Uncoupling the Hsp90 and DnaK chaperone activities revealed the in vivo relevance of their collaboration in bacteria.

Chaperone proteins are essential in all living cells to ensure protein homeostasis. Hsp90 is a major adenosine triphosphate (ATP)-dependent chaperone highly conserved from bacteria to eukaryotes. Recent studies have shown that bacterial Hsp90 is essential in some bacteria in stress conditions and that it participates in the virulence of pathogenic bacteria. In vitro, bacterial Hsp90 directly interacts and collaborates with the Hsp70 chaperone DnaK to reactivate model substrate proteins; however, it is still unknown whether this collaboration is relevant in vivo with physiological substrates. Here, we used site-directed mutagenesis on Hsp90 to impair DnaK binding, thereby uncoupling the chaperone activities. We tested the mutants in vivo in two bacterial models in which Hsp90 has known physiological functions. We found that the Hsp90 point mutants were defective to support (1) growth under heat stress and activation of an essential Hsp90 client in the aquatic bacterium Shewanella oneidensis and (2) biosynthesis of the colibactin toxin involved in the virulence of pathogenic Escherichia coli. Our study therefore demonstrates the essentiality of the direct collaboration between Hsp90 and DnaK in vivo in bacteria to support client folding. It also suggests that this collaboration already functional in bacteria has served as an evolutionary basis for a more complex Hsp70-Hsp90 collaboration found in eukaryotes.

Identification of bacterial lipopeptides as key players in IBS.

OBJECTIVES: Clinical studies revealed that early-life adverse events contribute to the development of IBS in adulthood. The aim of our study was to investigate the relationship between prenatal stress (PS), gut microbiota and visceral hypersensitivity with a focus on bacterial lipopeptides containing γ-aminobutyric acid (GABA). DESIGN: We developed a model of PS in mice and evaluated, in adult offspring, visceral hypersensitivity to colorectal distension (CRD), colon inflammation, barrier function and gut microbiota taxonomy. We quantified the production of lipopeptides containing GABA by mass spectrometry in a specific strain of bacteria decreased in PS, in PS mouse colons, and in faeces of patients with IBS and healthy volunteers (HVs). Finally, we assessed their effect on PS-induced visceral hypersensitivity. RESULTS: Prenatally stressed mice of both sexes presented visceral hypersensitivity, no overt colon inflammation or barrier dysfunction but a gut microbiota dysbiosis. The dysbiosis was distinguished by a decreased abundance of Ligilactobacillus murinus, in both sexes, inversely correlated with visceral hypersensitivity to CRD in mice. An isolate from this bacterial species produced several lipopeptides containing GABA including C14AsnGABA. Interestingly, intracolonic treatment with C14AsnGABA decreased the visceral sensitivity of PS mice to CRD. The concentration of C16LeuGABA, a lipopeptide which inhibited sensory neurons activation, was decreased in faeces of patients with IBS compared with HVs. CONCLUSION: PS impacts the gut microbiota composition and metabolic function in adulthood. The reduced capacity of the gut microbiota to produce GABA lipopeptides could be one of the mechanisms linking PS and visceral hypersensitivity in adulthood.

Uropathogenic E. coli induces DNA damage in the bladder.

Urinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of the cleavage product released during the synthesis of colibactin, a bacterial genotoxin, in 55 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells.

From ribosome to ribotoxins: understanding the toxicity of deoxynivalenol and Shiga toxin, two food borne toxins.

Ribosomes that synthesize proteins are among the most central and evolutionarily conserved organelles. Given the key role of proteins in cellular functions, prokaryotic and eukaryotic pathogens have evolved potent toxins to inhibit ribosomal functions and weaken their host. Many of these ribotoxin-producing pathogens are associated with food. For example, food can be contaminated with bacterial pathogens that produce the ribotoxin Shiga toxin, but also with the fungal ribotoxin deoxynivalenol. Shiga toxin cleaves ribosomal RNA, while deoxynivalenol binds to and inhibits the peptidyl transferase center. Despite their distinct modes of action, both groups of ribotoxins hinder protein translation, but also trigger other comparable toxic effects, which depend or not on the activation of the ribotoxic stress response. Ribotoxic stress response-dependent effects include inflammation and apoptosis, whereas ribotoxic stress response-independent effects include endoplasmic reticulum stress, oxidative stress, and autophagy. For other effects, such as cell cycle arrest and cytoskeleton modulation, the involvement of the ribotoxic stress response is still controversial. Ribotoxins affect one organelle yet induce multiple toxic effects with multiple consequences for the cell. The ribosome can therefore be considered as the cellular "Achilles heel" targeted by food borne ribotoxins. Considering the high toxicity of ribotoxins, they pose a substantial health risk, as humans are highly susceptible to widespread exposure to these toxins through contaminated food sources.

Wild Boars as Reservoir of Highly Virulent Clone of Hybrid Shiga Toxigenic and Enterotoxigenic Escherichia coli Responsible for Edema Disease, France.

Edema disease is an often fatal enterotoxemia caused by specific strains of Shiga toxin-producing Escherichia coli (STEC) that affect primarily healthy, rapidly growing nursery pigs. Recently, outbreaks of edema disease have also emerged in France in wild boars. Analysis of STEC strains isolated from wild boars during 2013-2019 showed that they belonged to the serotype O139:H1 and were positive for both Stx2e and F18 fimbriae. However, in contrast to classical STEC O139:H1 strains circulating in pigs, they also possessed enterotoxin genes sta1 and stb, typical of enterotoxigenic E. coli. In addition, the strains contained a unique accessory genome composition and did not harbor antimicrobial-resistance genes, in contrast to domestic pig isolates. These data thus reveal that the emergence of edema disease in wild boars was caused by atypical hybrid of STEC and enterotoxigenic E. coli O139:H1, which so far has been restricted to the wildlife environment.

Large-scale genome analysis of bovine commensal Escherichia coli reveals that bovine-adapted E. coli lineages are serving as evolutionary sources of the emergence of human intestinal pathogenic strains.

How pathogens evolve their virulence to humans in nature is a scientific issue of great medical and biological importance. Shiga toxin (Stx)-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are the major foodborne pathogens that can cause hemolytic uremic syndrome and infantile diarrhea, respectively. The locus of enterocyte effacement (LEE)-encoded type 3 secretion system (T3SS) is the major virulence determinant of EPEC and is also possessed by major STEC lineages. Cattle are thought to be the primary reservoir of STEC and EPEC. However, genome sequences of bovine commensal E. coli are limited, and the emerging process of STEC and EPEC is largely unknown. Here, we performed a large-scale genomic comparison of bovine commensal E. coli with human commensal and clinical strains, including EPEC and STEC, at a global level. The analyses identified two distinct lineages, in which bovine and human commensal strains are enriched, respectively, and revealed that STEC and EPEC strains have emerged in multiple sublineages of the bovine-associated lineage. In addition to the bovine-associated lineage-specific genes, including fimbriae, capsule, and nutrition utilization genes, specific virulence gene communities have been accumulated in stx- and LEE-positive strains, respectively, with notable overlaps of community members. Functional associations of these genes probably confer benefits to these E. coli strains in inhabiting and/or adapting to the bovine intestinal environment and drive their evolution to highly virulent human pathogens under the bovine-adapted genetic background. Our data highlight the importance of large-scale genome sequencing of animal strains in the studies of zoonotic pathogens.

Screening for ß-lactam resistance by penicillin G in the Streptococcus anginosus group challenged by rare strains with altered PBPs.

BACKGROUND: Streptococcus anginosus group (SAG) strains are common pathogens causing abscesses and bacteraemia. They are generally susceptible to ß-lactams, which constitute first-line treatment. EUCAST recommends testing penicillin G susceptibility to screen for ß-lactam resistance. Isolates categorized as susceptible (negative screening) can be reported as susceptible to aminopenicillins and third-generation cephalosporins. OBJECTIVES: To assess the reliability of penicillin G resistance screening in predicting ß-lactam resistance in SAG blood culture isolates, and to investigate isolates for which this test would be unreliable. METHODS: We determined the susceptibility to penicillin G, amoxicillin and ceftriaxone of 90 SAG blood culture isolates, all with negative penicillin G resistance screening. ß-Lactam-resistant strains were sequenced and compared with susceptible reference SAG strains. RESULTS: We detected two isolates displaying ß-lactam resistance, especially to third-generation cephalosporins, despite negative screening for penicillin G resistance. For these isolates, amino acid substitutions were identified next to the essential PBP motifs SxxK, SxN and/or KS/TGS/T. Changes in these motifs have been previously linked to ß-lactam resistance in Streptococcus pneumoniae. CONCLUSIONS: Our study suggests that aminopenicillin and third-generation cephalosporin susceptibility should be determined for SAG strains in the event of severe infection as screening for penicillin G resistance might not be sufficient to detect resistance mechanisms that predominantly affect cephalosporins. The PBP sequencing of resistant SAG strains allowed us to detect amino acid changes potentially linked to ß-lactam resistance.

Bacteria-derived long chain fatty acid exhibits anti-inflammatory properties in colitis.

OBJECTIVE: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action. DESIGN: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis. RESULTS: Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum, Holdemanella and Parabacteroides. In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma. CONCLUSION: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions.

Deciphering the interplay between the genotoxic and probiotic activities of Escherichia coli Nissle 1917.

Although Escherichia coli Nissle 1917 (EcN) has been used therapeutically for over a century, the determinants of its probiotic properties remain elusive. EcN produces two siderophore-microcins (Mcc) responsible for an antagonistic activity against other Enterobacteriaceae. EcN also synthesizes the genotoxin colibactin encoded by the pks island. Colibactin is a virulence factor and a putative pro-carcinogenic compound. Therefore, we aimed to decouple the antagonistic activity of EcN from its genotoxic activity. We demonstrated that the pks-encoded ClbP, the peptidase that activates colibactin, is required for the antagonistic activity of EcN. The analysis of a series of ClbP mutants revealed that this activity is linked to the transmembrane helices of ClbP and not the periplasmic peptidase domain, indicating the transmembrane domain is involved in some aspect of Mcc biosynthesis or secretion. A single amino acid substitution in ClbP inactivates the genotoxic activity but maintains the antagonistic activity. In an in vivo salmonellosis model, this point mutant reduced the clinical signs and the fecal shedding of Salmonella similarly to the wild type strain, whereas the clbP deletion mutant could neither protect nor outcompete the pathogen. The ClbP-dependent antibacterial effect was also observed in vitro with other E. coli strains that carry both a truncated form of the Mcc gene cluster and the pks island. In such strains, siderophore-Mcc synthesis also required the glucosyltransferase IroB involved in salmochelin production. This interplay between colibactin, salmochelin, and siderophore-Mcc biosynthetic pathways suggests that these genomic islands were co-selected and played a role in the evolution of E. coli from phylogroup B2. This co-evolution observed in EcN illustrates the fine margin between pathogenicity and probiotic activity, and the need to address both the effectiveness and safety of probiotics. Decoupling the antagonistic from the genotoxic activity by specifically inactivating ClbP peptidase domain opens the way to the safe use of EcN.

Escherichia coli†ClbS is a colibactin resistance protein.

The genomic pks island codes for the biosynthetic machinery that produces colibactin, a peptide-polyketide metabolite. Colibactin is a genotoxin that contributes to the virulence of extra-intestinal pathogenic Escherichia coli and promotes colorectal cancer. In this work, we examined whether the pks-encoded clbS gene of unknown function could participate in the self-protection of E. coli-producing colibactin. A clbS mutant was not impaired in the ability to inflict DNA damage in HeLa cells, but the bacteria activated the SOS response and ceased to replicate. This autotoxicity phenotype was markedly enhanced in a clbS uvrB double mutant inactivated for DNA repair by nucleotide excision but was suppressed in a clbS clbA double mutant unable to produce colibactin. In addition, ectopic expression of clbS protected infected HeLa cells from colibactin. Thus, ClbS is a resistance protein blocking the genotoxicity of colibactin both in the procaryotic and the eucaryotic cells.

Interplay between siderophores and colibactin genotoxin in Escherichia coli.

Highly pathogenic Escherichia coli strains that belong to the phylogenetic group B2 have developed a greater ability to acquire iron (heme receptor and numerous siderophores), to produce the genotoxin colibactin and to synthesize antimicrobial siderophore-microcins. There is an increased prevalence of these E. coli strains over the last 30 years in the intestinal microbiota in industrialized countries. Integrating the regulation of fitness/virulence factors, such as siderophores, colibactin and siderophore-microcins into networks that respond to specific environmental signals, such as the local iron concentration, could result in an accurate production of specific fitness/virulence factors, so that the E. coli can adapt to the competitive environment that is the gut and/or the blood. Iron deficiency is common in infancy, even in industrialized countries. Usual strategies for anemia correction are iron supplementation and iron fortification of foods. The long-term consequences and risks associated with high iron supply in the light of this iron-dependent network described in this review could explain at least in part the increased prevalence of E. coli B2 in the gut of people in industrialized countries. © 2017 IUBMB Life, 69(6):435-441, 2017.

HlyF Produced by Extraintestinal Pathogenic Escherichia coli Is a Virulence Factor That Regulates Outer Membrane Vesicle Biogenesis.

Escherichia coli can cause extraintestinal infections in humans and animals. The hlyF gene is epidemiologically associated with virulent strains of avian pathogenic E. coli and human neonatal meningitis-associated E. coli. We demonstrated that culture supernatants of E. coli expressing HlyF induced autophagy in eukaryotic cells. This phenotype coincided with an enhanced production of outer membrane vesicles (OMVs) by bacteria expressing HlyF. The HlyF protein displays a predicted catalytic domain of the short-chain dehydrogenase/reductase superfamily. This conserved domain was involved the ability of HlyF to promote the production of OMVs. The increased production of OMVs was associated with the release of toxins. hlyF was shown to be expressed during extraintestinal infection and to play a role in the virulence of extraintestinal pathogenic E. coli in a chicken model of colibacillosis. This is the first evidence that pathogenic bacteria produce a virulence factor directly involved in the production of OMVs.

The Bacterial Stress-Responsive Hsp90 Chaperone (HtpG) Is Required for the Production of the Genotoxin Colibactin and the Siderophore Yersiniabactin in Escherichia coli.

The genotoxin colibactin, synthesized by Escherichia coli, is a secondary metabolite belonging to the chemical family of hybrid polyketide/nonribosomal peptide compounds. It is produced by a complex biosynthetic assembly line encoded by the pks pathogenicity island. The presence of this large cluster of genes in the E. coli genome is invariably associated with the high-pathogenicity island, encoding the siderophore yersiniabactin, which belongs to the same chemical family as colibactin. The E. coli heat shock protein HtpG (Hsp90Ec) is the bacterial homolog of the eukaryotic molecular chaperone Hsp90, which is involved in the protection of cellular proteins against a variety of environmental stresses. In contrast to eukaryotic Hsp90, the functions and client proteins of Hsp90Ec are poorly known. Here, we demonstrated that production of colibactin and yersiniabactin is abolished in the absence of Hsp90Ec We further characterized an interplay between the Hsp90Ec molecular chaperone and the ClpQ protease involved in colibactin and yersiniabactin synthesis. Finally, we demonstrated that Hsp90Ec is required for the full in vivo virulence of extraintestinal pathogenic E. coli This is the first report highlighting the role of heat shock protein Hps90Ec in the production of two secondary metabolites involved in E. coli virulence.

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.

Iron Homeostasis Regulates the Genotoxicity of Escherichia coli That Produces Colibactin.

The genotoxin colibactin is a secondary metabolite produced by a variety of pathogenic enterobacteria. Its biosynthesis requires the enzymatic activity of the phosphopantetheinyl transferase (PPTase) ClbA. We previously showed that ClbA can also contribute to the production of siderophores. Because the biosynthesis of siderophores is regulated by iron availability, we hypothesized that iron could also modulate the production of colibactin through the transcriptional regulation of clbA This study revealed an increased transcription of clbA under iron-limiting conditions and a decrease of clbA expression in iron-rich media. We demonstrate that clbA transcription is regulated by both the ferric uptake regulator (Fur) and the small regulatory noncoding RNA RyhB. We evidenced that the regulation of the transcription of clbA by Fur and RyhB leads to the regulation of colibactin production. This work highlights the complex mechanism of regulation of an important virulence factor by the two major regulators of bacterial iron homeostasis, making iron a key environmental factor contributing to bacterial virulence and carcinogenesis.

Early settlers: which E. coli strains do you not want at birth?

The intestinal microbiota exerts vital biological processes throughout the human lifetime, and imbalances in its composition have been implicated in both health and disease status. Upon birth, the neonatal gut moves from a barely sterile to a massively colonized environment. The development of the intestinal microbiota during the first year of life is characterized by rapid and important changes in microbial composition, diversity, and magnitude. The pioneer bacteria colonizing the postnatal intestinal tract profoundly contribute to the establishment of the host-microbe symbiosis, which is essential for health throughout life. Escherichia coli is one of the first colonizers of the gut after birth. E. coli is a versatile population including harmless commensal, probiotic strains as well as frequently deadly pathogens. The prevalence of the specific phylogenetic B2 group, which encompasses both commensal and extra- or intraintestinal pathogenic E. coli strains, is increasing among E. coli strains colonizing infants quickly after birth. Fifty percent of the B2 group strains carry in their genome the pks gene cluster encoding the synthesis of a nonribosomal peptide-polyketide hybrid genotoxin named colibactin. In this review, we summarize both clinical and experimental evidence associating the recently emerging neonatal B2 E. coli population with several pathology and discuss how the expression of colibactin by both normal inhabitants of intestinal microflora and virulent strains may darken the borderline between commensalism and pathogenicity.

Divergent Evolution of the repFII Replicon of IncF Plasmids Carrying Cytotoxic Necrotizing Factor cnf2, Cytolethal Distending Toxin cdtIII, and f17Ae Fimbrial Variant Genes in Type 2 Necrotoxigenic Escherichia coli Isolates from Calves.

Among the pathovars of Escherichia coli in cattle, necrotoxigenic E. coli (NTEC) is defined by the production of cytotoxic necrotizing factors (CNFs). In particular, type 2 NTEC (NTEC2) strains are frequent in diarrheic and septicemic calves and usually coproduce CNF type 2 (CNF2), cytolethal distending toxin type III (CDTIII), and fimbrial adhesins of the F17 family, whose genetic determinants have frequently been reported on the same Vir-like plasmid. In this study, we investigated the genetic environment of the cnf2, f17Ae, and cdtIII genes in a collection of fecal E. coli isolates recovered from 484 French and 58 Iranian calves. In particular, we highlighted the spread of cnf2, f17Ae, and cdtIII on similar 150-kb IncF plasmids harboring the newly assigned repFII replicon allele F74 in NTEC2 isolates. Interestingly, this 150-kb IncF plasmid differed from the 140-kb IncF plasmid harboring the newly assigned repFII replicon allele F75 and carrying cnf2 alone. These results suggest two divergent lineages of cnf2-carrying IncF plasmids depending on the presence of the f17Ae and cdtIII genes. This partition was observed in E. coli strains of unrelated backgrounds, suggesting two different evolutionary paths of cnf2-carrying IncF plasmids rather than divergent evolutions of NTEC2 clones. The driving forces for such divergent evolutions are not known, and further studies are required to clarify the selection of plasmid subtypes spreading virulence determinants in E. coli, in particular, plasmids of the IncF family.

Characterization of Ciprofloxacin-Resistant and Ciprofloxacin-Susceptible Uropathogenic Escherichia coli Obtained from Patients with Gynecological Cancer.

The objective of this work was to assess the genetic characteristics of uropathogenic Escherichia coli, ciprofloxacin resistance or susceptibility, obtained from patients with gynecological cancer and urinary tract infection (UTI). Seventy-seven E. coli ciprofloxacin-resistant isolates and 38 ciprofloxacin-susceptible were analyzed by polymerase chain reaction (PCR) to determine the phylogenetic groups, virulence factors as iucC, fyuA, hlyC, cnf1 genes, and pks pathogenicity island. The presence of genes related to ciprofloxacin resistance such as qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA, and the sequencing of DNA gyrase genes and topoisomerase IV were determined. The genetic profile of the isolates was determined by pulsed-field gel electrophoresis (PFGE). Statistical analysis was performed using Fisher's exact test and Chi-square test. Phylogenetic group B2 was the most prevalent although a great genetic diversity was observed by PFGE. Only genes associated to siderophores were found in ciprofloxacin-resistant isolates; however, in ciprofloxacin-susceptible isolates, genes related to siderophores and toxin, were detected. Additionally qnrB was detected in both populations, ciprofloxacin resistant and susceptible. DNA mutations in gyrA were Ser-83-Leu and Asp-87-Asn and in parC were Ser-80-Ile and Glu-84-Val, Glu-84-Lys. In conclusion, it was observed a high prevalence of qnrB in the population studied; in addition, it was the first time the pks island was observed only in ciprofloxacin-susceptible isolates.

The Genotoxin Colibactin Is a Determinant of Virulence in Escherichia coli K1 Experimental Neonatal Systemic Infection.

Escherichia coli strains expressing the K1 capsule are a major cause of sepsis and meningitis in human neonates. The development of these diseases is dependent on the expression of a range of virulence factors, many of which remain uncharacterized. Here, we show that all but 1 of 34 E. coli K1 neonatal isolates carried clbA and clbP, genes contained within the pks pathogenicity island and required for the synthesis of colibactin, a polyketide-peptide genotoxin that causes genomic instability in eukaryotic cells by induction of double-strand breaks in DNA. Inactivation of clbA and clbP in E. coli A192PP, a virulent strain of serotype O18:K1 that colonizes the gastrointestinal tract and translocates to the blood compartment with very high frequency in experimental infection of the neonatal rat, significantly reduced the capacity of A192PP to colonize the gut, engender double-strand breaks in DNA, and cause invasive, lethal disease. Mutation of clbA, which encodes a pleiotropic enzyme also involved in siderophore synthesis, impacted virulence to a greater extent than mutation of clbP, encoding an enzyme specific to colibactin synthesis. Restoration of colibactin gene function by complementation reestablished the fully virulent phenotype. We conclude that colibactin contributes to the capacity of E. coli K1 to colonize the neonatal gastrointestinal tract and to cause invasive disease in the susceptible neonate.

Oral tolerance failure upon neonatal gut colonization with Escherichia coli producing the genotoxin colibactin.

The intestinal barrier controls the balance between tolerance and immunity to luminal antigens. When this finely tuned equilibrium is deregulated, inflammatory disorders can occur. There is a concomitant increase, in urban populations of developed countries, of immune-mediated diseases along with a shift in Escherichia coli population from the declining phylogenetic group A to the newly dominant group B2, including commensal strains producing a genotoxin called colibactin that massively colonized the gut of neonates. Here, we showed that mother-to-offspring early gut colonization by colibactin-producing E. coli impairs intestinal permeability and enhances the transepithelial passage of luminal antigen, leading to an increased immune activation. Functionally, this was accompanied by a dramatic increase in local and systemic immune responses against a fed antigen, decreased regulatory T cell population, tolerogenic dendritic cells, and enhanced mucosal delayed-type hypersensitivity response. Conversely, the abolition of colibactin expression by mutagenesis abrogates the alteration of oral tolerance induced by neonatal colonization by E. coli. In conclusion, the vertical colonization by E. coli producing the genotoxin colibactin enhances intestinal translocation and subsequently alters oral tolerance. Thus, early colonization by E. coli from the newly dominant phylogenetic group B2, which produces colibactin, may represent a risk factor for the development of immune-mediated diseases.