMV production and reduced OMV size, but did not further stimulate mlaA mutant OMV production or significantly alter mlaA mutant OMV size. qRT-PCR analysis demonstrated that the presence of ST decreased expression of both mlaA and mlaC in C. jejuni wild-type strains 11168 and 488. Collectively the data in this study suggests C. jejuni can regulate OMV production in response to host gut signals through changes in expression of the MLA pathway. As the gut bile composition is dependent on both diet and the microbiota, this study highlights the potential importance of diet and lifestyle factors on the varying disease presentations associated with gut pathogen infection.

Objectives: In this study we have investigated the in vitro and in vivo virulence characteristics of a new T6SS positive Campylobacter jejuni chicken isolate (SV12) originating from a poultry population in North Romania. A detailed phenotypic characterization was performed and compared to the T6SS negative C. jejuni 81-176 wild strain., Results: Our results indicate that the significantly higher capacity to attach and invade HCT-8 cells of C. jejuni SV12 isolate is associated with increased motility, increased resistance to bile salts and serum resistance, when compared to C. jejuni strain 81-76. Mice infected with the SV12 isolate showed statistically higher levels of colonization at both 7- and 14-days post-inoculation and in the stomach, caecum, duodenum and large intestine. Infection with the SV12 strain induced a stronger immune response as the gene transcript levels of IL-17, TNFα and IFNγ were more pronouncedly up-regulated compared to the C. jejuni strain 81-176. The present study showed that the new isolate SV12 had an enhanced virulence capacity compared to the wild strain which was evident in vivo as well. This work also provides an insight on the colonization pattern and host immune response differences between T6SS positive and T6SS negative C. jejuni.

Campylobacter jejuni is the leading cause of food-borne bacterial enteritis in humans, with contaminated poultry products considered the main source of infection. To survive the food chain, C. jejuni utilizes multiple defense mechanisms that counter oxidative and aerobic stresses. In this study, we phenotypically characterised 63 C. jejuni strains with oxidative stress survival and antimicrobial susceptibility testing to investigate correlations between these two phenotypes against the source of the strains and the presence of the MarR regulators RrpA and RrpB which have a role in regulating the response to oxidative and aerobic stress. C. jejuni strains isolated from meat and neck skin displayed the highest resistance to oxidative stress. In addition, C. jejuni strains that have an rrpA + rrpB - profile exhibit increased resistance to oxidative stress and to antimicrobials. Here we establish a preliminary link between the distribution of RrpA and RrpB and the increased resistance to antimicrobials. This study provides insight into how the genotypic make up of C. jejuni can influence the ability of the bacterium to survive within areas of high oxygen stress, such as the food chain, and subsequently can have a potential negative impact on human health., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence-associated proteins within outer membrane vesicles (OMVs). C. jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E-cadherin, promoting enhanced C. jejuni adhesion to and invasion of IECs. C. jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence-associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C. jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C. jejuni growth until the early stationary phase. Coculture of C. jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C. jejuni ST-OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST-OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs-associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs-induced cleavage of E-cadherin and occludin. C. jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C. jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence-associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis., (© 2017 John Wiley & Sons Ltd.)

Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram-negative bacteria. Campylobacter jejuni produces OMVs that trigger IL-8, IL-6, hBD-3 and TNF-α responses from T84 intestinal epithelial cells and are cytotoxic to Caco-2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E-cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co-incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E-cadherin and occludin. The addition of 11168H OMVs to the co-culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time-dependent and dose-dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells., (© 2015 John Wiley & Sons Ltd.)

Campylobacter jejuni is a leading cause of acute gastroenteritis. C. jejuni lipooligosaccharide (LOS) is a potent activator of Toll-like receptor (TLR) 4-mediated innate immunity. Structural variations of the LOS have been previously reported in the oligosaccharide (OS) moiety, the disaccharide lipid A (LA) backbone, and the phosphorylation of the LA. Here, we studied LOS structural variation between C. jejuni strains associated with different ecological sources and analyzed their ability to activate TLR4 function. MALDI-TOF MS was performed to characterize structural variation in both the OS and LA among 15 different C. jejuni isolates. Cytokine induction in THP-1 cells and primary monocytes was correlated with LOS structural variation in each strain. Additionally, structural variation was correlated with the source of each strain. OS sialylation, increasing abundance of LA d-glucosamine versus 2,3-diamino-2,3-dideoxy-d-glucose, and phosphorylation status all correlated with TLR4 activation as measured in THP-1 cells and monocytes. Importantly, LOS-induced inflammatory responses were similar to those elicited by live bacteria, highlighting the prominent contribution of the LOS component in driving host immunity. OS sialylation status but not LA structure showed significant association with strains clustering with livestock sources. Our study highlights how variations in three structural components of C. jejuni LOS alter TLR4 activation and consequent monocyte activation.

Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world; however, the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However, C. jejuni lacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilize outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the coordinated delivery of C. jejuni proteins into host cells. Proteomic analysis of C. jejuni 11168H OMVs identified 151 proteins, including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis, including the cytolethal distending toxin (CDT). C. jejuni OMVs contained 16 N-linked glycoproteins, indicating a delivery mechanism by which these periplasm-located yet immunogenic glycoproteins can interact with host cells. C. jejuni OMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pretreatment with proteinase K or polymyxin B prior to coincubation with IECs. Pretreatment of IECs with methyl-beta-cyclodextrin partially blocks OMV-induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions with C. jejuni OMVs. OMVs isolated from a C. jejuni 11168H cdtA mutant induced interleukin-8 (IL-8) to the same extent as did wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT.

Campylobacter jejuni infection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. While C. jejuni infection can be reproduced in vitro using intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performing in vitro assays under atmospheric oxygen conditions neither is optimal for microaerophilic C. jejuni nor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing an in vitro system that closely mimics in vivo conditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellular C. jejuni 11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotile C. jejuni 11168H rpoN mutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent the in vivo situation and will allow novel insights into C. jejuni pathogenic mechanisms.

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide. Despite stringent microaerobic growth requirements, C. jejuni is ubiquitous in the aerobic environment and so must possess regulatory systems to sense and adapt to external stimuli, such as oxidative and aerobic (O(2)) stress. Reannotation of the C. jejuni NCTC11168 genome sequence identified Cj1556 (originally annotated as a hypothetical protein) as a MarR family transcriptional regulator, and further analysis indicated a potential role in regulating the oxidative stress response. A C. jejuni 11168H Cj1556 mutant exhibited increased sensitivity to oxidative and aerobic stress, decreased ability for intracellular survival in Caco-2 human intestinal epithelial cells and J774A.1 mouse macrophages, and a reduction in virulence in the Galleria mellonella infection model. Microarray analysis of gene expression changes in the Cj1556 mutant indicated negative autoregulation of Cj1556 expression and downregulation of genes associated with oxidative and aerobic stress responses, such as katA, perR, and hspR. Electrophoretic mobility shift assays confirmed the binding of recombinant Cj1556 to the promoter region upstream of the Cj1556 gene. cprS, which encodes a sensor kinase involved in regulation of biofilm formation, was also upregulated in the Cj1556 mutant, and subsequent studies showed that the mutant had a reduced ability to form biofilms. This study identified a novel C. jejuni transcriptional regulator, Cj1556, that is involved in oxidative and aerobic stress responses and is important for the survival of C. jejuni in the natural environment and in vivo.

Background: Campylobacter jejuni is the leading bacterial cause of human gastroenteritis in the developed world. To improve our understanding of this important human pathogen, the C. jejuni NCTC11168 genome was sequenced and published in 2000. The original annotation was a milestone in Campylobacter research, but is outdated. We now describe the complete re-annotation and re-analysis of the C. jejuni NCTC11168 genome using current database information, novel tools and annotation techniques not used during the original annotation., Results: Re-annotation was carried out using sequence database searches such as FASTA, along with programs such as TMHMM for additional support. The re-annotation also utilises sequence data from additional Campylobacter strains and species not available during the original annotation. Re-annotation was accompanied by a full literature search that was incorporated into the updated EMBL file [EMBL: AL111168]. The C. jejuni NCTC11168 re-annotation reduced the total number of coding sequences from 1654 to 1643, of which 90.0% have additional information regarding the identification of new motifs and/or relevant literature. Re-annotation has led to 18.2% of coding sequence product functions being revised., Conclusions: Major updates were made to genes involved in the biosynthesis of important surface structures such as lipooligosaccharide, capsule and both O- and N-linked glycosylation. This re-annotation will be a key resource for Campylobacter research and will also provide a prototype for the re-annotation and re-interpretation of other bacterial genomes.

Campylobacter jejuni is the predominant cause of bacterial gastroenteritis worldwide, but traditional typing methods are unable to discriminate strains from different sources that cause disease in humans. We report the use of genomotyping (whole-genome comparisons of microbes using DNA microarrays) combined with Bayesian-based algorithms to model the phylogeny of this major food-borne pathogen. In this study 111 C. jejuni strains were examined by genomotyping isolates from humans with a spectrum of C. jejuni-associated disease (70 strains), chickens (17 strains), bovines (13 strains), ovines (5 strains), and the environment (6 strains). From these data, the Bayesian phylogeny of the isolates revealed two distinct clades unequivocally supported by Bayesian probabilities (P = 1); a livestock clade comprising 31/35 (88.6%) of the livestock isolates and a "nonlivestock" clade comprising further clades of environmental isolates. Several genes were identified as characteristic of strains in the livestock clade. The most prominent was a cluster of six genes (cj1321 to cj1326) within the flagellin glycosylation locus, which were confirmed by PCR analysis as genetic markers in six additional chicken-associated strains. Surprisingly these studies show that the majority (39/70, 55.7%) of C. jejuni human isolates were found in the nonlivestock clade, suggesting that most C. jejuni infections may be from nonlivestock (and possibly nonagricultural) sources. This study has provided insight into a previously unidentified reservoir of C. jejuni infection that may have implications in disease-control strategies. The comparative phylogenomics approach described provides a robust methodological prototype that should be applicable to other microbes.

In recent years, there has been an unprecedented advancement in in situ analytical approaches that contribute to the mechanistic understanding of microbial communities by explicitly incorporating ecology and studying their assembly. In this study, we have analyzed the temporal profiles of the healthy broiler cecal microbiome from day 3 to day 35 to recover the stable and varying components of microbial communities. During this period, the broilers were fed three different diets chronologically, and therefore, we have recovered signature microbial species that dominate during each dietary regime. Since broilers were raised in multiple pens, we have also parameterized these as an environmental condition to explore microbial niches and their overlap. All of these analyses were performed in view of different parameters such as body weight (BW-mean), feed intake (FI), feed conversion ratio (FCR), and age (days) to link them to a subset of microbes that these parameters have a bearing upon. We found that gut microbial communities exhibited strong and statistically significant specificity for several environmental variables. Through regression models, genera that positively/negatively correlate with the bird's age were identified. Some shortchain fatty acids (SCFAs)-producing bacteria, including Izemoplasmatales, Gastranaerophilales, and Roseburia, have a positive correlation with age. Certain pathogens, such as Escherichia-Shigella, Sporomusa, Campylobacter, and Enterococcus, negatively correlated with the bird's age, which indicated a high disease risk in the initial days. Moreover, the majority of pathways involved in amino acid biosynthesis were also positively correlated with the bird's age. Some probiotic genera associated with improved performance included Oscillospirales; UCG-010, Shuttleworthia, Bifidobacterium, and Butyricicoccaceae; UCG-009. In general, predicted antimicrobial resistance genes (piARGs) contributed at a stable level, but there was a slight increase in abundance when the diet was changed. To the best of the authors' knowledge, this is one of the first studies looking at the stability, complexity, and ecology of natural broiler microbiota development in a temporal setting. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. Mechanistically, how this pathogen interacts with intrinsic defence machinery of human intestinal epithelial cells (IECs) remains elusive. To address this, we investigated how C. jejuni counteracts the intracellular and extracellular reactive oxygen species (ROS) in IECs. Our work shows that C. jejuni differentially regulates intracellular and extracellular ROS production in human T84 and Caco-2 cells. C. jejuni downregulates the transcription and translation of nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX1), a key ROS-generating enzyme in IECs and antioxidant defence genes CAT and SOD1. Furthermore, inhibition of NOX1 by diphenylene iodonium (DPI) and siRNA reduced C. jejuni ability to interact, invade, and intracellularly survive within T84 and Caco-2 cells. Collectively, these findings provide mechanistic insight into how C. jejuni modulates the IEC defence machinery. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Type VI Secretion System (T6SS) has important roles relating to bacterial antagonism, subversion of host cells, and niche colonisation. Campylobacter jejuni is one of the leading bacterial causes of human gastroenteritis worldwide and is a commensal coloniser of birds. Although recently discovered, the T6SS biological functions and identities of its effectors are still poorly defined in C. jejuni. Here, we perform a comprehensive bioinformatic analysis of the C. jejuni T6SS by investigating the prevalence and genetic architecture of the T6SS in 513 publicly available genomes using C. jejuni 488 strain as reference. A unique and conserved T6SS cluster associated with the Campylobacter jejuni Integrated Element 3 (CJIE3) was identified in the genomes of 117 strains. Analyses of the T6SS-positive 488 strain against the T6SS-negative C. jejuni RM1221 strain and the T6SS-positive plasmid pCJDM202 carried by C. jejuni WP2-202 strain defined the "T6SS-containing CJIE3" as a pathogenicity island, thus renamed as Campylobacter jejuni Pathogenicity Island-1 (CJPI-1). Analysis of CJPI-1 revealed two canonical VgrG homologues, CJ488_0978 and CJ488_0998, harbouring distinct C-termini in a genetically variable region downstream of the T6SS operon. CJPI-1 was also found to carry a putative DinJ-YafQ Type II toxin-antitoxin (TA) module, conserved across pCJDM202 and the genomic island CJIE3, as well as several open reading frames functionally predicted to encode for nucleases, lipases, and peptidoglycan hydrolases. This comprehensive in silico study provides a framework for experimental characterisation of T6SS-related effectors and TA modules in C. jejuni. [ABSTRACT FROM AUTHOR]

Background: The classification of natural antimicrobials as potential antibiotic replacements is still hampered by the absence of clear biological mechanisms behind their mode of action. This study investigated the mechanisms underlying the anti-bacterial effect of a mixture of natural antimicrobials (maltodextrin, citric acid, sodium citrate, malic acid, citrus extract and olive extract) against Campylobacter jejuni RC039, Salmonella enterica SE 10/72 and Clostridium perfringens ATCC® 13124 invasion of Madin–Darby Canine Kidney cells (MDCK). Results: Minimum sub-inhibitory concentrations were determined for Campylobacter jejuni (0.25%), Salmonella enterica (0.50%) and Clostridium perfringens (0.50%) required for the in vitro infection assays with MDCK cells. The antimicrobial mixture significantly reduced the virulence of all three pathogens towards MDCK cells and restored the integrity of cellular tight junctions through increased transepithelial resistance (TEER) and higher expression levels of ZO-1 (zonula occludens 1) and occludin. This study also identified the ERK (external regulated kinase) signalling pathway as a key mechanism in blocking the pro-inflammatory cytokine production (IL-1β, IL-6, IL-8, TNF-α) in infected cells. The reduction in hydrogen peroxide (H2O2) production and release by infected MDCK cells, in the presence of the antimicrobial mixture, was also associated with less tetrathionate formed by oxidation of thiosulphate (p < 0.0001). Conclusion: The present study describes for the first time that mixtures of natural antimicrobials can prevent the formation of substrates used by bacterial pathogens to grow and survive in anaerobic environments (e.g. tetrathionate). Moreover, we provide further insights into pathogen invasion mechanisms through restoration of cellular structures and describe their ability to block the ERK–MAPK kinase pathway responsible for inflammatory cytokine release [ABSTRACT FROM AUTHOR]

The role of the Type VI secretion system (T6SS) in Campylobacter jejuni is poorly understood despite an increasing prevalence of the T6SS in recent C. jejuni isolates in humans and chickens. The T6SS is a contractile secretion machinery capable of delivering effectors that can play a role in host colonization and niche establishment. During host colonization, C. jejuni is exposed to oxidative stress in the host gastrointestinal tract, and in other bacteria the T6SS has been linked with the oxidative stress response. In this study, comparisons of whole genome sequences of a novel human isolate 488 with previously sequenced strains revealed a single highly conserved T6SS cluster shared between strains isolated from humans and chickens. The presence of a functional T6SS in the 488 wild-type strain is indicated by expression of T6SS genes and secretion of the effector TssD. Increased expression of oxidative stress response genes katA , sodB , and ahpC , and increased oxidative stress resistance in 488 wild-type strain suggest T6SS is associated with oxidative stress response. The role of the T6SS in interactions with host cells is explored using in vitro and in vivo models, and the presence of the T6SS is shown to increase C. jejuni cytotoxicity in the Galleria mellonella infection model. In biologically relevant models, the T6SS enhances C. jejuni interactions with and invasion of chicken primary intestinal cells and enhances the ability of C. jejuni to colonize chickens. This study demonstrates that the C. jejuni T6SS provides defense against oxidative stress and enhances host colonization, and highlights the importance of the T6SS during in vivo survival of T6SS-positive C. jejuni strains. [ABSTRACT FROM AUTHOR]

Editorial: Developments in Campylobacter, Helicobacter & Related Organisms Research - CHRO 2019 Keywords: Campylobacter; Helicobacter; CHRO 2019; gastrointestinal pathogen; Campylobacter Helicobacter and Related Organisms EN Campylobacter Helicobacter CHRO 2019 gastrointestinal pathogen Campylobacter Helicobacter and Related Organisms N.PAG N.PAG 3 01/13/21 20210108 NES 210108 I Campylobacter i spp. and I Helicobacter i spp. are important gastrointestinal pathogens that are major causes of acute gastroenteritis and gastric disease, respectively (Polk and Peek, [11]; Gundogdu and Wren, [6]). Both I Campylobacter i spp. and I Helicobacter i spp. possess a plethora of survival and virulence factors that have allowed them to survive and persist successfully (Gundogdu et al., [5]; Hathroubi et al., [7]; Capurro et al., [3]; El Abbar et al., [4]; Liaw et al., [9]). This Research Topic will increase the knowledge base and understanding of the processes of survival of I Campylobacter i spp. and I Helicobacter i spp. within the environment, in particular, relating to food safety, and to host-pathogen interactions. [Extracted from the article]

Human campylobacteriosis is considered one of the most common foodborne diseases worldwide with poultry identified as the main source of infection accounting for 50–80% of human cases. Highly virulent Campylobacter spp., positive for the Type VI secretion system (T6SS), which have an increased ability to adhere to and invade the host gastrointestinal epithelium are highly prevalent in poultry. Multidrug resistant strains of bacteria are rapidly evolving and therefore, new antimicrobials to supplement animal feed that are able to control Campylobacter species, are in great need. The work presented herein indicates that a novel phenolic antimicrobial, Auranta 3001, is able to reduce the adhesion and invasion of human intestinal epithelial cells (HCT-8) by two T6SS positive chicken isolates, C. jejuni RC039 (p < 0.05) and C. coli RC013 (p < 0.001). Exposure of C. jejuni RC039 and C. coli RC013 to Auranta 3001 downregulated the expression of hcp and cetB genes, known to be important in the functionality of T6SS. Furthermore, the reduced adhesion and invasion is associated with a significant decrease in bacterial motility of both isolates (p < 0.05– p < 0.001) in vitro. Most importantly our in vivo results show that Auranta 3001 is able to reduce cecum colonization levels from log 8 CFU/ml to log 2 CFU/ml for C. jejuni RC039 and from log 7 CFU/ml to log 2 CFU/ml for C. coli RC013. In conclusion, this novel antimicrobial is able to reduce the pathogenic properties of T6SS campylobacters in vitro and also to decrease colonization in vivo. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is the leading cause of bacterial foodborne diarrhoeal disease worldwide. Despite the microaerophilic nature of the bacterium, C. jejuni can survive the atmospheric oxygen conditions in the environment. Bacteria that can survive either within a host or in the environment like C. jejuni require variable responses to survive the stresses associated with exposure to different levels of reactive oxygen species. The MarR-type transcriptional regulators RrpA and RrpB have recently been shown to play a role in controlling both the C. jejuni oxidative and aerobic stress responses. Analysis of 3,746 C. jejuni and 486 C. coli genome sequences showed that whilst rrpA is present in over 99% of C. jejuni strains, the presence of rrpB is restricted and appears to correlate with specific MLST clonal complexes (predominantly ST-21 and ST-61). C. coli strains in contrast lack both rrpA and rrpB. In C. jejuni rrpB+ strains, the rrpB gene is located within a variable genomic region containing the IF subtype of the type I Restriction-Modification (hsd) system, whilst this variable genomic region in C. jejuni rrpB- strains contains the IAB subtype hsd system and not the rrpB gene. C. jejuni rrpB- strains exhibit greater resistance to peroxide and aerobic stress than C. jejuni rrpB+ strains. Inactivation of rrpA resulted in increased sensitivity to peroxide stress in rrpB+ strains, but not in rrpB- strains. Mutation of rrpA resulted in reduced killing of Galleria mellonella larvae and enhanced biofilm formation independent of rrpB status. The oxidative and aerobic stress responses of rrpB- and rrpB+ strains suggest adaptation of C. jejuni within different hosts and niches that can be linked to specific MLST clonal complexes. [ABSTRACT FROM AUTHOR]

Background: Recently the Type VI secretion system (T6SS), which can play a significant role in bacterial survival and pathogenesis, was reported in Campylobacter spp., having the hcp gene as a key component. Methods: Campylobacteriosis is associated with the consumption of infected chicken meat. Our study aimed to explore the presence of T6SS in C. jejuni (n = 59) and C coli (n = 57) isolates, from retail raw chicken and to investigate their pathogenic potential. The hcp gene was used as an indicator for the T6SS presence. Results: Using multiplex PCR we have identified a significantly higher prevalence of hcp in C coli isolates (56.1%) than in C. jejuni (28.8%) and AFLP analysis of the isolates showed a high degree of genetic similarity between the isolates carrying the hcp gene. Genome sequencing data showed that 84.3% of the C coli and 93.7% of the C. jejuni isolates had all 13 T6SS open reading frames. Moreover, the virulence characteristics of hcp + isolates, including motility and the ability to invade human intestinal epithelial cells in vitro, were significantly greater than in the control strain C. jejuni 12502; a human isolate which is hcp positive. Conclusion: Overall, it was discovered that hcp+ C coli and C. jejuni isolated from retail chicken isolates posses genetic and phenotypic properties associated with enhanced virulence. However, since human infections with C coli are significantly less frequent than those of C. jejuni, the relationship between virulence factors and pathogenesis requires further study. [ABSTRACT FROM AUTHOR]

The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarRtype transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide) respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2) stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA) expression. However, a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wildtype strain. Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2) stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the G mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment. [ABSTRACT FROM AUTHOR]

Keywords: Campylobacter jejuni; Type VI Secretion System; T6SS effectors; T6SS immunity proteins; toxin-antitoxin; pathogenicity island EN Campylobacter jejuni Type VI Secretion System T6SS effectors T6SS immunity proteins toxin-antitoxin pathogenicity island 1 1 1 11/19/21 20211116 NES 211116 In the original article, there was a mistake in Supplementary Data 2 (Data Sheet 2.ZIP) as published. Campylobacter jejuni, Type VI Secretion System, toxin-antitoxin, T6SS effectors, T6SS immunity proteins, pathogenicity island. [Extracted from the article]

Pumtang-on et al. [[20]] perform a systematic review of I C. jejuni i vaccine candidates for chickens, highlighting the need for increased consistency in the way I C. jejuni i vaccine studies in poultry are designed and reported in order to be able to undertake a robust comparison of I C. jejuni i vaccine candidates. I Campylobacter i is the most common bacterial cause of human gastroenteritis in the world, with the species I Campylobacter jejuni i being responsible for over 80% of I Campylobacter i infections [[1]]. A number of research articles investigate infection models and/or immunological aspects of I C. jejuni i infection. The Special Issue aims to look at novel infection models, immunological responses of I Campylobacter i infection and epidemiological studies with the overarching aim of intervention and control strategies. [Extracted from the article]

The paralogues RrpA and RrpB, which are members of the MarR family of DNA binding proteins, are important for the survival of the global bacterial foodborne pathogen Campylobacter jejuni under redox stress. We report that RrpA is a positive regulator of mdaB, encoding a flavin-dependent quinone reductase that contributes to the protection from redox stress mediated by structurally diverse quinones, while RrpB negatively regulates the expression of cj1555c (renamed nfrA for NADPH-flavin reductase A), encoding a flavin reductase. NfrA reduces riboflavin at a greater rate than its derivatives, suggesting that exogenous free flavins are the natural substrate. MdaB and NfrA both prefer NADPH as an electron donor. Cysteine substitution and posttranslational modification analyses indicated that RrpA and RrpB employ a cysteine-based redox switch. Complete genome sequence analyses revealed that mdaB is frequently found in Campylobacter and related Helicobacter spp., while nfrA is predominant in C. jejuni strains. Quinones and flavins are redox cycling agents secreted by a wide range of cell types that can form damaging superoxide by one-electron reactions. We propose a model for stress adaptation where MdaB and NfrA facilitate a two-electron reduction mechanism to the less toxic hydroquinones, thus aiding survival and persistence of this major pathogen. [ABSTRACT FROM AUTHOR]