Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119:H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells.

Enteropathogenic Escherichia coli (EPEC) induce attaching and effacing (A/E) lesions in enterocytes and produce the bundle-forming pilus (BFP) contributing to the localized adherence (LA) pattern formation on HeLa cells. Enteroaggregative E. coli (EAEC) produce aggregative adherence (AA) on HeLa cells and form prominent biofilms. The ability to produce LA or AA is an important hallmark to classify fecal E. coli isolates as EPEC or EAEC, respectively. E. coli strains of serotype O119:H6 exhibit an LA+ phenotype and have been considered as comprising a clonal group of EPEC strains. However, we have recently identified O119:H6 EPEC strains that produce LA and an AA-like pattern concurrently (LA/AA-like+). In this study, we evaluated the relatedness of three LA/AA-like+ and three LA+ O119:H6 strains by comparing their virulence and genotypic properties. We first found that the LA/AA-like+ strains induced actin accumulation in HeLa cells (indicative of A/E lesions formation) and formed biofilms on abiotic surfaces more efficiently than the LA+ strains. MLST analysis showed that the six strains all belong to the ST28 complex. All strains carried multiple plasmids, but as plasmid profiles were highly variable, this cannot be used to differentiate LA/AA-like+ and LA+ strains. We further obtained their draft genome sequences and the complete sequences of four plasmids harbored by one LA/AA-like+ strain. Analysis of these sequences and comparison with 37 fully sequenced E. coli genomes revealed that both O119:H6 groups belong to the E. coli phylogroup B2 and are very closely related with only 58-67 SNPs found between LA/AA-like+ and LA+ strains. Search of the draft sequences of the six strains for adhesion-related genes known in EAEC and other E. coli pathotypes detected no genes specifically present in LA/AA-like+ strains. Unexpectedly however, we found that a large plasmid distinct from pEAF is responsible for the AA-like phenotype of the LA/AA-like+ strains. Although we have not identified any plasmid genes specifically present in all LA/AA-like+ strains and absent in the LA+ strains, these results suggest the presence of an unknown mechanism to promote the AA-like pattern production and biofilm formation by the LA/AA-like+ strains. Because their ability to produce A/E lesions and biofilm concomitantly could exacerbate the clinical condition of the patient and lead to persistent diarrhea, the mechanism underlying the enhanced biofilm formation by the LA/AA-like+ O119:H6 strains and their spread and involvement in severe diarrheal diseases should be more intensively investigated.

Influence of environmental factors in the adherence of an atypical enteropathogenic Escherichia coli strain to epithelial cells.

BACKGROUND: Attachment is essential to maintain bacteria at their preferential intestinal colonization sites. There is little information on the influence of different environmental conditions in the interaction of atypical enteropathogenic Escherichia coli (aEPEC) strains with epithelial cells. In this study, we evaluated the effect of different glucose (5 and 25 mM) and CO2 (0.03 and 5%) concentrations and presence of bile salts on the adhesiveness of the aEPEC strain 1551-2. RESULTS: We found that a CO2-enriched atmosphere enhanced the adhesiveness of the aEPEC 1551-2 strain independently of glucose concentrations or presence of bile salts. Conversely, the presence of high glucose concentration altered the original localized adherence (LA) pattern observed at 5 mM glucose, which is characterized by the formation of compact bacterial clusters, to a hybrid adherence pattern (LA and an aggregative adherence-like pattern). In addition, at high glucose concentration, there was increased expression of the fimA gene, which encodes the major subunit of type 1 pilus (T1P), and an isogenic fimA mutant displayed only LA. The presence of bile salts did not interfere with the adhesion properties of the 1551-2 strain to HeLa cells. CONCLUSIONS: Our data suggest that a CO2-enriched atmosphere could favor aEPEC adhesion to the host cells, whereas enhanced T1P production under high glucose concentration could allow bacteria to access more extensive intestinal colonization sites in the host at the beginning of the infectious process.

Dissection of the role of pili and type 2 and 3 secretion systems in adherence and biofilm formation of an atypical enteropathogenic Escherichia coli strain.

Atypical enteropathogenic Escherichia coli (aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2 eae mutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion of fimA in 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2 sslE mutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a double eae espA mutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2 eae mutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains.

Bj-PRO-10c, as an allosteric regulator of argininosuccinate synthase, is a potential therapy for neuroblastoma metastasis.

Cancer is a global public health issue. Neuroblastoma (NB) originates from any tissue of the sympathetic nervous system, and the most affected site is the abdomen. The adrenal gland is the primary site in 38% of cases. Approximately 50% of patients have metastatic disease at diagnosis, and bone marrow is often affected. Metastatic disease is characterized by the spreading of cancer cells that are frequently resistant to chemotherapy and radiotherapy from the primary tumor to other specific parts of the body and is responsible for 90% of cancer-related deaths. Increasing evidence has indicated that nitric oxide (NO) signaling is implicated in the pathophysiology of many types of cancer, particularly in tumorigenesis and cancer progression. However, the effect of NO on metastasis cannot be easily classified as prometastatic or antimetastatic. An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease. Here, the proline-rich decapeptide isolated from Bothrops jararaca venom (Bj-PRO-10c) that enhances and sustains the generation of NO was used to unravel the role of metabolic NO in steps of metastasis. Bj-PRO-10c showed an antimetastatic effect, mainly by interfering with actin cytoskeleton rearrangement, controlling cell proliferation, and decreasing the seeding efficiency of NB in metastatic niches. Therefore, we proposed that an approach for controlled NO induction with the right molecular strategies can hopefully inhibit metastasis and increase the lifespan of NB patients.

Characterization of Escherichia coli strains isolated from patients with diarrhea in Sao Paulo, Brazil: identification of intermediate virulence factor profiles by multiplex PCR.

Intestinal pathogenic Escherichia coli is a major causative agent of severe diarrhea. In this study the prevalences of different pathotypes among 702 E. coli isolates from Brazilian patients with diarrhea were determined by multiplex PCR. Interestingly, most strains were enteroaggregative E. coli (EAEC) strains, followed by atypical EPEC (ATEC) strains. Classical enteropathogenic E. coli (EPEC) strains were not detected.

Escherichia albertii Pathogenesis.

Escherichia albertii is an emerging enteropathogen of humans and many avian species. This bacterium is a close relative of Escherichia coli and has been frequently misidentified as enteropathogenic or enterohemorrhagic E. coli due to their similarity in phenotypic and genetic features, such as various biochemical properties and the possession of a type III secretion system encoded by the locus of enterocyte effacement. This pathogen causes outbreaks of gastroenteritis, and some strains produce Shiga toxin. Although many genetic and phenotypic studies have been published and the genome sequences of more than 200 E. albertii strains are now available, the clinical significance of this species is not yet fully understood. The apparent zoonotic nature of the disease requires a deeper understanding of the transmission routes and mechanisms of E. albertii to develop effective measures to control its transmission and infection. Here, we review the current knowledge of the phylogenic relationship of E. albertii with other Escherichia species and the biochemical and genetic properties of E. albertii, with particular emphasis on the repertoire of virulence factors and the mechanisms of pathogenicity, and we hope this provides a basis for future studies of this important emerging enteropathogen.

Genomic Properties and Temporal Analysis of the Interaction of an Invasive Escherichia albertii With Epithelial Cells.

Diarrhea is one of the main causes of infant mortality worldwide, mainly in the developing world. Among the various etiologic agents, Escherichia albertii is emerging as an important human enteropathogen. E. albertii promote attaching and effacing (AE) lesions due to the presence of the locus of enterocyte effacement (LEE) that encodes a type three secretion system (T3SS), the afimbrial adhesin intimin and its translocated receptor, Tir, and several effector proteins. We previously showed that E. albertii strain 1551-2 invades several epithelial cell lineages by a process that is dependent on the intimin-Tir interaction. To understand the contribution of T3SS-dependent effectors present in E. albertii 1551-2 during the invasion process, we performed a genetic analysis of the LEE and non-LEE genes and evaluated the expression of the LEE operons in various stages of bacterial interaction with differentiated intestinal Caco-2 cells. The kinetics of the ability of the 1551-2 strain to colonize and form AE lesions was also investigated in epithelial HeLa cells. We showed that the LEE expression was constant during the early stages of infection but increased at least 4-fold during bacterial persistence in the intracellular compartment. An in silico analysis indicated the presence of a new tccP/espFU subtype, named tccP3. We found that the encoded protein colocalizes with Tir and polymerized F-actin during the infection process in vitro. Moreover, assays performed with Nck null cells demonstrated that the 1551-2 strain can trigger F-actin polymerization in an Nck-independent pathway, despite the fact that TccP3 is not required for this phenotype. Our study highlights the importance of the T3SS during the invasion process and for the maintenance of E. albertii 1551-2 inside the cells. In addition, this work may help to elucidate the versatility of the T3SS for AE pathogens, which are usually considered extracellular and rarely reach the intracellular environment.

Invasiveness as a putative additional virulence mechanism of some atypical Enteropathogenic Escherichia coli strains with different uncommon intimin types.

BACKGROUND: Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPEC are sub-grouped into typical (tEPEC) and atypical (aEPEC). We have recently demonstrated that aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. In this study, we evaluated whether aEPEC strains expressing other intimin sub-types are also invasive using the quantitative gentamicin protection assay. We also evaluated whether aEPEC invade differentiated intestinal T84 cells. RESULTS: Five of six strains invaded HeLa and T84 cells in a range of 13.3%-20.9% and 5.8%-17.8%, respectively, of the total cell-associated bacteria. The strains studied were significantly more invasive than prototype tEPEC strain E2348/69 (1.4% and 0.5% in HeLa and T84 cells, respectively). Invasiveness was confirmed by transmission electron microscopy. We also showed that invasion of HeLa cells by aEPEC 1551-2 depended on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface. CONCLUSION: Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.

The localized adherence pattern of an atypical enteropathogenic Escherichia coli is mediated by intimin omicron and unexpectedly promotes HeLa cell invasion.

Enteropathogenic Escherichia coli (EPEC) forms attaching and effacing lesions in the intestinal mucosa characterized by intimate attachment to the epithelium by means of intimin (an outer membrane adhesin encoded by eae). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC); only tEPEC carries the EAF (EPEC adherence factor) plasmid that encodes the bundle-forming pilus (BFP). Characteristically, after 3 h of incubation, tEPEC produces localized adherence (LA) (with compact microcolonies) in HeLa/HEp-2 cells by means of BFP, whereas most aEPEC form looser microcolonies. We have previously identified nine aEPEC strains displaying LA in extended (6 h) assays (LA6). In this study, we analysed the kinetics of LA6 pattern development and the role of intimin in the process. Transmission electron microscopy and confocal laser microscopy showed that the invasive process of strain 1551-2 displays a LA phenotype. An eae-defective mutant of strain 1551-2 prevented the invasion although preserving intense diffused adherence. Sequencing of eae revealed that strain 1551-2 expresses the omicron subtype of intimin. We propose that the LA phenotype of aEPEC strain 1551-2 is mediated by intimin omicron and hypothesize that this strain expresses an additional novel adhesive structure. The present study is the first to report the association of compact microcolony formation and an intense invasive ability in aEPEC.

HeLa-cell adherence patterns and actin aggregation of enteropathogenic Escherichia coli (EPEC) and Shiga-toxin-producing E. coli (STEC) strains carrying different eae and tir alleles.

A collection of 69 eae-positive strains expressing 29 different intimin types and eight tir alleles was characterized with respect to their adherence patterns to HeLa cells, ability to promote actin accumulation in vitro, the presence of bfpA alleles in positive strains, and bundle-forming pilus (BFP) expression. All of the nine typical enteropathogenic Escherichia coli (tEPEC) studied harbored the enteropathogenic E. coli adherence factor (EAF) plasmid, as shown by PCR and/or EAF probe results. In addition, they were positive for bfpA, as shown by PCR, and BFP expression, as confirmed by immunofluorescence (IFL) and/or immunoblotting (IBL) assays. Localized adherence (LA) was exclusively displayed by those nine tEPEC, while localized-adherence-like (LAL) was the most frequent pattern among atypical EPEC (aEPEC) and Shiga-toxinproducing E. coli (STEC). All LA and LAL strains were able to cause attaching and effacing (AE) lesions, as established by means of the FAS test. There was a significant association between the presence of tir allele alpha1 and bfpA-positive strains, and consequently, with the LA pattern. However, intimin type or bfpA was not associated with the adherence pattern displayed in HeLa cells. Among the eight bfpA alleles detected, a new type (beta10; accession number FN391178) was identified in a strain of serotype O157:H45, and a truncated variant (beta3.2-t; accession number FN 391181) in four strains belonging to different pathotypes.

Phenotypic characterization and virulence-related properties of Escherichia albertii strains isolated from children with diarrhea in Brazil.

Escherichia albertii are emerging enteropathogens, whose identification is difficult, as they share biochemical characteristics and some virulence-related genes with diarrheagenic Escherichia coli (DEC). Studies on phylogeny, phenotypic characteristics and potential virulence factors of human E. albertii strains are scarce. In this study, we identified by multiplex PCR five E. albertii among 106 strains isolated from diarrheic children in São Paulo, Brazil, which were previously classified as atypical enteropathogenic E. coli. All strains were investigated regarding their phylogeny, biochemical properties, virulence-related properties, antimicrobial resistance and presence of putative virulence-related genes. All strains belonged to different E. albertii lineages and adhered to and produced attaching and effacing lesions on HeLa cells. Three strains invaded Caco-2 cells, but did not persist intracellularly, and three formed biofilms on polystyrene surfaces. All strains were resistant to few antibiotics and only one carried a self-transmissible resistance plasmid. Finally, among 38 DEC and 18 extraintestinal pathogenic E. coli (ExPEC) virulence-related genes searched, six and three were detected, respectively, with paa and cdtB being found in all strains. Despite the limited number of strains, this study provided additional knowledge on human E. albertii virulence potential, showing that they share important virulence factors with DEC and ExPEC.

The Type III Secretion System (T3SS)-Translocon of Atypical Enteropathogenic Escherichia coli (aEPEC) Can Mediate Adherence.

The intimin protein is the major adhesin involved in the intimate adherence of atypical enteropathogenic Escherichia coli (aEPEC) strains to epithelial cells, but little is known about the structures involved in their early colonization process. A previous study demonstrated that the type III secretion system (T3SS) plays an additional role in the adherence of an Escherichia albertii strain. Therefore, we assumed that the T3SS could be related to the adherence efficiency of aEPEC during the first stages of contact with epithelial cells. To test this hypothesis, we examined the adherence of seven aEPEC strains and their eae (intimin) isogenic mutants in the standard HeLa adherence assay and observed that all wild-type strains were adherent while five isogenic eae mutants were not. The two eae mutant strains that remained adherent were then used to generate the eae/escN double mutants (encoding intimin and the T3SS ATPase, respectively) and after the adherence assay, we observed that one strain lost its adherence capacity. This suggested a role for the T3SS in the initial adherence steps of this strain. In addition, we demonstrated that this strain expressed the T3SS at significantly higher levels when compared to the other wild-type strains and that it produced longer translocon-filaments. Our findings reveal that the T3SS-translocon can play an additional role as an adhesin at the beginning of the colonization process of aEPEC.

Draft Whole-Genome Sequences of 10 Atypical Enteropathogenic Escherichia coli Strains Isolated in Brazil.

The number of diarrhea cases caused by atypical enteropathogenic Escherichia coli (aEPEC) has been increasing worldwide. Here, we report the draft whole-genome sequences of 10 aEPEC strains isolated in Brazil. These sequences will provide an important source for future studies concerning aEPEC pathogenicity and genetic markers of potentially virulent strains.

Escherichia albertii, a novel human enteropathogen, colonizes rat enterocytes and translocates to extra-intestinal sites.

Diarrhea is the second leading cause of death of children up to five years old in the developing countries. Among the etiological diarrheal agents are atypical enteropathogenic Escherichia coli (aEPEC), one of the diarrheagenic E. coli pathotypes that affects children and adults, even in developed countries. Currently, genotypic and biochemical approaches have helped to demonstrate that some strains classified as aEPEC are actually E. albertii, a recently recognized human enteropathogen. Studies on particular strains are necessary to explore their virulence potential in order to further understand the underlying mechanisms of E. albertii infections. Here we demonstrated for the first time that infection of fragments of rat intestinal mucosa is a useful tool to study the initial steps of E. albertii colonization. We also observed that an E. albertii strain can translocate from the intestinal lumen to Mesenteric Lymph Nodes and liver in a rat model. Based on our finding of bacterial translocation, we investigated how E. albertii might cross the intestinal epithelium by performing infections of M-like cells in vitro to identify the potential in vivo translocation route. Altogether, our approaches allowed us to draft a general E. albertii infection route from the colonization till the bacterial spreading in vivo.

Antisense intronic non-coding RNA levels correlate to the degree of tumor differentiation in prostate cancer.

A large fraction of transcripts are expressed antisense to introns of known genes in the human genome. Here we show the construction and use of a cDNA microarray platform enriched in intronic transcripts to assess their biological relevance in pathological conditions. To validate the approach, prostate cancer was used as a model, and 27 patient tumor samples with Gleason scores ranging from 5 to 10 were analyzed. We find that a considerably higher fraction (6.6%, [23/346]) of intronic transcripts are significantly correlated (P< or =0.001) to the degree of prostate tumor differentiation (Gleason score) when compared to transcripts from unannotated genomic regions (1%, [6/539]) or from exons of known genes (2%, [27/1369]). Among the top twelve transcripts most correlated to tumor differentiation, six are antisense intronic messages as shown by orientation-specific RT-PCR or Northern blot analysis with strand-specific riboprobe. Orientation-specific real-time RT-PCR with six tumor samples, confirmed the correlation (P=0.024) between the low/high degrees of tumor differentiation and antisense intronic RASSF1 transcript levels. The need to use intron arrays to reveal the transcriptome profile of antisense intronic RNA in cancer has clearly emerged.

Genomic properties and temporal analysis of the interaction of an invasive Escherichia albertii with epithelial cells

Diarrhea is one of the main causes of infant mortality worldwide, mainly in the developing world. Among the various etiologic agents, Escherichia albertii is emerging as an important human enteropathogen. E. albertii promote attaching and effacing (AE) lesions due to the presence of the locus of enterocyte effacement (LEE) that encodes a type three secretion system (T3SS), the afimbrial adhesin intimin and its translocated receptor, Tir, and several effector proteins. We previously showed that E. albertii strain 1551-2 invades several epithelial cell lineages by a process that is dependent on the intimin-Tir interaction. To understand the contribution of T3SS-dependent effectors present in E. albertii 1551-2 during the invasion process, we performed a genetic analysis of the LEE and non-LEE genes and evaluated the expression of the LEE operons in various stages of bacterial interaction with differentiated intestinal Caco-2 cells. The kinetics of the ability of the 1551-2 strain to colonize and form AE lesions was also investigated in epithelial HeLa cells. We showed that the LEE expression was constant during the early stages of infection but increased at least 4-fold during bacterial persistence in the intracellular compartment. An in silico analysis indicated the presence of a new tccP/espFU subtype, named tccP3. We found that the encoded protein colocalizes with Tir and polymerized F-actin during the infection process in vitro. Moreover, assays performed with Nck null cells demonstrated that the 1551-2 strain can trigger F-actin polymerization in an Nck-independent pathway, despite the fact that TccP3 is not required for this phenotype. Our study highlights the importance of the T3SS during the invasion process and for the maintenance of E. albertii 1551-2 inside the cells. In addition, this work may help to elucidate the versatility of the T3SS for AE pathogens, which are usually considered extracellular and rarely reach the intracellular environment.

The type III secretion system (T3SS)-Translocon of atypical enteropathogenic Escherichia coli (aEPEC) can mediate adherence

The intimin protein is the major adhesin involved in the intimate adherence of atypicalenteropathogenicEscherichia coli(aEPEC) strains to epithelial cells, but little is knownabout the structures involved in their early colonization process. A previous studydemonstrated that the type III secretion system (T3SS) plays an additional role in theadherence of anEscherichia albertiistrain. Therefore, we assumed that the T3SS couldbe related to the adherence efficiency of aEPEC during the first stages of contactwith epithelial cells. To test this hypothesis, we examined the adherence of sevenaEPEC strains and theireae(intimin) isogenic mutants in the standard HeLa adherenceassay and observed that all wild-type strains were adherent while five isogeniceaemutants were not. The twoeaemutant strains that remained adherent were then usedto generate theeae/escNdouble mutants (encoding intimin and the T3SS ATPase,respectively) and after the adherence assay, we observed that one strain lost itsadherence capacity. This suggested a role for the T3SS in the initial adherence stepsof this strain. In addition, we demonstrated that this strain expressed the T3SS atsignificantly higher levels when compared to the other wild-type strains and that itproduced longer translocon-filaments. Our findings reveal that the T3SS-transloconcan play an additional role as an adhesin at the beginning of the colonization processof aEPEC.

Invasion of differentiated intestinal Caco-2 cells is a sporadic property among atypical enteropathogenic Escherichia coli strains carrying common intimin subtypes.

Atypical enteropathogenic Escherichia coli (aEPEC) strains produce attaching-effacing (AE) lesions on enterocytes due to the interaction of the adhesin intimin with its translocated receptor. aEPEC strain 1551-2 was previously shown to invade HeLa and T84 cells by means of the uncommon intimin subtype omicron. Other aEPEC strains carrying uncommon intimin subtypes have also been shown to invade differentiated T84 intestinal cells. In this study, seven aEPEC strains carrying the most common EPEC intimin subtypes (alpha, beta, and gamma) were evaluated regarding the ability to invade differentiated intestinal Caco-2 cells. Although all strains adhered to and promoted AE lesions, the numbers of cell-associated bacteria varied significantly between the different strains regardless of the intimin subtype (P < 0.05). Gentamicin protection assay and transmission electron microscopy analyses showed that in comparison with the invasive strain 1551-2, only one strain (aEPEC EC423/03, intimin beta) was invasive (P = 0.05). Although both strains persisted intracellularly until 48 h, the number of viable bacteria of EC423/03 decreased, whereas that of 1551-2 increased significantly up to 24 h and then decreased. In conclusion, invasiveness is a sporadic property among aEPEC strains carrying some common intimin subtypes.

Draft whole-genome sequences of 10 atypical enteropathogenic Escherichia coli strains isolated in Brazil

The number of diarrhea cases caused by atypical enteropathogenic Escherichia coli (aEPEC) has been increasing worldwide. Here, we report the draft whole-genome sequences of 10 aEPEC strains isolated in Brazil. These sequences will provide an important source for future studies concerning aEPEC pathogenicity and genetic markers of potentially virulent strains.

The type III secretion system (T3SS)-Translocon of atypical enteropathogenic Escherichia coli (aEPEC) can mediate adherence

The intimin protein is the major adhesin involved in the intimate adherence of atypicalenteropathogenicEscherichia coli(aEPEC) strains to epithelial cells, but little is knownabout the structures involved in their early colonization process. A previous studydemonstrated that the type III secretion system (T3SS) plays an additional role in theadherence of anEscherichia albertiistrain. Therefore, we assumed that the T3SS couldbe related to the adherence efficiency of aEPEC during the first stages of contactwith epithelial cells. To test this hypothesis, we examined the adherence of sevenaEPEC strains and theireae(intimin) isogenic mutants in the standard HeLa adherenceassay and observed that all wild-type strains were adherent while five isogeniceaemutants were not. The twoeaemutant strains that remained adherent were then usedto generate theeae/escNdouble mutants (encoding intimin and the T3SS ATPase,respectively) and after the adherence assay, we observed that one strain lost itsadherence capacity. This suggested a role for the T3SS in the initial adherence stepsof this strain. In addition, we demonstrated that this strain expressed the T3SS atsignificantly higher levels when compared to the other wild-type strains and that itproduced longer translocon-filaments. Our findings reveal that the T3SS-transloconcan play an additional role as an adhesin at the beginning of the colonization processof aEPEC.