Helicobacter pullorum, a bacterium initially isolated from poultry, has been associated with human digestive disorders. However, the factor responsible for its cytopathogenic effects on epithelial cells has not been formally identified. The cytopathogenic alterations induced by several human and avian H. pullorum strains were investigated on human intestinal epithelial cell lines. Moreover, the effects of the cytolethal distending toxin (CDT) were evaluated first by using a wild-type strain and its corresponding cdtB isogenic mutant and second by delivering the active CdtB subunit of the CDT directly into the cells. All of the H. pullorum strains induced cellular distending phenotype, actin cytoskeleton remodeling, and G2/M cell cycle arrest. These effects were dependent on the CDT, as they were (1) not observed in response to a cdtB isogenic mutant strain and (2) present in cells expressing CdtB. CdtB also induced an atypical delocalization of vinculin from focal adhesions to the perinuclear region, formation of cortical actin-rich large lamellipodia with an upregulation of cortactin, and decreased cellular adherence. In conclusion, the CDT of H. pullorum is responsible for major cytopathogenic effects in vitro, confirming its role as a main virulence factor of this emerging human pathogen.

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.

Enterohepatic Helicobacter species are associated with several digestive diseases. Helicobacter pullorum is an emerging human foodborne pathogen, and Helicobacter hepaticus is a mouse pathogen; both species are associated with intestinal and/or hepatic diseases. They possess virulence factors, such as cytolethal distending toxin (CDT). Data indicate that CDT may be involved in chronic inflammatory responses, via its active subunit, CdtB. The proinflammatory properties of the CdtB of H. pullorum and H. hepaticus were assessed on human intestinal and hepatic epithelial cells in vitro. Interleukin 8 expression was evaluated by using wild-type strains and their corresponding CdtB isogenic mutants and by delivering CdtB directly into the cells. Nuclear factor κB nuclear translocation and transcriptomic characteristics in response to CdtB were also evaluated. The CdtB of these Helicobacter species induced nuclear factor κB nuclear translocation and exhibited proinflammatory properties, mainly the expression of T-helper type 17–related genes and genes encoding antimicrobial products also involved in cancer. The Histidine residue in position 265 of the CdtB catalytic site appeared to play a role in the regulation of most of these genes. As for flagellin or lipopolysaccharides, CdtB also induced expression of inflammation-associated genes related to antimicrobial activity.

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. Humans are frequently exposed to bacterial genotoxins linked to cancers, colibactin and Cytolethal Distending Toxin (CDT). These genotoxins induce DNA damage and can promote formation of nucleoplasmic reticulum (NR), deeply invaginated in the nucleoplasm of giant nuclei both in vivo and in vitro. Our cellular models showed that these structures can be observed together with profound nuclear reorganization corresponding to remodeling of nuclear material. The core of the genotoxin-induced NRs concentrates protein production machinery of the cell as well as controlling elements of protein turnover. These genotoxin-induced dynamic structures may also be signaling hubs controlling mRNA turnover and translation of selected mRNAs and thus correspond to a privileged gateway for the synthesis of selected mRNA which are preferentially transported from the nucleus through pores and translated therein. These transient and reversible hubs allow the cell to pause and repair the DNA damage caused by bacterial genotoxins in order to maintain cell survival. As NR formation is a common feature of many cancers, similar mechanism could occur and contribute to the resistance of cancer cells to radiotherapies and some chemotherapies aimed at inducing DNA damage.