Transient infection of eukaryotic cells with commensal and extraintestinal pathogenic Escherichia coli of phylogenetic group B2 blocks mitosis and induces megalocytosis. This trait is linked to a widely spread genomic island that encodes giant modular nonribosomal peptide and polyketide synthases. Contact with E. coli expressing this gene cluster causes DNA double-strand breaks and activation of the DNA damage checkpoint pathway, leading to cell cycle arrest and eventually to cell death. Discovery of hybrid peptide-polyketide genotoxins in E. coli will change our view on pathogenesis and commensalism and open new biotechnological applications.

Escherichia coli is a normal inhabitant of the human gut. However, E. coli strains of phylogenetic group B2 harbor a genomic island called "pks" that codes for the production of a polyketide-peptide genotoxin, Colibactin. Here we report that in vivo infection with E. coli harboring the pks island, but not with a pks isogenic mutant, induced the formation of phosphorylated H2AX foci in mouse enterocytes. We show that a single, short exposure of cultured mammalian epithelial cells to live pks + E. coli at low infectious doses induced a transient DNA damage response followed by cell division with signs of incomplete DNA repair, leading to anaphase bridges and chromosome aberrations. Micronuclei, aneuploidy, ring chromosomes, and anaphase bridges persisted in dividing cells up to 21 d after infection, indicating occurrence of breakage-fusion-bridge cycles and chromosomal instability. Exposed cells exhibited a significant increase in gene mutation frequency and anchorage-independent colony formation, demonstrating the infection mutagenic and transforming potential. Therefore, colon colonization with these E. coli strains harboring the pks island could contribute to the development of sporadic colorectal cancer.T he dense bacterial consortium, called "microbiota," that inhabits the intestinal tract is recognized increasingly as playing a major role in human health and disease. The microbiota generally influences the host in a beneficial fashion by shaping gastrointestinal and immune functions, exerting protection against pathogens, and contributing to metabolic pathways (1). Escherichia coli is a consistent member of the human intestinal microbiota, colonizing the intestine within a few days after birth and persisting throughout the life of the host. The E. coli strain population can be categorized in at least four major phylogenetic groups (A, B1, B2, and D), each group being more specifically associated with certain ecological niches. E. coli strains belonging to group B2 are recovered from the environment less frequently but can persist longer in the colon than other groups and represent 30-50% of strains isolated from the feces of healthy humans living in high-income countries (2, 3). We recently discovered that up to 34% of commensal E. coli strains of the phylogenetic group B2 carry a conserved genomic island named "pks island" (4-6). This gene cluster codes for nonribosomal peptide synthetases (NRPS) and polyketide synthetases (PKS) that allow production of a putative hybrid peptide-polyketide genotoxin, Colibactin. In vitro infection with these strains induces DNA double-strand breaks (DSBs) in cultivated human cells, but the pks island was not proved to cause DNA damage in vivo (4).In this study, we wished to explore whether those bacteria were able to induce genetic damage in vivo on the colonic mucosa and to characterize the consequences of this damage on mammalian cells in relation with the number of infecting bacteria. We report that pks + E. coli induced DSBs in vivo. In addition, infection of various ma...

SummaryEnteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. Both use a type III secretion system (TTSS) encoded by the 'locus of enterocyte effacement' (LEE) to subvert and attach to epithelial cells through the injection of a repertoire of effector molecules. Here, we report the identification of a new TTSS translocated effector molecule called Cif, which blocks cell cycle G 2 /M transition and induces the formation of stress fibres through the recruitment of focal adhesions. Cif is not encoded by the LEE but by a lambdoid prophage present in EPEC and EHEC. A cif mutant causes localized effacement of microvilli and intimately attaches to the host cell surface, but is defective in the ability to block mitosis. When expressed in TTSS competent LEE-positive pathogens, Cif is injected into the infected epithelial cells. These cells arrested at the G 2 / M phase displayed accumulation of inactive phosphorylated Cdk1. In conclusion, Cif is a new member of a growing family of bacterial cyclomodulins that subvert the host eukaryotic cell cycle.

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.Although Escherichia coli belongs to the normal microflora present in the gastrointestinal tracts of most mammals and birds, certain E. coli strains have been associated with intestinal or extraintestinal infections. Among these pathogenic E. coli strains, enteropathogenic E. coli (EPEC) is a major cause of infant diarrhea in developing countries (for a recent review, see reference 51) and is a significant category of diarrheagenic E. coli in different animal species. In addition, EPEC is an important cause of morbidity and mortality in weaned rabbits (5,54,56). EPEC is also pathogenic in neonatal calves (20,53) and seems to be isolated more frequently in farms with recurrent diarrhea (7). In swine, EPEC is involved in cases of postweaning diarrhea (67). There is also increasing evidence for a diarrheagenic role of EPEC in dogs (16,64). Finally, EPEC has been isolated ...

Oral administration of the probiotic bacterium Escherichia coli Nissle 1917 improves chronic inflammatory bowel diseases, but the molecular basis for this therapeutic efficacy is unknown. E. coli Nissle 1917 harbors a cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s). This biosynthetic pathway confers the ability for bacteria to induce DNA double strand breaks in eukaryotic cells. Here we reveal that inactivation of the clbA gene within this genomic island abrogated the ability for the strain to induce DNA damage and chromosomal abnormalities in non-transformed cultured rat intestinal epithelial cells but is required for the probiotic activity of E. coli Nissle 1917. Thus, evaluation of colitis severity induced in rodent fed with E. coli Nissle 1917 or an isogenic non-genotoxic mutant demonstrated the need for a functional biosynthetic pathway both in the amelioration of the disease and in the modulation of cytokine expression. Feeding rodents with a complemented strain for which genotoxicity was restored confirmed that this biosynthetic pathway contributes to the health benefits of the probiotic by modulating its immunomodulatory properties. Our data provide additional evidence for the benefit of this currently used probiotic in colitis but remind us that an efficient probiotic may also have side effects as any other medication.

The pks genomic island of Escherichia coli encodes polyketide (PK) and nonribosomal peptide (NRP) synthases that allow assembly of a putative hybrid PK-NRP compound named colibactin that induces DNA double-strand breaks in eukaryotic cells. The pks-encoded machinery harbors an atypical essential protein, ClbP. ClbP crystal structure and mutagenesis experiments revealed a serine-active site and original structural features compatible with peptidase activity, which was detected by biochemical assays. Ten ClbP homologs were identified in silico in NRP genomic islands of closely and distantly related bacterial species. All tested ClbP homologs were able to complement a clbP-deficient E. coli mutant. ClbP is therefore a prototype of a new subfamily of extracytoplasmic peptidases probably involved in the maturation of NRP compounds. Such peptidases will be powerful tools for the manipulation of NRP biosynthetic pathways.

In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin) and of a genotoxin (colibactin) are known to require a 4′-phosphopantetheinyl transferase (PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies.

The neonatal gut is rapidly colonized by a newly dominant group of commensal Escherichia coli strains among which a large proportion produces a genotoxin called colibactin. In order to analyze the short- and long-term effects resulting from such evolution, we developed a rat model mimicking the natural transmission of E. coli from mothers to neonates. Genotoxic and non-genotoxic E. coli strains were equally transmitted to the offspring and stably colonized the gut across generations. DNA damage was only detected in neonates colonized with genotoxic E. coli strains. Signs of genotoxic stress such as anaphase bridges, higher occurrence of crypt fission and accelerated renewal of the mature epithelium were detected at adulthood. In addition, we observed alterations of secretory cell populations and gut epithelial barrier. Our findings illustrate how critical is the genotype of E. coli strains acquired at birth for gut homeostasis at adulthood.