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Objectives: Urinary tract infections (UTIs) are primarily caused by uropathogenic Escherichia coli (UPEC). This study aims to elucidate the role of the virulence factor HlyF in the epidemiology and pathophysiology of UTIs and investigate the dissemination of plasmids carrying the hlyF gene., Methods: An epidemiological analysis was conducted on a representative collection of 225 UPEC strains isolated from community-acquired infections. Selected hlyF+ strains were fully sequenced using a combination of Illumina and Nanopore technologies. To investigate the impact of HlyF, a murine model of UTI was utilized to compare clinical signs, bacterial loads in the bladder, kidney, and spleen, onset of bacteraemia, and inflammation through cytokine quantification among wild-type hlyF+ strains, isogenic mutants, and complemented mutants., Results: Our findings demonstrate that 20% of UPEC encode the HlyF protein. These hlyF+ UPEC strains exhibited enhanced virulence, frequently leading to pyelonephritis accompanied by bloodstream infections. Unlike typical UPEC strains, hlyF+ UPEC strains demonstrate a broader phylogroup distribution and possess a unique array of virulence factors and antimicrobial resistance genes, primarily carried by ColV-like plasmids. In the murine UTI model, expression of HlyF was linked to the UPECs' capacity to induce urosepsis and elicit an exacerbated inflammatory response, setting them apart from typical UPEC strains., Discussion: Overall, our results strongly support the notion that HlyF serves as a significant virulence factor for UPECs, and the dissemination of ColV-like plasmids encoding HlyF warrants further investigation., (Copyright © 2023 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Shiga toxin (Stx)-producing Escherichia coli (STEC) belonging to the "top 7″ serotypes (i.e. O157:H7, O26:H11, O45:H2, O103:H2, O111:H8, O121:H19 and O145:H28) are considered as the main pathogenic enterohemorrhagic E. coli (EHEC). As ruminants, including calves, are a reservoir of pathogenic STEC, we investigated the prevalence, major virulence genes and genetic relatedness of top7 STEC in veal calves slaughtered in France, through the analysis of 500 fecal samples collected over one year. Thirty top7 STEC isolates were recovered from 28 calves. The two serotypes O103:H2 and O26:H11 accounted for 73% of STEC strains, followed by O145:H28 and O157:H7. STEC super-shedding levels were identified for two calves carrying STEC O103:H2 and O157:H7, respectively. Thirty-nine atypical enteropathogenic E. coli (aEPEC) were also recovered from calves. Overall, a prevalence of 5.6% top7 STEC-positive calves was found, thus higher than that previously determined for the French slaughtered adult cattle (1.8%), confirming the impact of animals age on STEC carriage. Most top7 STEC strains carried the stx1a subtype suggesting a low pathogenicity for humans. Seasonal variation in STEC carriage was also observed, with two peaks of higher prevalence during spring and fall. Genetic similarity of top7 STEC isolates was found for calves originating from the same fattening facilities, reflecting STEC circulation between animals kept in groups. This study indicates that veal calves grown for meat production are at higher risk of shedding top7 STEC compared to adult cattle. They thus represent ideal targets for the implementation of farm interventions aimed at reducing STEC burden in cattle and the food chain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The structure and mode of action of colibactin with its potential involvement in cancer have been extensively studied but little is known about the intrinsic function of the biosynthetic gene cluster, coding for colibactin, as a bacterial genotoxin. Paradoxically, this pathogenicity island is also found in commensal and probiotic strains of Escherichia coli and in bacterial species colonizing olive trees and the digestive tract of bees. In this review, we summarize the available literature to address the following key questions. What does this genomic island really encode? What explains the extensive dissemination of this genetically mobile element? What do we really know about the biosynthetic and secretory pathways of colibactin? What is its inherent target/function?, (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

The probiotic Escherichia coli strain Nissle 1917 (DSM 6601, Mutaflor), generally considered beneficial and safe, has been used for a century to treat various intestinal diseases. However, Nissle 1917 hosts in its genome the pks pathogenicity island that codes for the biosynthesis of the genotoxin colibactin. Colibactin is a potent DNA alkylator, suspected to play a role in colorectal cancer development. We show in this study that Nissle 1917 is functionally capable of producing colibactin and inducing interstrand cross-links in the genomic DNA of epithelial cells exposed to the probiotic. This toxicity was even exacerbated with lower doses of the probiotic, when the exposed cells started to divide again but exhibited aberrant anaphases and increased gene mutation frequency. DNA damage was confirmed in vivo in mouse models of intestinal colonization, demonstrating that Nissle 1917 produces the genotoxin in the gut lumen. Although it is possible that daily treatment of adult humans with their microbiota does not produce the same effects, administration of Nissle 1917 as a probiotic or as a chassis to deliver therapeutics might exert long-term adverse effects and thus should be considered in a risk-versus-benefit evaluation. IMPORTANCE Nissle 1917 is sold as a probiotic and considered safe even though it has been known since 2006 that it harbors the genes for colibactin synthesis. Colibactin is a potent genotoxin that is now linked to causative mutations found in human colorectal cancer. Many papers concerning the use of this strain in clinical applications ignore or elude this fact or misleadingly suggest that Nissle 1917 does not induce DNA damage. Here, we demonstrate that Nissle 1917 produces colibactin in vitro and in vivo and induces mutagenic DNA damage. This is a serious safety concern that must not be ignored in the interests of patients, the general public, health care professionals, and ethical probiotic manufacturers.

Bacteria have to process several levels of gene regulation and coordination of interconnected regulatory networks to ensure the most adequate cellular response to specific growth conditions. Especially, expression of complex and costly fitness and pathogenicity-associated traits is coordinated and tightly regulated at multiple levels. We studied the interconnected regulation of the expression of the colibactin and yersiniabactin polyketide biosynthesis machineries, which are encoded by two pathogenicity islands found in many phylogroup B2 Escherichia coli isolates. Comparative phenotypic and genotypic analyses identified the BarA-UvrY two-component system as an important regulatory element involved in colibactin and yersiniabactin expression. The carbon storage regulator (Csr) system controls the expression of a wide range of central metabolic and virulence-associated traits. The availability of CsrA, the key translational regulator of the Csr system, depends on BarA-UvrY activity. We employed reporter gene fusions to demonstrate UvrY- and CsrA-dependent expression of the colibactin and yersiniabactin determinants and confirmed a direct interaction of CsrA with the 5' untranslated leader transcripts of representative genes of the colibactin and yersiniabactin operons by RNA electrophoretic mobility shift assays. This posttranscriptional regulation adds an additional level of complexity to control mechanisms of polyketide expression, which is also orchestrated at the level of ferric uptake regulator (Fur)-dependent regulation of transcription and phosphopantetheinyl transferase-dependent activation of polyketide biosynthesis. Our results emphasize the interconnection of iron- and primary metabolism-responsive regulation of colibactin and yersiniabactin expression by the fine-tuned action of different regulatory mechanisms in response to variable environmental signals as a prerequisite for bacterial adaptability, fitness, and pathogenicity in different habitats. IMPORTANCE Secondary metabolite expression is a widespread strategy among bacteria to improve their fitness in habitats where they constantly compete for resources with other bacteria. The production of secondary metabolites is associated with a metabolic and energetic burden. Colibactin and yersiniabactin are two polyketides, which are expressed in concert and promote the virulence of different enterobacterial pathogens. To maximize fitness, they should be expressed only in microenvironments in which they are required. Accordingly, precise regulation of colibactin and yersiniabactin expression is crucial. We show that the expression of these two polyketides is also interconnected via primary metabolism-responsive regulation at the posttranscriptional level by the CsrA RNA-binding protein. Our findings may help to optimize (over-)expression and further functional characterization of the polyketide colibactin. Additionally, this new aspect of concerted colibactin and yersiniabactin expression extends our knowledge of conditions that favor the expression of these virulence- and fitness-associated factors in different Enterobacterales members.

Siderophore-microcins are antimicrobial peptides produced by enterobacteria, especially Escherichia coli and Klebsiella pneumoniae strains. The antibiotic peptide is post-translationally modified by the linkage of a siderophore moiety. Therefore, it can enter and kill phylogenetically related bacteria by a "Trojan Horse" stratagem, by mimicking the iron-siderophore complexes. Consequently, these antimicrobial peptides are key determinants of bacterial competition within the intestinal niche, which is the reservoir for pathogenic E. coli . The most frequent extraintestinal infections caused by E. coli are urinary tract infections. Uropathogenic E. coli (UPEC) can produce many virulence factors, including siderophore-microcins. Siderophore-microcins are chromosomally encoded by small genomic islands that exhibit conserved organization. In UPEC, the siderophore-microcin gene clusters and biosynthetic pathways differ from the "archetypal" models described in fecal strains. The gene cluster is shorter. Thus, active siderophore-microcin production requires proteins from two other genomic islands that also code for virulence factors. This functional and modular synergy confers a strong selective advantage for the domination of the colonic niche, which is the first step toward infection. This optimization of genetic resources might favor the selection of additional virulence factors, which are essential in the subsequent steps of pathogenesis in E. coli infection., (Copyright © 2020 Massip and Oswald.)

Colibactin is a nonribosomal peptide/polyketide hybrid natural product expressed by different members of the Enterobacteriaceae which can be correlated with induction of DNA double-strand breaks and interference with cell cycle progression in eukaryotes. Regulatory features of colibactin expression are only incompletely understood. We used Escherichia coli strain M1/5 as a model to investigate regulation of expression of the colibactin determinant at the transcriptional level and to characterize regulatory elements located within the colibactin pathogenicity island itself. We measured clbR transcription in vitro and observed that cultivation in defined minimal media led to increased colibactin expression relative to rich media. Transcription of clbR directly responds to iron availability. We also characterized structural DNA elements inside the colibactin determinant involved in ClbR-dependent regulation, i.e., ClbR binding sites and a variable number of tandem repeats located upstream of clbR We investigated the impact of clbR overexpression or deletion at the transcriptome and proteome levels. Moreover, we compared global gene regulation under these conditions with that occurring upon overexpression or deletion of clbQ , which affects the flux of colibactin production. Combining the results of the transcriptome and proteome analyses with indirect measurements of colibactin levels by cell culture assays and an approximate quantification of colibactin via the second product of colibactin cleavage from precolibactin, N-myristoyl-d-asparagine, we demonstrate that the variable number of tandem repeats plays a significant regulatory role in colibactin expression. We identify ClbR as the only transcriptional activator known so far that is specific and essential for efficient regulation of colibactin production. IMPORTANCE The nonribosomal peptide/polyketide hybrid colibactin can be considered a bacterial virulence factor involved in extraintestinal infection and also a procarcinogen. Nevertheless, and despite its genotoxic effect, colibactin expression can also inhibit bacterial or tumor growth and correlates with probiotic anti-inflammatory and analgesic properties. Although the biological function of this natural compound has been studied extensively, our understanding of the regulation of colibactin expression is still far from complete. We investigated in detail the role of regulatory elements involved in colibactin expression and in the growth conditions that promote colibactin expression. In this way, our data shed light on the regulatory mechanisms involved in colibactin expression and may support the expression and purification of this interesting nonribosomal peptide/polyketide hybrid for further molecular characterization., (Copyright © 2020 Wallenstein et al.)

Cells employ specific and nonspecific mechanisms to protect their genome integrity against exogenous and endogenous factors. The clbS gene is part of the polyketide synthase machinery (pks genomic island) encoding colibactin, a genotoxin implicated in promoting colorectal cancer. The pks is found among the Enterobacteriaceae, in particular Escherichia coli strains of the B2 phylogenetic group. Several resistance mechanisms protect toxin producers against toxicity of their products. ClbS, a cyclopropane hydrolase, was shown to confer colibactin resistance by opening its electrophilic cyclopropane ring. Here we report that ClbS sustained viability and enabled growth also of E. coli expressing another genotoxin, the Usp nuclease. The recA::gfp reporter system showed that ClbS protects against Usp induced DNA damage. To elucidate the mechanism of ClbS mediated protection, we studied the DNA binding ability of the ClbS protein. We show that ClbS directly interacts with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), whereas ssDNA seems to be the preferred substrate. Thus, the ClbS DNA-binding characteristics may serve bacteria to protect their genomes against DNA degradation., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

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 Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Various forms of cancer have been linked to the carcinogenic activities of microorganisms(1-3). The virulent gene island polyketide synthase (pks) produces the secondary metabolite colibactin, a genotoxic molecule(s) causing double-stranded DNA breaks(4) and enhanced colorectal cancer development(5,6). Colibactin biosynthesis involves a prodrug resistance strategy where an N-terminal prodrug scaffold (precolibactin) is assembled, transported into the periplasm and cleaved to release the mature product(7-10). Here, we show that ClbM, a multidrug and toxic compound extrusion (MATE) transporter, is a key component involved in colibactin activity and transport. Disruption of clbM attenuated pks+ E. coli-induced DNA damage in vitro and significantly decreased the DNA damage response in gnotobiotic Il10(-/-) mice. Colonization experiments performed in mice or zebrafish animal models indicate that clbM is not implicated in E. coli niche establishment. The X-ray structure of ClbM shows a structural motif common to the recently described MATE family. The 12-transmembrane ClbM is characterized as a cation-coupled antiporter, and residues important to the cation-binding site are identified. Our data identify ClbM as a precolibactin transporter and provide the first structure of a MATE transporter with a defined and specific biological function.

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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

During 2007-2010, 13 545 confirmed human verocytotoxin (VT)-producing Escherichia coli (VTEC) infections were reported in the European Union, including 777 haemolytic uraemic syndrome (HUS) cases. Clinical manifestations were reported for 53% of cases, 64% of which presented with diarrhoea alone and 10% with HUS. Isolates from 85% of cases were not fully serotyped and could not be classified on the basis of the Karmali seropathotype concept. There is no single or combination of phenotypic or genetic marker(s) that fully define 'pathogenic' VTEC. Isolates which contain the vtx2 (verocytotoxin 2) gene in combination with the eae (intimin-encoding) gene or aaiC (secreted protein of enteroaggregative E. coli) and aggR (plasmid-encoded regulator) genes have been associated with a higher risk of more severe illness. A molecular approach targeting genes encoding VT and other virulence determinants is thus proposed to allow an assessment of the potential severity of disease that may be associated with a given VTEC isolate.

F17 fimbriae are produced by pathogenic Escherichia coli involved in diarrhea and septicemia outbreaks in calves and lambs. These proteins result from the expression of four different clustered genes, namely f17A, f17D, f17C and f17G, encoding a pilin protein, a periplasmic protein, an anchor protein and an adhesin protein, respectively. Several variants of f17A and f17G genes have been reported and found genetically associated with typical virulence factors of bovine pathogenic E. coli strains. In this study, a new F17e-A variant, closely related to F17b-A, was identified from a collection of 58 E. coli isolates from diarrheic calves in Iran. While highly prevalent in Iranian F17-producing clinical isolates from calves, this variant was rare among E. coli from a French healthy adult bovine population, suggesting a possible association with virulence. The f17Ae gene was also found in the genome of the Shiga-like toxin variant Stx1d-producing bovine E. coli strain MHI813, and belonged to a gene cluster also encoding a new F17-G3 variant, which greatly differed from F17-G1 and F17-G2. This gene cluster was located on a pathogenicity island integrated in the tRNA pheV gene. The gene coding for a third new F17f-A variant corresponding to a combination of F17c-A and F17d-A was also identified on the pVir68 plasmid in the bovine pathogenic E. coli strain 6.0900. In conclusion, we identified three new F17-A and F17-G variants in cattle E. coli, which may also have significant impact on the development of new diagnostics and vaccination tools.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae, and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

The role of the post-transcriptional carbon storage regulator (Csr) system in nutrient utilization and in the control of the central metabolism in E. coli reference commensal strain Nissle 1917 was investigated. Analysis of the growth capabilities of mutants altered for various components of the Csr system (csrA51, csrB, csrC and csrD mutations) showed that only the protein CsrA - the key component of the system - exerts a marked role in carbon nutrition. Attenuation of CsrA activity in the csrA51 mutant affects the growth efficiency on a broad range of physiologically relevant carbon sources, including compounds utilized by the Entner-Doudoroff (ED) pathway. Detailed investigations of the metabolomes and fluxomes of mutants and wild-type cells grown on carbon sources representative of glycolysis and of the ED pathway (glucose and gluconate, respectively), revealed significant re-adjusting of central carbon metabolism for both compounds in the csrA51 mutant. However, the metabolic re-adjusting observed on gluconate was strikingly different from that observed on glucose, indicating a nutrient-specific control of metabolism by the Csr system.

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.

In order to improve the identification of avian pathogenic Escherichia coli (APEC) strains, an extensive characterization of 1,491 E. coli isolates was conducted, based on serotyping, virulence genotyping, and experimental pathogenicity for chickens. The isolates originated from lesions of avian colibacillosis (n = 1,307) or from the intestines of healthy animals (n = 184) from France, Spain, and Belgium. A subset (460 isolates) of this collection was defined according to their virulence for chicks. Six serogroups (O1, O2, O5, O8, O18, and O78) accounted for 56.5% of the APEC isolates and 22.5% of the nonpathogenic isolates. Thirteen virulence genes were more frequently present in APEC isolates than in nonpathogenic isolates but, individually, none of them could allow the identification of an isolate as an APEC strain. In order to take into account the diversity of APEC strains, a statistical analysis based on a tree-modeling method was therefore conducted on the sample of 460 pathogenic and nonpathogenic isolates. This resulted in the identification of four different associations of virulence genes that enables the identification of 70.2% of the pathogenic strains. Pathogenic strains were identified with an error margin of 4.3%. The reliability of the link between these four virulence patterns and pathogenicity for chickens was validated on a sample of 395 E. coli isolates from the collection. The genotyping method described here allowed the identification of more APEC isolates with greater reliability than the classical serotyping methods currently used in veterinary laboratories.

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.

Cycle inhibiting factors (Cifs) are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are "cyclomodulins" that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL) complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions.

Aims: To develop real-time PCR assays targeting genes encoding the flagellar antigens (fliC) and intimin subtypes (eae) associated with the five most clinically important serotypes of enterohaemorrhagic Escherichia coli (EHEC), i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7., Methods and Results:   Primers and probes specific to fliC(H2) , fliC(H7) , fliC(H8) , fliC(H11) , fliC(H28) , eae-β1, eae-γ1, eae-ε and eae-θ were combined in simplex and multiplex 5'-nuclease PCR assays. The specificity of the assays was assessed on 201 bacterial strains and the sensitivity determined on serially diluted EHEC genomes. The developed PCR assays were found to be highly specific and detected as few as five EHEC genome equivalents per reaction. Furthermore, it was possible to detect the five major EHEC serotypes in cheese samples inoculated at concentration levels of ≤5CFU per 25g after overnight enrichment using the PCR assays., Conclusions: The PCR assays developed here were found to be sensitive and specific for the reliable detection of genes encoding the flagellar antigens and intimin variants belonging to the five most clinically relevant EHEC serotypes., Significance and Impact of the Study: Application of real-time PCR assays should improve the identification of foods contaminated by EHEC and facilitate the molecular typing of these organisms., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G₁/S and G₂/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21(waf1) and p27(kip1). Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria.

Determinants of urosepsis in Escherichia coli remain incompletely defined. Cyclomodulins (CMs) are a growing functional family of toxins that hijack the eukaryotic cell cycle. Four cyclomodulin types are actually known in E. coli: cytotoxic necrotizing factors (CNFs), cycle-inhibiting factor (Cif), cytolethal distending toxins (CDTs), and the pks-encoded toxin. In the present study, the distribution of CM-encoding genes and the functionality of these toxins were investigated in 197 E. coli strains isolated from patients with community-acquired urosepsis (n = 146) and from uninfected subjects (n = 51). This distribution was analyzed in relation to the phylogenetic background, clinical origin, and antibiotic resistance of the strains. It emerged from this study that strains harboring the pks island and the cnf1 gene (i) were strongly associated with the B2 phylogroup (P, <0.001), (ii) frequently harbored both toxin-encoded genes in phylogroup B2 (33%), and (iii) were predictive of a urosepsis origin (P, <0.001 to 0.005). However, the prevalences of the pks island among phylogroup B2 strains, in contrast to those of the cnf1 gene, were not significantly different between fecal and urosepsis groups, suggesting that the pks island is more important for the colonization process and the cnf1 gene for virulence. pks- or cnf1-harboring strains were significantly associated with susceptibility to antibiotics (amoxicillin, cotrimoxazole, and quinolones [P, <0.001 to 0.043]). Otherwise, only 6% and 1% of all strains harbored the cdtB and cif genes, respectively, with no particular distribution by phylogenetic background, antimicrobial susceptibility, or clinical origin.

The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins, the cyclomodulins, which modulate the host cell cycle. Upon injection into the host cell by the type III secretion system of enteropathogenic Escherichia coli (EPEC), Cif induces both G(2) and G(1) cell cycle arrests. The cell cycle arrests correlate with the accumulation of p21(waf1) and p27(kip1) proteins that inhibit CDK-cyclin complexes, whose activation is required for G(1)/S and G(2)/M transitions. Increases of p21 and p27 levels are independent of p53 transcriptional induction and result from protein stabilization through inhibition of the ubiquitin/proteasome degradation pathway. In this study, we show that Cif not only induces cell cycle arrest but also eventually provokes a delayed cell death. Indeed, 48 h after infection with EPEC expressing Cif, cultured IEC-6 intestinal cells were positive for extracellular binding of annexin V and exhibited high levels of cleaved caspase-3 and lactate dehydrogenase release, indicating evidence of apoptosis. Cif was necessary and sufficient for inducing this late apoptosis, and the cysteine residue of the catalytic site was required for Cif activity. These results highlight a more complex role of Cif than previously thought, as a cyclomodulin but also as an apoptosis inducer.

A genomic island encoding the biosynthesis and secretion pathway of putative hybrid nonribosomal peptide-polyketide colibactin has been recently described in Escherichia coli. Colibactin acts as a cyclomodulin and blocks the eukaryotic cell cycle. The origin and prevalence of the colibactin island among enterobacteria are unknown. We therefore screened 1,565 isolates of different genera and species related to the Enterobacteriaceae by PCR for the presence of this DNA element. The island was detected not only in E. coli but also in Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter koseri isolates. It was highly conserved among these species and was always associated with the yersiniabactin determinant. Structural variations between individual strains were only observed in an intergenic region containing variable numbers of tandem repeats. In E. coli, the colibactin island was usually restricted to isolates of phylogenetic group B2 and inserted at the asnW tRNA locus. Interestingly, in K. pneumoniae, E. aerogenes, C. koseri, and three E. coli strains of phylogenetic group B1, the functional colibactin determinant was associated with a genetic element similar to the integrative and conjugative elements ICEEc1 and ICEKp1 and to several enterobacterial plasmids. Different asn tRNA genes served as chromosomal insertion sites of the ICE-associated colibactin determinant: asnU in the three E. coli strains of ECOR group B1, and different asn tRNA loci in K. pneumoniae. The detection of the colibactin genes associated with an ICE-like element in several enterobacteria provides new insights into the spread of this gene cluster and its putative mode of transfer. Our results shed light on the mechanisms of genetic exchange between members of the family Enterobacteriaceae.

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) inject a repertoire of effector proteins into host cells via a type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE). OspG is an effector protein initially identified in Shigella that was shown to inhibit the host innate immune response. In this study, we found ospG homologues in EHEC (mainly of serogroup O111) and in Yersinia enterocolitica. The T3SS encoded by the LEE was able to inject these different OspG homologues into host cells. Infection of HeLa cells with EHEC O111 inhibited the NF-kappaB-dependent innate immune response via a T3SS-dependent mechanism. However, an EHEC O111 ospG mutant was still able to inhibit NF-kappaB p65 transfer to the nucleus in infected cells stimulated by tumour necrosis factor alpha (TNF-alpha). In addition, no difference in the inflammatory response was observed between wild-type EHEC O111 and the isogenic ospG mutant in the rabbit ligated intestinal loop model. These results suggest that OspG is not the sole effector protein involved in the inactivation of the host innate immune system during EHEC O111 infection.

Virulence factors are associated with the capacity of E. coli strains to cause intestinal and extraintestinal infections. Thirty one E. coli isolates were obtained from heart blood or internal organs of septicemic calves. The O serogroups of isolates were determined. PCR assays were performed to determine the phylogenetic groups and presence of specific virulence genes. Fourteen (45.16%) isolates belonged to seven O serogroups (O8, O15, O20, O45, O78, O101 and O103) and 17 (54.83%) isolates were O-nontypeable. E. coli isolates fall into three phylogenetic groups included 15 isolates belonged to B1, 9 to A and 7 to D phylogenetic groups. Nineteen (61.29%) isolates exhibited at least one of the virulence genes. F17 family (5 isolates f17b, 3 isolates f17c, 1 isolate f17a) genes and aerobactin encoding gene of iucD (5 isolates) were the two most prevalent virulence genes. Three isolates were positive for cnf2 and cdtIII genes in combination and they were O-nontypeable. AfaE-VIII, CS31A gene (clpG) and hemolysin encoding gene (hly) were detected in 3, 4 and 3 isolates respectively. None of the isolates contained the ipaH sequences and the genes encoding fimbria (F5, F41, S, P), AfaI adesin, toxins (LT-I, ST-I, SLT-I, SLT-II, CNF1 and CDT-IV) and intimin.

Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. The T3SS effector cycle inhibiting factor (Cif) produced by EPEC and EHEC is able to block host eukaryotic cell-cycle progression. We present here a crystal structure of Cif, revealing it to be a divergent member of the superfamily of enzymes including cysteine proteases and acetyltransferases that share a common catalytic triad. Mutation of these conserved active site residues abolishes the ability of Cif to block cell-cycle progression. Finally, we demonstrate that irreversible cysteine protease inhibitors do not abolish the Cif cytopathic effect, suggesting that another enzymatic activity may underlie the biological activity of this virulence factor.

The cycle inhibiting factor (Cif) is a cyclomodulin produced by enteropathogenic and enterohemorrhagic Escherichia coli. Upon injection into the host cell by the bacterial type III secretion system, Cif inhibits the G2/M transition via sustained inhibition of the mitosis inducer CDK1 independently of the DNA damage response. In this study, we show that Cif induces not only G2, but also G1 cell cycle arrest depending on the stage of cells in the cell cycle during the infection. In various cell lines including differentiated and untransformed enterocytes, the cell cycle arrests are correlated with the accumulation of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Cif-induced cyclin-dependent kinase inhibitor accumulation is independent of the p53 pathway but occurs through inhibition of their proteasome-mediated degradation. Our results provide a direct link between the mode of action of Cif and the host cell cycle control.

Many pathogenic bacteria subvert normal host cell processes by delivering effector proteins which mimic eukaryotic functions directly into target cells. EspF is a multifunctional protein injected into host cells by attaching and effacing pathogens, but its mechanism of action is not understood completely. In silico analyses of EspF revealed two key motifs: proline-rich domains and PDZ domain binding motifs. Such functional domains may allow EspF to act as an actin nucleation-promoting factor by mimicking host proteins. In agreement with these predictions, we found that EspF from rabbit enteropathogenic Escherichia coli (E22) participates in the regulation of actin polymerization by binding to a complex of proteins at the tight junctions (TJ). EspF bound to actin and profilin throughout the course of infection. However, after 2 h of infection, EspF also bound to the neural Wiskott-Aldrich syndrome protein and to the Arp2/3, zonula occludens-1 (ZO-1), and ZO-2 proteins. Moreover, EspF caused occludin, claudin, ZO-1, and ZO-2 redistribution and loss of transepithelial electrical resistance, suggesting that actin sequestration by EspF may cause local actin depolymerization leading to EspF-induced TJ disruption. Furthermore, EspF caused recruitment of these TJ proteins into the pedestals. An E22 strain lacking EspF did not cause TJ disruption and pedestals were smaller than those induced by the wild-type strain. Additionally, the pedestals were located mainly in the TJ. The overexpression of EspF caused bigger pedestals located along the length of the cells. Thus, actin sequestration by EspF allows the recruitment of junctional proteins into the pedestals, leading to the maturation of actin pedestals and the disruption of paracellular permeability.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049 E. coli strains of human, animal, and environmental origins. A total of 115 E. coli isolates from diverse origins and geographic locations carried cif. The presence of cif was tightly associated with the LEE, since all the cif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population of E. coli but positively selected within the population of LEE-positive strains. Nonetheless, 66% of cif-positive E. coli strains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in the cif gene. The passenger region of the prophages carrying cif was highly variable and showed various combinations of IS elements and genes coding for other effectors such as nleB, nleC, nleH, nleG, espJ, and nleA/espI (some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism.

A rapid two-step identification method based on PCR-RFLP analysis of the intimin gene was developed to differentiate specific alleles in pathogenic Escherichia coli. This technique, tested on isolates eae-positive, accurately detects eae and resolves alleles encoding the alpha1, alpha2, beta, gamma1, gamma2/theta, kappa, epsilon, zeta, and iota intimin variants.

The recently described hybrid nonribosomal peptide-polyketide colibactin, found in various Escherichia coli strains, invokes a cytopathic effect in HeLa cells upon cocultivation with these bacteria. However, not much is known so far about the transcriptional organization of the colibactin genes (clb) or the regulation of their transcription. Here, the operon structure of the colibactin gene cluster of E. coli strain Nissle 1917 was investigated by means of reverse transcriptase (RT)-PCR and seven transcripts were found of which four are transcribed polycistronically. The polycistrons comprise the genes clbC to clbG, clbI to clbN, clbO to clbP, and clbR to clbA and span 6.3, 23.3, 3.9, and 0.9 kb, respectively. Furthermore, transcript levels for different cultivation conditions were determined by RT-PCR of the whole cluster as well as by luciferase reporter gene assays of the genes clbA, clbB, clbQ, and clbR. RT-PCR revealed an overall increased transcription in shaking cultures as well as of the genes clbA to clbH in general. Luciferase reporter gene fusions indicated an influence of the carbon source on clb gene expression.

The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins and effector proteins, the cyclomodulins, that deregulate the host cell cycle. Upon injection into HeLa cells by the enteropathogenic Escherichia coli (EPEC) type III secretion system, Cif induces a cytopathic effect characterized by the recruitment of focal adhesion plates and the formation of stress fibres, an irreversible cell cycle arrest at the G(2)/M transition, and sustained inhibitory phosphorylation of mitosis inducer, CDK1. Here, we report that the reference typical EPEC strain B171 produces a functional Cif and that lipid-mediated delivery of purified Cif into HeLa cells induces cell cycle arrest and actin stress fibres, implying that Cif is necessary and sufficient for these effects. EPEC infection of intestinal epithelial cells (Caco-2, IEC-6) also induces cell cycle arrest and CDK1 inhibition. The effect of Cif is strikingly similar to that of cytolethal distending toxin (CDT), which inhibits the G(2)/M transition by activating the DNA-damage checkpoint pathway. However, in contrast to CDT, Cif does not cause phosphorylation of histone H2AX, which is associated with DNA double-stranded breaks. Following EPEC infection, the checkpoint effectors ATM/ATR, Chk1 and Chk2 are not activated, the levels of the CDK-activating phosphatases Cdc25B and Cdc25C are not affected, and Cdc25C is not sequestered in host cell cytoplasm. Hence, Cif activates a DNA damage-independent signalling pathway that leads to inhibition of the G(2)/M transition.

Enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains carry a pathogenicity island termed locus of enterocyte effacement (LEE) responsible for attaching and effacing lesions on epithelial cells. The expression of LEE varies among isolates and is dependent on environmental cues. In the EHEC O157:H7 Sakaï isolate (RIMD-0509952 strain), we found that the non-coding RNA, DsrA, activates the expression of the LEE. This activation requires RpoS, the stress sigma factor. The DsrA/RpoS regulatory pathway mediates its positive effect by stimulating the transcription of ler, a positive regulatory gene encoded by the LEE. A second regulatory pathway, repressed by HNS, is also able to activate the transcription of ler and requires GrlA, another LEE-encoded regulator. Both regulatory pathways, DsrA/RpoS and HNS/GrlA, affect the activity of the ler distal promoter and require the Ler protein to be functional. Our data demonstrate that the LEE expression can be turned on by at least two separate pathways acting on the transcription of ler.

Wastewater samples from 12 slaughterhouses located in different regions in France were tested for the presence of stx-positive and eae-positive Escherichia coli isolates, and characteristics of the isolates obtained were determined. A total of 224 wastewater samples were collected in wastewater treatment plants at different stages of wastewater processing. Altogether, 5,001 E. coli isolates were obtained by colony counting and screened for the presence of stx and eae genes by multiplex PCR. stx-positive and eae-positive E. coli isolates were detected in 25% of the samples collected; they were found in 13% and 3% of the samples obtained from treated effluent and sludge, respectively, suggesting that they could be spread into the environment. Screening of the samples collected by immunomagnetic separation allowed us to isolate 31 additional E. coli serogroup O157 isolates. Four of these isolates harbored stx and eae genes. All stx-positive and eae-positive E. coli isolates were analyzed for eae and stx genetic variants, as well as for additional virulence factors and serotypes. Our results suggest that the majority of the stx- and eae-positive E. coli isolates from wastewater have low virulence for humans. However, the diversity of the enterohemorrhagic E. coli-associated virulence factors in the strains indicates that the environment may play an important role in the emergence of new pathogenic enterohemorrhagic E. coli strains.

Enteropathogenic Escherichia coli (EPEC) that are known to cause severe diarrhoea in children and young rabbits are well characterized, but there are few reports on the serotypes and intimin (eae) types of EPEC in weaned pigs. Based on detection of the eae gene by PCR and by DNA-hybridisation with LEE specific gene probes, 20 intestinal and 17 faecal eae(+) strains from diarrhoeal (164) and non-diarrhoeal (57) weaned pigs from 13 Hungarian farms, representing 12.8% of diarrhoeal and 14.0% of non-diarrhoeal pigs, were identified. The dominant serotype was O123:H11 (40%) among intestinal, and O108:H9 (23%) among faecal strains. The majority (85%) of the intestinal strains possessed eae-beta and 10% carried eae-gamma gene. In contrast, significantly (p<0.025) fewer faecal strains (53%) harboured the eae-beta gene, and 23% were eae-gamma positive. In vitro adhesion tests of intestinal and faecal eae(+) strains indicated adhesion of 20/37 of the strains to PK15 (porcine kidney) cells while only 3/37 strains adhered to HeLa cells. The ultrastructure of intimate bacterial attachment of representative porcine eae(+) strains to PK15 cells showed no pedestal formation, in contrast to the human EPEC (O127:H5, eae-alpha) strain. In conclusion, the data do not demonstrate a significant role for the eae(+)E. coli in porcine post-weaning diarrhoea, but provide new information on a dominant porcine serotype (O123:H11, eae-beta), and on differences of serotypes and intimin types of porcine eae(+) strains according to their site of isolation. Furthermore there was an indication that the PK15 cell line could be used as a model to study in vitro adherence of eae(+)E. coli of some human and porcine origin.

We investigated the genetic relationships of 54 Escherichia coli O103 strains from humans, animals, and meat by molecular typing of housekeeping and virulence genes and by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) of seven housekeeping genes revealed seven profiles, I through VII. MLST profiles I plus III cover 45 Shiga toxin-producing E. coli (STEC) O103:H2 strains from Australia, Canada, France, Germany, and Northern Ireland that are characterized by the intimin (eae) epsilon gene and carry enterohemorrhagic E. coli (EHEC) virulence plasmids. MLST profile II groups five human and animal enteropathogenic E. coli (EPEC) O103:H2 strains that were positive for intimin (eae) beta. Although strains belonging to MLST groups II and I plus III are closely related to each other (92.6% identity), major differences were found in the housekeeping icdA gene and in the virulence-associated genes eae and escD. E. coli O103 strains with MLST patterns IV to VII are genetically distant from MLST I, II, and III strains, as are the non-O103 E. coli strains EDL933 (O157), MG1655 (K-12), and CFT073 (O6). Comparison of MLST results with those of PFGE and virulence typing demonstrated that E. coli O103 STEC and EPEC have recently acquired different virulence genes and DNA rearrangements, causing alterations in their PFGE patterns. PFGE typing was very useful for identification of genetically closely related subgroups among MLST I strains, such as Stx2-producing STEC O103 strains from patients with hemolytic uremic syndrome. Analysis of virulence genes contributed to grouping of E. coli O103 strains into EPEC and STEC. Novel virulence markers, such as efa (EHEC factor for adherence), paa (porcine adherence factor), and cif (cell cycle-inhibiting factor), were found widely associated with E. coli O103 EPEC and STEC strains.

PCR tests designed in these studies identified three rabbit adhesive factor genes among 43 enteropathogenic E. coli (EPEC) strains: afr1 (2 strains), the F4(K88)/CS31A-related afr2 (10 strains), and ral (15 strains). Several EPEC strains (i.e., O153:H7 and O157:H2) lacked these genes but did adhere to HeLa cells and produced attaching and effacing lesions in rabbits.

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions and watery diarrhea, both of which are intimin and EspA dependent. In this work, we explored the mucosal immune response by detecting cytokine induction in rabbits with diarrhea caused by rabbit EPEC (REPEC). Orally inoculated rabbits exhibited weight loss and mucosal inflammation, developed watery diarrhea, and died (day 7). At day 6 postinoculation, animals were analyzed for the induction of proinflammatory cytokines in enterocytes. The role of lymphocyte-dependent immunity was determined through the expression of proinflammatory cytokines by lymphocytes from Peyer's patches (PP) and the spleen. EspA and intimin mutants were used to explore the role of A/E lesions in the expression of these cytokines. REPEC-infected rabbit enterocytes showed increased interleukin 1beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) mRNA expression, but that of anti-inflammatory IL-10 was increased only slightly. In contrast, intimin mutant-infected rabbits were unable to produce this proinflammatory cytokine profile but did produce a remarkable increase in IL-10 expression. Bacteria lacking EspA increased the expression of IL-8 and TNF-alpha, but that of IL-10 was increased only slightly. PP lymphocytes also produced proinflammatory cytokines, which were dependent on EspA (except for TNF-alpha) and intimin, while IL-10 was induced by EspA and intimin mutants. In contrast, spleen lymphocytes (systemic compartment) were unable to produce IL-1beta and TNF-alpha. These data show the importance of the proinflammatory cytokines secreted by enterocytes and those expressed locally by PP lymphocytes, which can activate effector mechanisms at the epithelium. Furthermore, this cytokine profile, including IL-6 and IL-1beta, which may be involved in the diarrhea produced by EPEC, depends on intimin.

Subversion of host cell actin microfilaments is the hallmark of enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli infections. Both pathogens translocate the trans-membrane receptor protein-translocated intimin receptor (Tir), which links the extracellular bacterium to the cell cytoskeleton. While both converge on neural Wiskott-Aldrich syndrome protein (N-WASP), Tir-mediated actin accretion by EPEC and EHEC differ in that Tir(EPEC) requires both tyrosine phosphorylation and the host adaptor protein Nck, whereas Tir(EHEC) is not phosphorylated and utilizes an unidentified linker. Here we report the identification of Tir-cytoskeleton coupling protein (TccP), a novel EHEC effector that displays an Nck-like coupling activity following translocation into host cells. A tccP mutant did not affect Tir translocation and focusing but failed to recruit alpha-actinin, Arp3, N-WASP and actin to the site of bacterial adhesion. When expressed in EPEC, bacterial-derived TccP restored actin polymerization activity following infection of an Nck-deficient cell line. TccP has a similar biological activity on infected human intestinal explants ex vivo. Purified TccP activates N-WASP stimulating, in the presence of Arp2/3, actin polymerization in vitro. These results show that EHEC translocates both its own receptor (Tir) and an Nck-like protein (TccP) to facilitate actin polymerization.

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS). Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton. Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains. In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 beta-lactamase. Translocation was detected directly in living host cells by using the fluorescent beta-lactamase substrate CCF2/AM. We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells. Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner. A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation. In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation. In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells.

Enteropathogenic 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 G2/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 G2/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.

Newborn colostrum-restricted calves were orally inoculated with an Escherichia coli strain, identified originally as non-pathogenic, and into which the plasmid pVir was conjugally transferred. This resulted in diarrhea, intestinal lesions and extra-intestinal invasion, suggesting that factors affecting these pathogenic properties are located on pVir. In order to analyze the respective roles of the toxins CNF2 and CDTIII in the pathogenesis, colostrum-restricted calves were inoculated with isogenic mutants in the cnf2 and the cdt-III genes. The loss of cnf2 is associated with a reduction in the pathogenicity, since diarrhea does not occur in calves challenged, in spite of successful colonization of the intestine. Nevertheless, the mutant strain remains able to invade the bloodstream and to localize in the internal organs. Conversely, the calves inoculated with mutant in the cdt-III gene evolved in the same way as wild-type strain-inoculated calves with regard to clinical signs and macroscopic or microscopic lesions.

Two multiplex PCRs were developed for the detection of necrotoxigenic Escherichia coli virulence genes. M1 contained the primers for the toxins and the aerobactin, and M2 contained the primers for the adhesins. They were validated by single PCRs performed with reference E. coli strains and by multiplex PCRs with necrotoxigenic E. coli strains isolated from different animal species.

Fecal samples from healthy children under 2 years of age living in Berlin, Germany (205 infants), and Melbourne, Australia (184 infants), were investigated for the presence of attaching and effacing (AE) Escherichia coli (AEEC) strains by screening for eae (intimin) genes. Twenty-seven AEEC strains were isolated from 14 children (7.6%) from Melbourne and from 12 children (5.9%) from Berlin. The 27 AEEC strains were classified as enterohemorrhagic E. coli (one strain, producing Shiga toxin 1), typical enteropathogenic E. coli (EPEC) (one strain carrying an EPEC adherence factor [EAF] plasmid), and atypical EPEC (25 strains negative for Shiga toxins and EAF plasmids). The AEEC were divided into 18 different serotypes, O-nontypeable and O-rough strains. Typing of their intimin genes revealed the presence of intimin alpha in 6 strains, intimin beta in 11 strains, intimin gamma in 7 strains, intimin zeta in 2 strains, and intimin eta in one strain. Analysis of HEp-2 cell adherence showed diffuse adherence or localized adherence-like patterns in 26 AEEC strains; local adherence was found only with the EAF-positive strain. Ten AEEC strains showed an AE property with the fluorescent actin staining (FAS) test. The introduction of an EAF plasmid (pMAR7) converted 11 FAS-negative AEEC strains to FAS positive and increased the FAS reaction in six FAS-positive AEEC strains, indicating that the genes needed for the AE phenotype were functional in these strains. Our finding indicates that atypical EPEC strains could play a double role as strains that naturally immunize against intimin in humans and as reservoirs for new emerging human pathogenic EPEC strains.

In this study, we determined the sequences of four intimin variant genes detected in attaching and effacing Escherichia coli isolates of human origin. Three of them were novel and were designated eae-eta (eta), eae-iota (iota), and eae-kappa (kappa). The fourth was identical to the recently described eae-zeta (zeta), isolated from a bovine E. coli O84:NM isolate. We compared these sequences with those of published intimin-alpha, intimin-beta, intimin-gamma1, intimin-gamma2, intimin- epsilon, and intimin-theta alleles. Sequence analysis of these 10 intimin alleles confirmed extensive genetic diversity within the intimin gene family in E. coli. The genetic diversity was more prominent in the 3' region (starting at bp 2,112), which encodes the binding domain of intimin. Phylogenetic analyses revealed four groups of closely related intimin genes: alpha and zeta; beta and kappa; gamma1 and gamma2/theta; and epsilon and eta. Calculation of homoplasy ratios of sequences of the 5' region of eae (positions 1 to 2,111) revealed evidence for intragenic recombination. Split decomposition analysis also indicates that recombination events have played a role in the evolutionary history of eae. In conclusion, we recommend an eae nomenclature system based on the Greek alphabet and provide an updated PCR scheme for amplification and typing of E. coli eae.

Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle.

Rabbit enteropathogenic Escherichia coli (EPEC) O103 induces in HeLa cells an irreversible cytopathic effect characterized by the recruitment of focal adhesions, formation of stress fibers, and inhibition of cell proliferation. We have characterized the modalities of the proliferation arrest and investigated its underlying mechanisms. We found that HeLa cells that were exposed to the rabbit EPEC O103 strain E22 progressively accumulated at 4C DNA content and did not enter mitosis. A significant proportion of the cells were able to reinitiate DNA synthesis without division, leading to 8C DNA content. This cell cycle inhibition by E22 was abrogated in mutants lacking EspA, -B, and -D and was restored by transcomplementation. In contrast, intimin and Tir mutants retained the antiproliferative effect. The cell cycle arrest was not a direct consequence of the formation of stress fibers, since their disruption by toxins during exposure to E22 did not reverse the cell cycle inhibition. Likewise, the cell cycle arrest was not dependent on the early tyrosine dephosphorylation events triggered by E22 in the cells. Two key partner effectors controlling entry into mitosis were also investigated: cyclin B1 and the associated cyclin-dependent kinase 1 (Cdk1). Whereas cyclin B1 was not detectably affected in E22-exposed cells, Cdk1 was maintained in a tyrosine-phosphorylated inactive state and lost its affinity for p13(suc1)-agarose beads. This shows that Cdk1 is implicated in the G2/M arrest caused by EPEC strain E22.