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ABSTRACT Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population; in a subset of this population, its presence is associated with development of severe disease, such as gastric cancer. Genomic analysis of several strains has revealed an extensive H. pylori pan-genome, likely to grow as more genomes are sampled. Here we describe the draft genome sequence (63 contigs; 26× mean coverage) of H. pylori strain B45, isolated from a patient with gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The major finding was a 24.6-kb prophage integrated in the bacterial genome. The prophage shares most of its genes (22/27) with prophage region II of Helicobacter acinonychis strain Sheeba. After UV treatment of liquid cultures, circular DNA carrying the prophage integrase gene could be detected, and intracellular tailed phage-like particles were observed in H. pylori cells by transmission electron microscopy, indicating that phage production can be induced from the prophage. PCR amplification and sequencing of the integrase gene from 341 H. pylori strains from different geographic regions revealed a high prevalence of the prophage (21.4%). Phylogenetic reconstruction showed four distinct clusters in the integrase gene, three of which tended to be specific for geographic regions. Our study implies that phages may play important roles in the ecology and evolution of H. pylori. IMPORTANCE Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population, and while most of the infected individuals do not develop disease, H. pylori infection doubles the risk of developing gastric cancer. An abundance and diversity of viruses (phages) infect microbial populations in most environments and are important mediators of microbial diversity. Our finding of a 24.6-kb prophage integrated inside an H. pylori genome and the observation of circular integrase gene-containing DNA and phage-like particles inside cells upon UV treatment demonstrate that we have discovered a viable H. pylori phage. The additional finding of integrase genes in a large proportion of screened isolates of diverse geographic origins indicates that the prevalence of prophages may have been underestimated in H. pylori. Since phages are important drivers of microbial evolution, the discovery should be important for understanding and predicting genetic diversity in H. pylori.

ABSTRACT The present study describes the first isolation of a recently described Campylobacter species, Campylobacter vicugnae, in humans. The isolates were recovered by two independent French laboratories in 2020 and 2022 from a man and a woman suffering from gastroenteritis. Biochemical and growth characteristics, and electron microscopy for these two strains indicated that they belong to Campylobacter genus. 16S rDNA and GyrA-based phylogeny, as well as average nucleotide identity and in silico DNA-DNA Hybridization analyses revealed that both strains belong to the Campylobacter vicugnae species. Both isolates possess a complete cytolethal distending toxin (CDT) locus with cdtA, cdtB, and cdtC, and features of CDT activity were demonstrated in vitro with Caco-2 intestinal epithelial cells. Our data suggest that these two isolates of C. vicugnae were associated with gastroenteritis in humans and induced major cytopathogenic effects in vitro. C. vicugnae is likely to be a novel human pathogen, with a source of foodborne infection that needs to be determined.IMPORTANCECampylobacter species that display toxicity features are a worldwide public health issue. In clinical contexts, it is crucial to identify which isolate could be an urgent threat to a patient. Actual and widely used laboratory methods such as mass spectrometry or PCR may be flawed in the field of species identification. In contrast, the present study shows that next-generation sequencing allows to precisely identify isolates to species level that may have been omitted otherwise. Moreover, it helps to identify emerging species before they become a threat to human health. Recovery of a new Campylobacter species in human sample, such as the new species “Campylobacter vicugnae,” is an important step for the identification of emerging pathogens posing threat to global health.

Abstract Background Lyme borreliosis is the most prevalent arthropod-borne infection in the Northern Hemisphere. In Europe, Borrelia afzelii is predominantly involved in cutaneous manifestations, Borrelia garinii and Borrelia bavariensis in neurological manifestations, and Borrelia burgdorferi sensu stricto in articular ones. Liver impairement is not classical in Lyme borreliosis. Diagnosis is currently mainly based on serological testing, and is challenging in immunocompromised patients. Case presentation We report the first case of B. garinii infection revealed by liver involvement in an immunocompromised man. A 73-year-old man with marginal zone lymphoma, treated with bendamustine and rituximab, developed intermittent fever and inflammatory syndrome. Microbial investigations were all negative and FDG-PET showed complete remission of the lymphoma. Three months later, liver biopsy was performed and histology revealed spirochetes-like bacteria. Microbial diagnosis was performed by 16S rDNA sequencing, flagellin (flaB) gene sequencing and multi-locus sequence typing and identified B. garinii. The patient recovered successfully after a three weeks course of antibiotics. Diagnosis was challenging because Borrelia hepatic involvement is unusual and no erythema migrans nor tick bite were notified. Conclusion This case highlights that unexplained fever and inflammatory syndrome in immunocompromised patients warrants specific investigations to identify bacteria such as spirochetes.

Humans are frequently exposed to bacterial genotoxins of the gut microbiota, such as colibactin and cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro on HT29 intestinal cells while following the ectopic expression of the active CdtB subunit of Helicobacter hepaticus CDT. Microarray data showed a CdtB-dependent upregulation of transcripts involved in positive regulation of autophagy concomitant with the downregulation of transcripts involved in negative regulation of autophagy. CdtB promotes the activation of autophagy in intestinal and hepatic cell lines. Experiments with cells lacking autophagy related genes, ATG5 and ATG7 infected with CDT- and colibactin-producing bacteria revealed that autophagy protects cells against the genotoxin-induced apoptotic cell death. Autophagy induction could also be associated with nucleoplasmic reticulum (NR) formation following DNA damage induced by these bacterial genotoxins. In addition, both genotoxins promote the accumulation of the autophagic receptor P62/SQSTM1 aggregates, which colocalized with foci concentrating the RNA binding protein UNR/CSDE1. Some of these aggregates were deeply invaginated in NR in distended nuclei together or in the vicinity of UNR-rich foci. Interestingly, micronuclei-like structures and some vesicles containing chromatin and γH2AX foci were found surrounded with P62/SQSTM1 and/or the autophagosome marker LC3. This study suggests that autophagy and P62/SQSTM1 regulate the abundance of micronuclei-like structures and are involved in cell survival following the DNA damage induced by CDT and colibactin. Similar effects were observed in response to DNA damaging chemotherapeutic agents, offering new insights into the context of resistance of cancer cells to therapies inducing DNA damage.

Campylobacter is the leading cause of the human bacterial foodborne infections in the developed countries. The perception cues from biotic or abiotic environments by the bacteria are often related to bacterial surface and membrane proteins that mediate the cellular response for the adaptation of Campylobacter jejuni to the environment. These proteins function rarely as a unique entity, they are often organized in functional complexes. In C. jejuni, these complexes are not fully identified and some of them remain unknown. To identify putative functional multi-subunit entities at the membrane subproteome level of C. jejuni, a holistic non a priori method was addressed using two-dimensional blue native/Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in strain C. jejuni 81–176. Couples of acrylamide gradient/migration-time, membrane detergent concentration and hand-made strips were optimized to obtain reproducible extraction and separation of intact membrane protein complexes (MPCs). The MPCs were subsequently denatured using SDS-PAGE and each spot from each MPCs was identified by mass spectrometry. Altogether, 21 MPCs could be detected including multi homo-oligomeric and multi hetero-oligomeric complexes distributed in both inner and outer membranes. The function, the conservation and the regulation of the MPCs across C. jejuni strains were inspected by functional and genomic comparison analyses. In this study, relatedness between subunits of two efflux pumps, CmeABC and MacABputC was observed. In addition, a consensus sequence CosR-binding box in promoter regions of MacABputC was present in C. jejuni but not in Campylobacter coli. The MPCs identified in C. jejuni 81–176 membrane are involved in protein folding, molecule trafficking, oxidative phosphorylation, membrane structuration, peptidoglycan biosynthesis, motility and chemotaxis, stress signaling, efflux pumps and virulence.

The present study describes three putative novel species received at the French National Reference Center for Campylobacters & Helicobacters (CNRCH). The CNRCH 2005/566H strain was isolated in 2005 from the feces of a patient with a hepatocellular carcinoma and gastroenteritis. Strain 48519 was isolated in 2017 from the blood of a male patient suffering from a bacteremia. Strain Cn23e was isolated from a gastric biopsy from a dog suffering from chronic gastritis. Biochemical and growth characteristics and electron microscopy for these three strains were studied. Their genomes were also sequenced. gyrA based phylogeny built with 72 nucleotide sequences placed CNRCH 2005/566H among the unsheathed enterohepatic helicobacters, close to Helicobacter valdiviensis; strain 48519 among the sheathed enterohepatic helicobacters, close to Helicobacter cinaedi; and strain Cn23e among gastric helicobacters, close to Helicobacter felis. 16S rRNA gene phylogeny showed similar results, but with weak discriminant strength. Average nucleotide identity and in silico DNA–DNA hybridization analyses revealed that CNRCH 2005/566H and 48519 strains belong to new putative species, but confirmed that Cn23e corresponds to H. felis. Cn23e was able to infect C57BL6 mice and to induce gastric inflammation. The genomics data, together with their different morphological and biochemical characteristics, revealed that these two strains represent novel Helicobacter species. We propose the following names: ‘Helicobacter burdigaliensis,’ with the type strain CNRCH 2005/566H ( =CECT 8850 =CIP 111660), and ‘Helicobacter labetoulli,’ with the type strain 48519 ( =CCUG 73475 =CIP 1111659). This study highlights that the diversity of the Helicobacteraceae family remains to be fully explored.

Humans are frequently exposed to bacterial genotoxins involved in digestive cancers, colibactin and Cytolethal Distending Toxin (CDT), the latter being secreted by many pathogenic bacteria. Our aim was to evaluate the effects induced by these genotoxins on nuclear remodeling in the context of cell survival. Helicobacter infected mice, coculture experiments with CDT- and colibactin-secreting bacteria and hepatic, intestinal and gastric cells, and xenograft mouse-derived models were used to assess the nuclear remodeling in vitro and in vivo. Our results showed that CDT and colibactin induced-nuclear remodeling can be associated with the formation of deep cytoplasmic invaginations in the nucleus of giant cells. These structures, observed both in vivo and in vitro, correspond to nucleoplasmic reticulum (NR). The core of the NR was found to concentrate ribosomes, proteins involved in mRNA translation, polyadenylated RNA and the main components of the complex mCRD involved in mRNA turnover. These structures are active sites of mRNA translation, correlated with a high degree of ploidy, and involve MAPK and calcium signaling. Additional data showed that insulation and concentration of these adaptive ribonucleoprotein particles within the nucleus are dynamic, transient and protect the cell until the genotoxic stress is relieved. Bacterial genotoxins-induced NR would be a privileged gateway for selected mRNA to be preferably transported therein for local translation. These findings offer new insights into the context of NR formation, a common feature of many cancers, which not only appears in response to therapies-induced DNA damage but also earlier in response to genotoxic bacteria.

Enterohepatic Helicobacters, such as Helicobacter hepaticus and Helicobacter pullorum, are associated with several intestinal and hepatic diseases. Their main virulence factor is the cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro in HT-29 intestinal cells while following the ectopic expression of the active CdtB subunit of H. hepaticus CDT. A CdtB-dependent upregulation of the V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) gene encoding the MAFB oncoprotein was found, as well as the CdtB-dependent regulation of several MAFB target genes. The transduction and coculture experiments confirmed MAFB mRNA and protein induction in response to CDT and its CdtB subunit in intestinal and hepatic cell lines. An analysis of MAFB protein subcellular localization revealed a strong nuclear and perinuclear localization in the CdtB-distended nuclei in intestinal and hepatic cells. MAFB was also detected at the cell periphery of the CdtB-induced lamellipodia in some cells. The silencing of MAFB changed the cellular response to CDT with the formation of narrower lamellipodia, a reduction of the increase in nucleus size, and the formation of less γH2AX foci, the biomarker for DNA double-strand breaks. Taken together, these data show that the CDT of enterohepatic Helicobacters modulates the expression of the MAFB oncoprotein, which is translocated in the nucleus and is associated with the remodeling of the nuclei and actin cytoskeleton.

Cytolethal distending toxins (CDTs) are common among pathogenic bacteria of the human and animal microbiota. CDTs exert cytopathic effets, via their active CdtB subunit. No clear description of those cytopathic effects has been reported at the cellular level in the target organs in vivo. In the present study, xenograft mouse models of colon and liver cell lines were set up to study the effects of the CdtB subunit of Helicobacter hepaticus. Conditional transgenic cell lines were established, validated in vitro and then engrafted into immunodeficient mice. After successful engraftment, mice were treated with doxycyclin to induce the expression of transgenes (red fluorescent protein, CdtB, and mutated CdtB). For both engrafted cell lines, results revealed a delayed tumor growth and a reduced tumor weight in CdtB-expressing tumors compared to controls. CdtB-derived tumors showed γ-H2AX foci formation, an increase in apoptosis, senescence, p21 and Ki-67 nuclear antigen expression. No difference in proliferating cells undergoing mitosis (phospho-histone H3) was observed. CdtB intoxication was also associated with an overexpression of cytokeratins in cells at the invasive front of the tumor as well as an increase in ploidy. All these features are hallmarks of endoreplication, as well as aggressiveness in cancer. These effects were dependent on the histidine residue at position 265 of the CdtB, underlying the importance of this residue in CdtB catalytic activity. Taken together, these data indicate that the CdtB triggers senescence and cell endoreplication leading to giant polyploid cells in these xenograft mouse models.

Helicobacter pylori is one of the most successful human pathogens, which colonizes the mucus layer of the gastric epithelium of more than 50% of the world’s population. This curved, microaerophilic, Gram-negative bacterium induces a chronic active gastritis, often asymptomatic, in all infected individuals. In some cases, this gastritis evolves to more severe diseases such as peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. H. pylori has developed a unique set of factors, actively supporting its successful survival and persistence in its natural hostile ecological niche, the human stomach, throughout the individual’s life, unless treated. In the human stomach, the vast majority of H. pylori cells are motile in the mucus layer lining, but a small percentage adheres to the epithelial cell surfaces. Adherence to the gastric epithelium is important for the ability of H. pylori to cause disease because this intimate attachment facilitates: (1) colonization and persistence, by preventing the bacteria from being eliminated from the stomach, by mucus turnover and gastric peristalsis; (2) evasion from the human immune system and (3) efficient delivery of proteins into the gastric cell, such as the CagA oncoprotein. Therefore, bacteria with better adherence properties colonize the host at higher densities. H. pylori is one of the most genetically diverse bacterial species known and is equipped with an extraordinarily large set of outer membrane proteins, whose role in the infection and persistence process will be discussed in this review, as well as the different receptor structures that have been so far described for mucosal adherence.

Molecular methods applied to 2,855 strains of Campylobacter-like organisms received from a surveillance network of Campylobacter infections in France identified 29 Arcobacter butzleri infections. This species ranks fourth for Campylobacteraceae isolation and appears to have the same pathogenic potential as the other species in the genus.

Bone marrow-derived mesenchymal stem cells (MSC) have the ability to differentiate into a variety of cell types and are a potential source for epithelial tissue repair. Several studies have demonstrated their ability to repopulate the gastrointestinal tract (GIT) in bone marrow transplanted patients or in animal models of gastrointestinal carcinogenesis where they were the source of epithelial cancers. However, mechanism of MSC epithelial differentiation still remains unclear and controversial with trans-differentiation or fusion events being evoked. This study aimed to investigate the ability of MSC to acquire epithelial characteristics in the particular context of the gastrointestinal epithelium and to evaluate the role of cell fusion in this process. In vitro coculture experiments were performed with three gastrointestinal epithelial cell lines and MSC originating from two patients. After an 8 day coculture, MSC expressed epithelial markers. Use of a semi-permeable insert did not reproduce this effect, suggesting importance of cell contacts. Tagged cells coculture or FISH on gender-mismatched cells revealed clearly that epithelial differentiation resulted from cellular fusion events, while expression of mesenchymal markers on fused cells decreased over time. In vivo cell xenograft in immunodeficient mice confirmed fusion of MSC with gastrointestinal epithelial cells and self-renewal abilities of these fused cells. In conclusion, our results indicate that fusion could be the predominant mechanism by which human MSC may acquire epithelial characteristics when in close contact with epithelial cells from gastrointestinal origin . These results could contribute to a better understanding of the cellular and molecular mechanisms allowing MSC engraftment into the GIT epithelium.