Type 1 fimbria and P pili: regulatory mechanisms of the prototypical members of the chaperone-usher fimbrial family.

Adherence to both cellular and abiotic surfaces is a crucial step in the interaction of bacterial pathogens and commensals with their hosts. Bacterial surface structures known as fimbriae or pili play a fundamental role in the early colonization stages by providing specificity or tropism. Among the various fimbrial families, the chaperone-usher family has been extensively studied due to its ubiquity, diversity, and abundance. This family is named after the components that facilitate their biogenesis. Type 1 fimbria and P pilus, two chaperone-usher fimbriae associated with urinary tract infections, have been thoroughly investigated and serve as prototypes that have laid the foundations for understanding the biogenesis of this fimbrial family. Additionally, the study of the mechanisms regulating their expression has also been a subject of great interest, revealing that the regulation of the expression of the genes encoding these structures is a complex and diverse process, involving both common global regulators and those specific to each operon.

The osmoregulated metabolism of trehalose contributes to production of type 1 fimbriae and bladder colonization by extraintestinal Escherichia coli strain BEN2908.

In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.

E. coli Common pili promote the fitness and virulence of a hybrid aEPEC/ExPEC strain within diverse host environments.

Pathogenic subsets of Escherichia coli include diarrheagenic (DEC) strains that cause disease within the gut and extraintestinal pathogenic E. coli (ExPEC) strains that are linked with urinary tract infections, bacteremia, and other infections outside of intestinal tract. Among DEC strains is an emergent pathotype known as atypical enteropathogenic E. coli (aEPEC), which can cause severe diarrhea. Recent sequencing efforts revealed that some E. coli strains possess genetic features that are characteristic of both DEC and ExPEC isolates. BA1250 is a newly reclassified hybrid strain with characteristics of aEPEC and ExPEC. This strain was isolated from a child with diarrhea, but its genetic features indicate that it might have the capacity to cause disease at extraintestinal sites. The spectrum of adhesins encoded by hybrid strains like BA1250 are expected to be especially important in facilitating colonization of diverse niches. E. coli common pilus (ECP) is an adhesin expressed by many E. coli pathogens, but how it impacts hybrid strains has not been ascertained. Here, using zebrafish larvae as surrogate hosts to model both gut colonization and extraintestinal infections, we found that ECP can act as a multi-niche colonization and virulence factor for BA1250. Furthermore, our results indicate that ECP-related changes in activation of envelope stress response pathways may alter the fitness of BA1250. Using an in silico approach, we also delineated the broader repertoire of adhesins that are encoded by BA1250, and provide evidence that the expression of at least a few of these varies in the absence of functional ECP.

Role of aggregate-forming pilus (AFP) in adherence and colonization of both intestinal and urinary tracts.

Hybrid-pathogenic Escherichia coli represent an important group of strains associated with intestinal and extraintestinal infections. Recently, we described strain UPEC-46, a uropathogenic/enteroaggregative E. coli (UPEC/EAEC) strain presenting the aggregative adherence (AA) pattern on bladder and colorectal epithelial cells mediated by aggregate-forming pili (AFP). However, the role of AFP and other uninvestigated putative fimbriae operons in UPEC-46 pathogenesis remains unclear. Thus, this study evaluated the involvement of AFP and other adhesins in uropathogenicity and intestinal colonization using different in vitro and in vivo models. The strain UPEC-46 was able to adhere and invade intestinal and urinary cell lines. A library of transposon mutants also identified the involvement of type I fimbriae (TIF) in the adherence to HeLa cells, in addition to colorectal and bladder cell lines. The streptomycin-treated mouse in vivo model also showed an increased number of bacterial counts in the colon in the presence of AFP and TIF. In the mouse model of ascending urinary tract infection (UTI), AFP was more associated with kidney colonization, while TIF appears to mediate bladder colonization. Results observed in in vivo experiments were also confirmed by electron microscopy (EM) analyses. In summary, the in vitro and in vivo analyses show a synergistic role of AFP and TIF in the adherence and colonization of intestinal and urinary epithelia. Therefore, we propose that hybrid E. coli strains carrying AFP and TIF could potentially cause intestinal and urinary tract infections in the same patient.

The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri.

Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.

Atomic and Electronic Structure of Pilus from Geobacter sulfurreducens through QM/MM Calculations: Evidence for Hole Transfer in Aromatic Residues.

Long-range electron transport has been widely and experimentally reported in Geobacter sulfurreducens pilus protein. However, a better understanding of the still undefined molecular arrangement can bring to light the role of key residues in this phenomenon. We propose a theoretical investigation of the electronic structure of aromatic residue groups in the protein through a classical molecular dynamics (MD) simulation, followed by a quantum mechanics/molecular mechanics (QM/MM) electronic study of different frames sampled from MD trajectories, an electrostatic potential and electron density analysis, an analysis of the density of states, and an investigation of hole formation through Dyson orbital calculations. We observe a highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) energy gap in the ranges of 1.4-2.3 eV and 2.9-3.3 eV and a less intense dipole moment along the aromatic residues in the presence of water in comparison to the system in vacuum. HOMO and LUMO electron densities highlight the occupation of one tyrosine residue in every representation for HOMO and a delocalization along two to three rings for LUMO. The results show how the electronic structure of the aromatic residues is sensitive to the ring arrangement and the surrounding environment. In our study, we observe that slight rearrangements in the fiber geometry can create temporary conditions for hole transfer.

Defining chaperone-usher fimbriae repertoire in Serratia marcescens.

Chaperone-usher (CU) fimbriae are surface organelles particularly prevalent among the Enterobacteriaceae. Mainly associated to their adhesive properties, CU fimbriae play key roles in biofilm formation and host cell interactions. Little is known about the fimbriome composition of the opportunistic human pathogen Serratia marcescens. Here, by using a search based on consensus fimbrial usher protein (FUP) sequences, we identified 421 FUPs across 39 S. marcescens genomes. Further analysis of the FUP-containing loci allowed us to classify them into 20 conserved CU operons, 6 of which form the S. marcescens core CU fimbriome. A new systematic nomenclature is proposed according to FUP sequence phylogeny. We also established an in vivo transcriptional assay comparing CU promoter expression between an environmental and a clinical isolate of S. marcescens, which revealed that promoters from 3 core CU operons (referred as fgov, fpo, and fps) are predominantly expressed in the two strains and might represent key core adhesion appendages contributing to S. marcescens pathogenesis.

Diarrheagenic Escherichia coli isolates from neonatal alpacas mainly display F17 fimbriae adhesion gene.

Neonatal domestic South American Camelid llamas and alpacas suffer from an enteric disease complex characterized by abdominal distention, lethargy, dehydration, and eventual fatal septicemia. Analysis of rectal swabs from neonatal alpacas suffering clinical diarrheas has constantly isolated Escherichia coli, mainly the EPEC and EHEC pathotypes. The present communication reports the results of molecular analysis of 226 E. coli strains from neonatal alpaca rectal swabs. The isolates were initially tested by multiple PCR, to identify E. coli virulence genes eae, bfp, Lt, Stx1, Stx2, sta, stab, and lt genes and a similar test to detect F4, F5, F6, F17, and F41fimbriae adhesin genes. Forty-two of the 226 (18.5%) isolates tested positive for at least one pathogenic gene, 25 of the 42 were classified as EPEC (3 positives for only eae and 22 for both eae and bfp) and the remaining 17 were classified as EHEC. Twenty-four (57%) of the 42 isolates tested positive to F17 adhesin while one was positive for both F6 and F17.

Interfering with the Folding of Group A Streptococcal pili Proteins.

Gram-positive bacteria use their adhesive pili to attach to host cells during early stages of a bacterial infection. These extracellular hair-like appendages experience mechanical stresses of hundreds of picoNewtons; however, the presence of an internal isopeptide bond prevents the pilus protein from unfolding. Here, we describe a method to interfere with nascent pili proteins through a peptide that mimics one of the ß-strands of the molecule. By using AFM-based force spectroscopy, we study the isopeptide bond formation and the effect of the peptide in the elasticity of the pilus protein. This method could be used to afford a new strategy for mechanically targeted antibiotics by simply blocking the folding of the bacterial pilus protein.

c-di-GMP-related phenotypes are modulated by the interaction between a diguanylate cyclase and a polar hub protein.

c-di-GMP is a major player in the switch between biofilm and motile lifestyles. Several bacteria exhibit a large number of c-di-GMP metabolizing proteins, thus a fine-tuning of this nucleotide levels may occur. It is hypothesized that some c-di-GMP metabolizing proteins would provide the global c-di-GMP levels inside the cell whereas others would maintain a localized pool, with the resulting c-di-GMP acting at the vicinity of its production. Although attractive, this hypothesis has yet to be demonstrated in Pseudomonas aeruginosa. We found that the diguanylate cyclase DgcP interacts with the cytosolic region of FimV, a polar peptidoglycan-binding protein involved in type IV pilus assembly. Moreover, DgcP is located at the cell poles in wild type cells but scattered in the cytoplasm of cells lacking FimV. Overexpression of dgcP leads to the classical phenotypes of high c-di-GMP levels (increased biofilm and impaired motilities) in the wild-type strain, but not in a ΔfimV background. Therefore, our findings suggest that DgcP activity is regulated by FimV. The polar localization of DgcP might contribute to a local c-di-GMP pool that can be sensed by other proteins at the cell pole, bringing to light a specialized function for a specific diguanylate cyclase.

Synergistic mode of action of catechin, vanillic and protocatechuic acids to inhibit the adhesion of uropathogenic Escherichia coli on silicone surfaces.

AIMS: To study the individual and combined contribution of catechin, protocatechuic and vanillic acids to inhibit the adhesion of uropathogenic Escherichia coli (UPEC) on the surface of silicone catheters. METHODS AND RESULTS: The adhesion of UPEC to silicone catheters during the exposure to nonlethal concentrations of phenolic compounds was measured, as well as changes in motility, presence of fimbriae, extra-cellular polymeric substances, surface charge, hydrophobicity and membrane fluidity. The phenolic combination reduced 26-51% of motility, 1 log CFU per cm2 of adhered bacteria and 20-40% the carbohydrate and protein content in the biofilm matrix. Curli fimbriae, surface charge and cell hydrophobicity were affected to a greater extent by the phenolic combination. In the mixture, vanillic acid was the most effective for reducing bacterial adhesion, extra-polymeric substance production, motility, curli fimbriae and biofilm structure. Notwithstanding, protocatechuic acid caused major changes in the bacterial cell surface properties, whereas catechin affected the cell membrane functionality. CONCLUSION: Catechin, protocatechuic and vanillic acids have different bacterial cell targets, explaining the synergistic effect of their combination against uropathogenic E. coli. SIGNIFICANCE AND IMPACT OF STUDY: This study shows the contribution of catechin, protocatechuic and vanillic acids in producing a synergistic mixture against the adhesion of uropathogenic E. coli on silicone catheters. The action of catechin, vanillic and protocatechuic acids included specific contributions of each compound against the E. coli membrane's integrity, motility, surface properties and production of extracellular polymeric substances. Therefore, the studied mixture of phenolic compounds could be used as an antibiotic alternative to reduce urinary tract infections associated with silicone catheters.

Biofilm production and distribution of pilus variants among Streptococcus agalactiae isolated from human and animal sources.

Streptococcus agalactiae (group B Streptococcus, GBS) is a major pathogen in humans and animals. Pili and biofilm may be important virulence factors in this bacterial species. Here, biofilm production and the distribution of pilus variants among 134 GBS isolates from human and animal sources were evaluated. Biofilm production was significantly enhanced in 1% glucose-supplemented medium (p < 0.05). Using this medium, most GBS strains were strong biofilm producers. Biomass was mainly composed of proteins, followed by extracellular DNA, while polysaccharides represented a minor portion. All GBS strains presented at least one pilus variant. PI-2a was the most common among human GBS while PI-2b was the most common among animal isolates. Human GBS harboring PI-2b and animal GBS harboring PI-2a presented significantly reduced biofilm production (p = 0.0033). In conclusion, strong biofilm production seems to be a common characteristic in GBS, and association of the clinical source with the pilus variant may be crucial for this.

Transcriptional regulation of the Salmonella enterica std fimbrial operon by the RcsCDB system.

In Salmonella enterica serovar Typhimurium, the RcsCDB regulatory system controls the expression of genes involved in synthesis of colanic acid, formation of flagella and virulence. Here, we show that activation of the RcsCDB system downregulates expression of std, an operon that encodes fimbriae involved in Salmonella attachment to the mucus layer in the large intestine. Bioinformatic analysis predicts the existence of an RcsB-binding site located 180 bp upstream to the +1 transcription start site of the std promoter, and electrophoretic mobility shift assays confirm that RcsB binds the std promoter region in vitro. This study adds RcsB to the list of regulators of std transcription and provides an example of modulation of fimbriae synthesis by a signal transduction system.

QseC Signaling in the Outbreak O104:H4 Escherichia coli Strain Combines Multiple Factors during Infection.

Enteroaggregative Escherichia coli (EAEC) from the O104:H4 specific serotype caused a large outbreak of bloody diarrhea with some complicated cases of hemolytic-uremic syndrome (HUS) in Europe in 2011. The outbreak strain consisted in an EAEC capable to produce the Shiga toxin (Stx) subtype 2a, a characteristic from enterohemorrhagic E. coli QseBC two-component system detects AI-3/Epi/NE and mediates the chemical signaling between pathogen and mammalian host. This system coordinates a cascade of virulence genes expression in important human enteropathogens. The blocking of QseC of EAEC C227-11 (Stx+) strain by N-phenyl-4-{[(phenylamino) thioxomethyl]amino}-benzenesulfonamide (also known as LED209) in vivo demonstrated a lower efficiency of colonization. The periplasmic protein VisP, which is related to survival mechanisms in a colitis model of infection, bacterial membrane maintenance, and stress resistance, here presented high levels of expression during the initial infection within the host. Under acid stress conditions, visP expression levels were differentiated in an Stx-dependent way. Together, these results emphasize the important role of VisP and the histidine kinase sensor QseC in the C227-11 (Stx+) outbreak strain for the establishment of the infectious niche process in the C57BL/6 mouse model and of LED209 as a promising antivirulence drug strategy against these enteric pathogens.IMPORTANCE EAEC is a remarkable etiologic agent of acute and persistent diarrhea worldwide. The isolates harbor specific subsets of virulence genes and their pathogenesis needs to be better understood. Chemical signaling via histidine kinase sensor QseC has been shown as a potential target to elucidate the orchestration of the regulatory cascade of virulence factors.

TLR4 Participates in the Inflammatory Response Induced by the AAF/II Fimbriae From Enteroaggregative Escherichia coli on Intestinal Epithelial Cells.

Enteroaggregative Escherichia coli (EAEC) infections are one of the most frequent causes of persistent diarrhea in children, immunocompromised patients and travelers worldwide. The most prominent colonization factors of EAEC are aggregative adherence fimbriae (AAF). EAEC prototypical strain 042 harbors the AAF/II fimbriae variant, which mediates adhesion to intestinal epithelial cells and participates in the induction of an inflammatory response against this pathogen. However, the mechanism and the cell receptors implicated in eliciting this response have not been fully characterized. Since previous reports have shown that TLR4 recognize fimbriae from different pathogens, we evaluated the role of this receptor in the response elicited against EAEC by intestinal cells. Using a mutual antagonist against TLR2 and TLR4 (OxPAPC), we observed that blocking of these receptors significantly reduces the secretion of the inflammatory marker IL-8 in response to EAEC and AAF/II fimbrial extract in HT-29 cells. Using a TLR4-specific antagonist (TAK-242), we observed that the secretion of this cytokine was significantly reduced in HT-29 cells infected with EAEC or incubated with AAF/II fimbrial extract. We evaluated the participation of AAF/II fimbriae in the TLR4-mediated secretion of 38 cytokines, chemokines, and growth factors involved in inflammation. A reduction in the secretion of IL-8, GRO, and IL-4 was observed. Our results suggest that TLR4 participates in the secretion of several inflammation biomarkers in response to AAF/II fimbriae.

Diversity of strategies used by atypical enteropathogenic Escherichia coli to induce attaching and effacing lesion in epithelial cells.

PURPOSE: This study aimed to characterize 82 atypical enteropathogenic Escherichia coli (aEPEC) isolates, obtained from patients with diarrhea in Brazil, regarding their adherence patterns on HeLa cells and attaching and effacing (AE) lesion pathways. METHODOLOGY: The adherence and fluorescence-actin staining (FAS) assays were performed using HeLa cells. AE lesion pathways were determined through the detection of tyrosine residue 474 (Y474) phosphorylation in the Tir protein, after its translocation to host cells, and by PCR assays for tir genotyping and detection of Tir-cytoskeleton coupling protein (tccP) genes. RESULTS: Regarding the adherence pattern, determined in the presence of d-mannose, 12 isolates (14.6 %) showed the localized adherence (LA)-like pattern, 3 (3.7  %) the aggregative adherence pattern and 4 (4.9  %) a hybrid LA/diffuse adherence pattern. In addition, 36 (43.9  %) isolates displayed an undefined adherence, and 26 (31.7  %) were non-adherent (NA), while one (1.2 %) caused cell detachment. Among the 26 NA aEPEC isolates, 11 showed a type 1 pilus-dependent adherence in assays performed without d-mannose, while 15 remained NA. Forty-eight (58.5 %) aEPEC were able to trigger F-actin accumulation underneath adherent bacteria (FAS-positive), which is an important feature of AE lesions. The majority (58.3 %) of these used the Tir-Nck pathway, while 39.6  % may use both Tir-Nck and Tir-TccP pathways to induce AE lesions. CONCLUSION: Our results reveal the diversity of strategies used by aEPEC isolates to interact with and damage epithelial host cells, thereby causing diarrheal diseases.

Influence of the major pilA transcriptional regulator in electrochemical responses of Geobacter sulfureducens PilR-deficient mutant biofilm formed on FTO electrodes.

Geobacter sulfurreducens is a model organism for understanding the role of bacterial structures in extracellular electron transfer mechanism (EET). This kind of bacteria relies on different structures such as type IV pili and over 100 c-type cytochromes to perform EET towards soluble and insoluble electron acceptors, including electrodes. To our knowledge, this work is the first electrochemical study comparing a G. sulfurreducens PilR-deficient mutant and wild type biofilms developed on fluorine-doped tin oxide (FTO) electrodes. Open circuit potential (OCP), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), were used to evaluate the electroactive properties of biofilms grown without externally imposed potential. Parallel studies of Confocal Laser Scanning Microscopy (CLSM) correlated with the electrochemical results. PilR is a transcriptional regulator involved in the expression of a wide variety of genes, including pilA (pilus structural protein) relevant c-type cytochromes and some other genes involved in biofilm formation and EET processes. Our findings suggest that PilR-deficient mutant forms a thinner (CLSM analysis) and less conductive biofilm (EIS analysis) than wild type, exhibiting different and irreversible redox processes at the interface (CV analysis). Additionally, this work reinforces some of the remarkable features described in previous reports about this G. sulfurreducens mutant.

Characterization of intestinal Escherichia coli isolated from calves with diarrhea due to rotavirus and coronavirus.

PURPOSE: To address more information about changes in commensal Escherichia coli during virus intestinal infection, we characterized 30 faecal E. coli isolates from calves (21 to 60 days old) with diarrhea due to rotavirus and coronavirus, which received, before diagnosis, tetracycline, gentamicin and enrofloxacin drugs. METHODOLOGY: Clermont's phylogenetic classification; presence of genes for curli, cellulose, fimbriae (F4, F5, F6, F18, F41); and antimicrobial susceptibility were used to characterize the isolates. Disk diffusion technique and PCR were used as methodologies. RESULTS: E. coli isolates from calves with diarrhea were phylogenetically classified as B1 (70%, 21/30), B2 (3.33%, 1/30), C (3.33%, 1/30), D (3.33%, 1/30), E (13.33%, 4/30) and unknown (6.7 %; 2/30), whereas E. coli isolates from the control group were classified only as B1 (83.3%, 25/30), E (10 %; 3/30) and unknown (6,7 %; 2/30). E. coli isolates from calves with diarrhea showed a much higher resistance profile with 16 (53.3%) multiresistant isolates. Only isolates (30%-9/30) from diarrheic calves were also positive for fimbriae, specifically 16.7% (5/30) for F5 and 13.3% (4/30) for F18. CONCLUSION: To sum up, E. coli isolates from calves with diarrhea showed differences in relation to the control group, confirming changes in commensal E. coli during virus intestinal infection. It can be emphasized that some care should be taken to manage diarrheic calves: the pathological agent must be diagnosed prior to treatment; antibacterial treatment should be with antimicrobials with a different mechanism of action; and finally, treated animals should be maintained separately from others because they can carry micro-organisms with a resistant profile.

Biofilm Formation by Avian Pathogenic Escherichia coli is Not Related to In Vivo Pathogenicity.

Avian pathogenic Escherichia coli (APEC) is one of the pathogens that most concerns the poultry industry worldwide due to the economic losses it can cause. APEC persistence and survival, both in the environment and in the host, may be a consequence of biofilm-producing capabilities. The aim of this study was to evaluate APEC strains' biofilm production and its relationship to in vivo pathogenicity. Two hundred thirty-eight APEC isolates from three different origins (broiler bedding material, cellulite lesions, and respiratory diseases) were selected. The in vivo pathogenicity index (PI) was determined. Biofilm formation was evaluated using a microplate assay with analysis of colony morphology in Congo Red agar in order to detect the phenotypic expression of curli fimbriae and cellulose. Regarding biofilm production, it was observed that 55.8% of the strains produced biofilms. In the morphological test, 88.2% of the isolates expressed one or both components at one of the temperatures at least, and 11.8% of the isolates did not express curli or cellulose. Cellulose production was significantly higher at 25 °C. On the other hand, curli production was significantly higher at 37 °C. The study data indicate that there is no association between biofilm production and in vivo pathogenicity.

Distribution of the pilS gene in Escherichia coli pathovars, its transfer ability and influence in the typical enteropathogenic E. coli adherence phenotype.

Typical enteropathogenic Escherichia coli strains (tEPEC) cause attaching/effacing lesions in eukaryotic cells and produce the bundle-forming pilus (BFP), which interweaves and aggregates bacteria, resulting in the localized adherence (LA) pattern on eukaryotic cells. Previously, we identified tEPEC strains (serotype O119:H6) that exhibited LA simultaneously with an aggregative adherence (AA)-like pattern (LA/AA-like+). Remarkably, AA is characteristically produced by strains of enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathovar. In one LA/AA-like + strain (Ec404/03), we identified a conjugative plasmid containing the pil operon, which encodes the Pil fimbriae. Moreover, a pil operon associated with an AA pattern and plasmid transfer had been previously described in the EAEC C1096 strain. In this study, we investigated the occurrence of the two pilS alleles (pilSEc404 and pilSC1096) in tEPEC strains of different serotypes, origins and years of isolation. We also examined the potential relationship of pilS with the AA-like phenotype, its ability to be transferred by conjugation, and occurrence among strains of the other E. coli pathovars. The pilS alleles were found in 90 (55.2%) of 163 tEPEC strains, with pilSEc404 occurring more often (30.7%) than pilSC1096 (25.1%). About 21 tEPEC serotypes carried pilS. The pilS alleles were found in tEPEC strains from Chile, Peru and different Brazilian cities, with the oldest strain being isolated in 1966. No absolute correlation was found between the presence of pilS and the AA-like pattern. Conjugative pilS transfer was detected in 26.2% of pilSEc404+ strains and in 65.1% of pilSC1096+ strains, but only pilSEc404+ transconjugants were AA-like+, thus suggesting that the latter allele might need a different genetic background to express this phenotype. pilS was found in all other E. coli pathovars, where it was most prevalent in enterotoxigenic E. coli. More studies are needed to understand the mechanisms involved in the regulation of Pil expression and production.