Colibactin-producing Escherichia coli enhance resistance to chemotherapeutic drugs by promoting epithelial to mesenchymal transition and cancer stem cell emergence.

Human colorectal cancers (CRCs) are readily colonized by colibactin-producing E. coli (CoPEC). CoPEC induces DNA double-strand breaks, DNA mutations, genomic instability, and cellular senescence. Infected cells produce a senescence-associated secretory phenotype (SASP), which is involved in the increase in tumorigenesis observed in CRC mouse models infected with CoPEC. This study investigated whether CoPEC, and the SASP derived from CoPEC-infected cells, impacted chemotherapeutic resistance. Human intestinal epithelial cells were infected with the CoPEC clinical 11G5 strain or with its isogenic mutant, which is unable to produce colibactin. Chemotherapeutic resistance was assessed in vitro and in a xenograft mouse model. Expressions of cancer stem cell (CSC) markers in infected cells were investigated. Data were validated using a CRC mouse model and human clinical samples. Both 11G5-infected cells, and uninfected cells incubated with the SASP produced by 11G5-infected cells exhibited an increased resistance to chemotherapeutic drugs in vitro and in vivo. This finding correlated with the induction of the epithelial to mesenchymal transition (EMT), which led to the emergence of cells exhibiting CSC features. They grew on ultra-low attachment plates, formed colonies in soft agar, and overexpressed several CSC markers (e.g. CD133, OCT-3/4, and NANOG). In agreement with these results, murine and human CRC biopsies colonized with CoPEC exhibited higher expression levels of OCT-3/4 and NANOG than biopsies devoid of CoPEC. Conclusion: CoPEC might aggravate CRCs by inducing the emergence of cancer stem cells that are highly resistant to chemotherapy.

Summer diarrhea in children: a monocentric French epidemiological observational study.

Pediatric diarrhea is a major public health problem worldwide. In France, continuous surveillance shows a winter epidemic peak and a more modest summer recrudescence. Few studies describe the infectious agents responsible for pediatric summer diarrhea in France. The objectives were to estimate the prevalence of infectious diarrhea and describe the pathogens responsible for summer diarrhea in children; and to describe common factors that can be used as guidance on the etiology of these diarrheas. A cross-sectional, single-center, epidemiological observational study was conducted in the pediatric emergency department of a French hospital between June and September in 2019 and 2020. Multiplex gastrointestinal pathogen panels were used for diagnostics. A multiple correspondence analysis was used to determine profiles of patients. A total of 95 children were included, of whom 82.1% (78/95) were under five years old. The prevalence of infectious summer diarrhea was 81.1% (77/95, 95%CI 71.7-88.4%). A total of 126 infectious agents were detected (50.0% bacteria, 38.1% viruses, 11.9% parasites). The main enteric pathogens were enteropathogen Escherichia coli (24/126), rotavirus (17/126) and Salmonella (16/126). A co-detection was found in 51.9% (40/77) of cases. Four patient profiles, considering the severity and the pathogen involved, were highlighted.

Cytotoxic necrotizing factor 1 hinders colon tumorigenesis induced by colibactin-producing Escherichia coli in ApcMin/+ mice.

Colorectal cancer (CRC) patients are frequently colonized by colibactin-producing Escherichia coli (CoPEC) (>40%), which enhances tumorigenesis in mouse models of CRC. We observed that 50% of CoPEC also contains the cnf1 gene, which encodes cytotoxic necrotizing factor-1 (CNF1), an enhancer of the eukaryotic cell cycle. The impact of its co-occurrence with colibactin (Clb) has not yet been investigated. We evaluated the impact of CNF1 on colorectal tumorigenesis using human colonic epithelial HT-29 cells and CRC-susceptible ApcMin/+ mice inoculated with the CoPEC 21F8 clinical strain (Clb+Cnf+) or 21F8 isogenic mutants (Clb+Cnf-, Clb-Cnf+ and Clb-Cnf-). Infection with the Clb+Cnf- strain induced higher levels of inflammatory cytokines and senescence markers both in vitro and in vivo compared to those induced by infection with the Clb+Cnf+ strain. In contrast, the Clb+Cnf- and Clb+Cnf+ strains generated similar levels of DNA damage in HT-29 cells and in colonic murine tissues. Furthermore, the ApcMin/+ mice inoculated with the Clb+Cnf- strain developed significantly more tumors than the mice inoculated with the Clb+Cnf+ strain or the isogenic mutants, and the composition of their microbiota was changed. Finally, rectal administration of the CNF1 protein in ApcMin/+ mice inoculated with the Clb+Cnf- strain significantly decreased tumorigenesis and inflammation. Overall, this study provides evidence that CNF1 decreases the carcinogenic effects of CoPEC in ApcMin/+ mice by decreasing CoPEC-induced cellular senescence and inflammation.

Genes mcr improve the intestinal fitness of pathogenic E. coli and balance their lifestyle to commensalism.

BACKGROUND: The plasmid-mediated resistance gene mcr-1 confers colistin resistance in Escherichia coli and paves the way for the evolution to pan-drug resistance. We investigated the impact of mcr-1 in gut colonization in the absence of antibiotics using isogenic E. coli strains transformed with a plasmid encoding or devoid of mcr-1. RESULTS: In gnotobiotic and conventional mice, mcr-1 significantly enhanced intestinal anchoring of E. coli but impaired their lethal effect. This improvement of intestinal fitness was associated with a downregulation of intestinal inflammatory markers and the preservation of intestinal microbiota composition. The mcr-1 gene mediated a cross-resistance to antimicrobial peptides secreted by the microbiota and intestinal epithelial cells (IECs), enhanced E. coli adhesion to IECs, and decreased the proinflammatory activity of both E. coli and its lipopolysaccharides. CONCLUSION: Overall, mcr-1 changed multiple facets of bacterial behaviour and appeared as a factor enhancing commensal lifestyle and persistence in the gut even in the absence of antibiotics. Video Abstract.

Nitric Oxide Impacts Human Gut Microbiota Diversity and Functionalities.

The disruption of gut microbiota homeostasis has been associated with numerous diseases and with a disproportionate inflammatory response, including overproduction of nitric oxide (NO) in the intestinal lumen. However, the influence of NO on the human gut microbiota has not been well characterized yet. We used in vitro fermentation systems inoculated with human fecal samples to monitor the effect of repetitive NO pulses on the gut microbiota. NO exposure increased the redox potential and modified the fermentation profile and gas production. The overall metabolome was modified, reflecting less strict anaerobic conditions and shifts in amino acid and nitrogen metabolism. NO exposure led to a microbial shift in diversity with a decrease in Clostridium leptum group and Faecalibacterium prausnitzii biomass and an increased abundance of the Dialister genus. Escherichia coli, Enterococcus faecalis, and Proteus mirabilis operational taxonomic unit abundance increased, and strains from those species isolated after NO stress showed resistance to high NO concentrations. As a whole, NO quickly changed microbial fermentations, functions, and composition in a pulse- and dose-dependent manner. NO could shift, over time, the trophic chain to conditions that are unfavorable for strict anaerobic microbial processes, implying that a prolonged or uncontrolled inflammation has detrimental and irreversible consequences on the human microbiome. IMPORTANCE Gut microbiota dysbiosis has been associated with inflammatory diseases. The human inflammatory response leads to an overproduction of nitric oxide (NO) in the gut. However, so far, the influence of NO on the human gut microbiota has not been characterized. In this study, we used in vitro fermentation systems with human fecal samples to understand the effect of NO on the microbiota: NO modified the microbial composition and its functionality. High NO concentration depleted the microbiota of beneficial butyrate-producing species and favored potentially deleterious species (E. coli, E. faecalis, and P. mirabilis), which we showed can sustain high NO concentrations. Our work shows that NO may participate in the vicious circle of inflammation, leading to detrimental and irreversible consequences on human health.

Yersiniabactin Siderophore of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Is Involved in Autophagy Activation in Host Cells.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC) have been implicated in the etiology of Crohn's disease. The AIEC reference strain LF82 possesses a pathogenicity island similar to the high pathogenicity island of Yersinia spp., which encodes the yersiniabactin siderophore required for iron uptake and growth of the bacteria in iron-restricted environment. Here, we investigated the role of yersiniabactin during AIEC infection. METHODS: Intestinal epithelial T84 cells and CEABAC10 transgenic mice were infected with LF82 or its mutants deficient in yersiniabactin expression. Autophagy was assessed by Western blot analysis for p62 and LC3-II expression. RESULTS: Loss of yersiniabactin decreased the growth of LF82 in competitive conditions, reducing the ability of LF82 to adhere to and invade T84 cells and to colonize the intestinal tract of CEABAC10 mice. However, yersiniabactin deficiency increased LF82 intracellular replication. Mechanistically, a functional yersiniabactin is necessary for LF82-induced expression of HIF-1α, which is implicated in autophagy activation in infected cells. CONCLUSION: Our study highlights a novel role for yersiniabactin siderophore in AIEC-host interaction. Indeed, yersiniabactin, which is an advantage for AIEC to growth in a competitive environment, could be a disadvantage for the bacteria as it activates autophagy, a key host defense mechanism, leading to bacterial clearance.

Propionate catabolism by CD-associated adherent-invasive E. coli counteracts its anti-inflammatory effect.

Crohn's disease (CD) is a chronic and disabling inflammatory disorder of the gut that is profoundly influenced by intestinal microbiota composition, host genetics and environmental factors. Several groups worldwide have described an imbalance of the gut microbiome composition, called dysbiosis, in CD patients, with an increase in Proteobacteria and Bacteroidetes and a decrease in Firmicutes. A high prevalence of adherent-invasive Escherichia coli (AIEC) pathobionts has been identified in the intestinal mucosa of CD patients. A significant loss in the bacteria that produce short-chain fatty acids (SCFAs) with anti-inflammatory properties, such as propionate, is also a consequence of dysbiosis in CD patients. Here, the AIEC reference strain LF82 was able to degrade propionate in the gut, which was sufficient to counteract the anti-inflammatory effect of propionate both in in vitro models and in mice with DSS-induced colitis. The consumption of propionate by AIEC pathobionts leads to an increase in TNF-α production by macrophages upon infection through the bacterial methyl-citrate pathway. To induce the protective effects of SCFAs on the inflamed gut, we used a G-protein-coupled receptor 43 agonist (GPR43 agonist) that is not metabolizable by intestinal bacteria. Interestingly, this agonist showed anti-inflammatory properties and decreased the severity of colitis in AIEC-infected mice, as assessed by an improvement in the disease activity index (DAI) and a decrease in AIEC pathobiont encroachment. Taken together, these results highlight the effectiveness of GPR43 agonist treatment in the control of gut inflammation and improved our understanding of the ability of AIEC to modulate propionate availability to create an infectious niche to its advantage.

Pathogenicity Factors of Genomic Islands in Intestinal and Extraintestinal Escherichia coli.

Escherichia coli is a versatile bacterial species that includes both harmless commensal strains and pathogenic strains found in the gastrointestinal tract in humans and warm-blooded animals. The growing amount of DNA sequence information generated in the era of "genomics" has helped to increase our understanding of the factors and mechanisms involved in the diversification of this bacterial species. The pathogenic side of E. coli that is afforded through horizontal transfers of genes encoding virulence factors enables this bacterium to become a highly diverse and adapted pathogen that is responsible for intestinal or extraintestinal diseases in humans and animals. Many of the accessory genes acquired by horizontal transfers form syntenic blocks and are recognized as genomic islands (GIs). These genomic regions contribute to the rapid evolution, diversification and adaptation of E. coli variants because they are frequently subject to rearrangements, excision and transfer, as well as to further acquisition of additional DNA. Here, we review a subgroup of GIs from E. coli termed pathogenicity islands (PAIs), a concept defined in the late 1980s by Jörg Hacker and colleagues in Werner Goebel's group at the University of Würzburg, Würzburg, Germany. As with other GIs, the PAIs comprise large genomic regions that differ from the rest of the genome by their G + C content, by their typical insertion within transfer RNA genes, and by their harboring of direct repeats (at their ends), integrase determinants, or other mobility loci. The hallmark of PAIs is their contribution to the emergence of virulent bacteria and to the development of intestinal and extraintestinal diseases. This review summarizes the current knowledge on the structure and functional features of PAIs, on PAI-encoded E. coli pathogenicity factors and on the role of PAIs in host-pathogen interactions.

Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn's disease-associated adherent-invasive E. coli.

Adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of Crohn's disease (CD) patients, are able to adhere to and invade intestinal epithelial cells (IECs), survive and replicate within macrophages and induce a pro-inflammatory response. AIEC infection of IECs induces secretion of exosomes that increase AIEC replication in exosome-receiving IECs and macrophages. Here, we investigated the mechanism underlying the increased AIEC replication in cells receiving exosomes from AIEC-infected cells. Exosomes released by uninfected human intestinal epithelial T84 cells (Exo-uninfected) or by T84 cells infected with the clinical AIEC LF82 strain (Exo-LF82), the nonpathogenic E. coli K12 strain (Exo-K12) or the commensal E. coli HS strain (Exo-HS) were purified and used to stimulate T84 cells. Stimulation of T84 cells with Exo-LF82 inhibited autophagy compared with Exo-uninfected, Exo-K12 and Exo-HS. qRT-PCR analysis revealed increased levels of miR-30c and miR-130a in Exo-LF82 compared to Exo-uninfected, Exo-K12 and Exo-HS. These miRNAs were transferred via exosomes to recipient cells, in which they targeted and inhibited ATG5 and ATG16L1 expression and thereby autophagy response, thus favoring AIEC intracellular replication. Inhibition of these miRNAs in exosome-donor cells infected with AIEC LF82 abolished the increase in miR-30c and miR-130a levels in the released Exo-LF82 and in Exo-LF82-receiving cells, thus suppressing the inhibitory effect of Exo-LF82 on ATG5 and ATG16L1 expression and on autophagy-mediated AIEC clearance in Exo-LF82-receiving cells. Our study shows that upon AIEC infection, IECs secrete exosomes that can transfer specific miRNAs to recipient IECs, inhibiting autophagy-mediated clearance of intracellular AIEC.

Adaptation of adherent-invasive E. coli to gut environment: Impact on flagellum expression and bacterial colonization ability.

The pathogenesis of Crohn's disease (CD) is multifactorial and involves genetic susceptibility, environmental triggers and intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) are flagellated bacteria more prevalent in CD patients than in healthy subjects and promote chronic intestinal inflammation. We aim at deciphering the role of flagella and flagellin modulation by intestinal conditions. AIEC flagellum expression is required for optimal adhesion to and invasion of intestinal epithelial cells. Interestingly, differential flagellin regulation was observed between commensal E. coli (HS) and AIEC (LF82) strains: flagellum expression by AIEC bacteria, in contrast to that of commensal E. coli, is enhanced under intestinal conditions (the presence of bile acids and mucins). Flagella are involved in the ability of the AIEC LF82 strain to cross a mucus layer in vitro and in vivo, conferring a selective advantage in penetrating the mucus layer and reaching the epithelial surface. In a CEABAC10 mouse model, a non-motile mutant (LF82-ΔfliC) exhibits reduced colonization that is restored by a dextran sodium sulfate treatment that alters mucus layer integrity. Moreover, a mutant that continuously secretes flagellin (LF82-ΔflgM) triggers a stronger inflammatory response than the wild-type strain, and the mutant's ability to colonize the CEABAC10 mouse model is decreased. Overexpression of flagellin in bacteria in contact with epithelial cells can be detrimental to their virulence by inducing acute inflammation that enhances AIEC clearance. AIEC pathobionts must finely modulate flagellum expression during the infection process, taking advantage of their specific virulence gene regulation to improve their adaptability and flexibility within the gut environment.

Metabolic adaptation of adherent-invasive Escherichia coli to exposure to bile salts.

The adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of Crohn's disease patients, adhere to intestinal epithelial cells, invade them and exacerbate intestinal inflammation. The high nutrient competition between the commensal microbiota and AIEC pathobiont requires the latter to occupy their own metabolic niches to survive and proliferate within the gut. In this study, a global RNA sequencing of AIEC strain LF82 has been used to observe the impact of bile salts on the expression of metabolic genes. The results showed a global up-regulation of genes involved in degradation and a down-regulation of those implicated in biosynthesis. The main up-regulated degradation pathways were ethanolamine, 1,2-propanediol and citrate utilization, as well as the methyl-citrate pathway. Our study reveals that ethanolamine utilization bestows a competitive advantage of AIEC strains that are metabolically capable of its degradation in the presence of bile salts. We observed that bile salts activated secondary metabolism pathways that communicate to provide an energy benefit to AIEC. Bile salts may be used by AIEC as an environmental signal to promote their colonization.

Crohn's Disease-Associated Adherent-Invasive Escherichia coli Manipulate Host Autophagy by Impairing SUMOylation.

The intestinal mucosa of Crohn's disease (CD) patients is abnormally colonized with adherent-invasive Escherichia coli (AIEC) that are able to adhere to and to invade intestinal epithelial cells (IECs), to survive in macrophages, and to induce a pro-inflammatory response. AIEC persist in the intestine, and induce inflammation in CEABAC10 transgenic mice expressing human CAECAM6, the receptor for AIEC. SUMOylation is a eukaryotic-reversible post-translational modification, in which SUMO, an ubiquitin-like polypeptide, is covalently linked to target proteins. Here, we investigated the role of SUMOylation in host responses to AIEC infection. We found that infection with the AIEC LF82 reference strain markedly decreased the levels of SUMO-conjugated proteins in human intestinal epithelial T84 cells. This was also observed in IECs from LF82-infected CEABAC10 transgenic mice. LF82-induced deSUMOylation in IECs was due in part to increased level of microRNA (miR)-18, which targets PIAS3 mRNA encoding a protein involved in SUMOylation. Over-expression of SUMOs in T84 cells induced autophagy, leading to a significant decrease in the number of intracellular LF82. Consistently, a decreased expression of UBC9, a protein necessary for SUMOylation, was accompanied with a decrease of LF82-induced autophagy, increasing bacterial intracellular proliferation and inflammation. Finally, the inhibition of miR-18 significantly decreased the number of intracellular LF82. In conclusion, our results suggest that AIEC inhibits the autophagy response to replicate intracellularly by manipulating host SUMOylation.

Colibactin: More Than a New Bacterial Toxin.

Cyclomodulins are bacterial toxins that interfere with the eukaryotic cell cycle. A new cyclomodulin called colibactin, which is synthetized by the pks genomic island, was discovered in 2006. Despite many efforts, colibactin has not yet been purified, and its structure remains elusive. Interestingly, the pks island is found in members of the family Enterobacteriaceae (mainly Escherichia coli and Klebsiella pneumoniae) isolated from different origins, including from intestinal microbiota, septicaemia, newborn meningitis, and urinary tract infections. Colibactin-producing bacteria induce chromosomal instability and DNA damage in eukaryotic cells, which leads to senescence of epithelial cells and apoptosis of immune cells. The pks island is mainly observed in B2 phylogroup E. coli strains, which include extra-intestinal pathogenic E. coli strains, and pksE. coli are over-represented in biopsies isolated from colorectal cancer. In addition, pksE. coli bacteria increase the number of tumours in diverse colorectal cancer mouse models. Thus, colibactin could have a major impact on human health. In the present review, we will focus on the biological effects of colibactin, the distribution of the pks island, and summarize what is currently known about its synthesis and its structure.

Impact of CDT Toxin on Human Diseases.

Cytolethal distending toxin (CDT) is found in Gram-negative bacteria, especially in certain Proteobacteria such as the Pasteurellaceae family, including Haemophilus ducreyi and Aggregatibacter (Actinobacillus) actinomycetemcomitans, in the Enterobacteriaceae family and the Campylobacterales order, including the Campylobacter and Helicobacter species. In vitro and in vivo studies have clearly shown that this toxin has a strong effect on cellular physiology (inflammation, immune response modulation, tissue damage). Some works even suggest a potential involvement of CDT in cancers. In this review, we will discuss these different aspects.

The Vat-AIEC protease promotes crossing of the intestinal mucus layer by Crohn's disease-associated Escherichia coli.

The aetiology of Crohn's disease (CD) involves disorders in host genetic factors and intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) are receiving increased attention because in studies of mucosa-associated microbiota, they are more prevalent in CD patients than in healthy subjects. AIEC are associated both with ileal and colonic disease phenotypes. In this study, we reported a protease called Vat-AIEC from AIEC that favours the mucosa colonization. The deletion of the Vat-AIEC-encoding gene resulted in an adhesion-impaired phenotype in vitro and affected the colonization of bacteria in contact with intestinal epithelial cells in a murine intestinal loop model, and also their gut colonization in vivo. Furthermore, unlike LF82Δvat-AIEC, wild-type AIEC reference strain LF82 was able to penetrate a mucus column extensively and promoted the degradation of mucins and a decrease in mucus viscosity. Vat-AIEC transcription was stimulated by several chemical conditions found in the ileum environment. Finally, the screening of E. coli strains isolated from CD patients revealed a preferential vat-AIEC association with AIEC strains belonging to the B2 phylogroup. Overall, this study revealed a new component of AIEC virulence that might favour their implantation in the gut of CD patients.

Small-molecule inhibitors prevent the genotoxic and protumoural effects induced by colibactin-producing bacteria.

OBJECTIVE: Colorectal cancers (CRCs) are frequently colonised by colibactin toxin-producing Escherichia coli bacteria that induce DNA damage in host cells and exhibit protumoural activities. Our objective was to identify small molecules inhibiting the toxic effects induced by these colibactin-producing bacteria. DESIGN: A structural approach was adopted for the identification of a putative ligand for the ClbP enzyme involved in the synthesis of colibactin. Intestinal epithelial cells and a CRC mouse model were used to assess the activity of the selected compounds in vitro and in vivo. RESULTS: Docking experiments identified two boron-based compounds with computed ligand efficiency values (-0.8 and -0.9 kcal/mol/atom) consistent with data expected for medicinal chemistry leads. The crystalline structure of ClbP in complex with the compounds confirmed that the compounds were binding to the active site of ClbP. The two compounds (2 mM) suppressed the genotoxic activity of colibactin-producing E coli both in vitro and in vivo. The mean degree of suppression of DNA damage for the most efficient compound was 98±2% (95% CI). This compound also prevented cell proliferation and colibactin-producing E coli-induced tumourigenesis in mice. In a CRC murine model colonised by colibactin-producing E coli, the number of tumours decreased by 3.5-fold in animals receiving the compound in drinking water (p<0.01). CONCLUSIONS: These results demonstrate that targeting colibactin production controls the genotoxic and protumoural effects induced by this toxin.

The bacterial genotoxin colibactin promotes colon tumor growth by modifying the tumor microenvironment.

The gut microbiota is suspected to promote colorectal cancer (CRC). Escherichia coli are more frequently found in CCR biopsies than in healthy mucosa; furthermore, the majority of mucosa-associated E. coli isolated from CCR harbors the pks genomic island (pks+ E. coli) that is responsible for the synthesis of colibactin, a genotoxic compound. We have recently reported that transient contact of a few malignant cells with colibactin-producing E. coli increases tumor growth in a xenograft mouse model. Growth is sustained by cellular senescence that is accompanied by the production of growth factors. We demonstrated that cellular senescence is a consequence of the pks+ E. coli-induced alteration of p53 SUMOylation, an essential post-translational modification in eukaryotic cells. The underlying mechanisms for this process involve the induction of miR-20a-5p expression, which targets SENP1, a key protein in the regulation of the SUMOylation process. These results are consistent with the expression of SENP1, miR-20a-5p and growth factors that are observed in a CRC mouse model and in human CCR biopsies colonized by pks+ E. coli. Overall, the data reveal a new paradigm for carcinogenesis in which pks+ E. coli infection induces cellular senescence characterized by the production of growth factors that promote the proliferation of uninfected cells and, subsequently, tumor growth.