MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira, has an essential role during infection

Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch’s molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen’s ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease.

Adhesin related genes as potential markers for the enteroaggregative Escherichia coli category

Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea in children and adults worldwide. This pathotype is phenotypically characterized by the aggregative-adherence (AA) pattern in HEp-2 cells and genetically associated to the presence of the aatA gene. EAEC pathogenesis relies in different virulence factors. At least, three types of adhesins have been specifically associated with EAEC strains: the five variants of the aggregative adherence fimbriae (AAF), the aggregative forming pilus (AFP) and more recently, a fibrilar adhesin named CS22. Our study aimed to evaluate the presence of AAF, AFP and CS22-related genes among 110 EAEC strains collected from feces of children with diarrhea. The presence of aggR (EAEC virulence regulator) and genes related to AAFs (aggA, aafA, agg3A, agg4A, agg5A and agg3/4C), AFP (afpA1 and afpR) and CS22 (cseA) was detected by PCR, and the adherence patterns were evaluated on HeLa cells. aggR-positive strains comprised 83.6% of the collection; among them, 80.4% carried at least one AAF-related gene and presented the AA pattern. aggA was the most frequent AAF-related gene (28.4% of aggR+ strains). cseA was detected among aggR+ (16.3%) and aggR- strains (22.2%); non-adherent strains or strains presenting AA pattern were observed in both groups. afpR and afpA1 were exclusively detected among aggR- strains (77.8%), most of which (71.4%) also presented AA pattern. Our results indicate that AAF- and AFP-related genes may contribute to identify EAEC strains, while the presence of cseA and its importance as an EAEC virulence factor and genotypic marker needs to be further evaluated.

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 aggregate-forming pili (AFP) mediates the aggregative adherence of a hybrid-pathogenic Escherichia coli (UPEC/EAEC) isolated from a urinary tract infection

Enteroaggregative Escherichia coli (EAEC) comprises an important diarrheagenic pathotype, while uropathogenic E. coli (UPEC) is the most important agent of urinary tract infection (UTI). Recently, EAEC virulence factors have been detected in E. coli strains causing UTI, showing the importance of these hybrid-pathogenic strains. Previously, we detected an E. coli strain isolated from UTI (UPEC-46) presenting characteristics of EAEC, e.g., the aggregative adherence (AA) pattern and EAEC-associated genes (aatA, aap, and pet). In this current study, we analyzed the whole genomic sequence of UPEC-46 and characterized some phenotypic traits. The AA phenotype was observed in cell lineages of urinary and intestinal origin. The production of curli, cellulose, bacteriocins, and Pet toxin was detected. Additionally, UPEC-46 was not capable of forming biofilm using different culture media and human urine. The genome sequence analysis showed that this strain belongs to serotype O166:H12, ST10, and phylogroup A, harbors the tet, aadA, and dfrA/sul resistance genes, and is phylogenetically more related to EAEC strains isolated from human feces. UPEC-46 harbors three plasmids. Plasmid p46-1 (~135 kb) carries some EAEC marker genes and those encoding the aggregate-forming pili (AFP) and its regulator (afpR). A mutation in afpA (encoding the AFP major pilin) led to the loss of pilin production and assembly, and notably, a strongly reduced adhesion to epithelial cells. In summary, the genetic background and phenotypic traits analyzed suggest that UPEC-46 is a hybrid strain (UPEC/EAEC) and highlights the importance of AFP adhesin in the adherence to colorectal and bladder cell lines.

EspFu-mediated actin assembly enhances enteropathogenic escherichia coli adherence and activates host cell inflammatory signaling pathways

The translocation of effectors into the host cell through type 3 secretion systems (T3SS) is a sophisticated strategy employed by pathogenic bacteria to subvert host responses and facilitate colonization. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) utilize the Tir and EspFu (also known as TccP) effectors to remodel the host cytoskeleton, culminating in the formation of attaching and effacing (AE) lesions on enterocytes. While some EPEC strains require tyrosine phosphorylation of Tir and recruitment of the host Nck to trigger actin polymerization, EHEC and certain EPEC strains, whose Tir is not phosphorylated, rely on the effector EspFu for efficient actin remodeling. Here, we investigated the role played by Tir-Nck and Tir-EspFu actin polymerization pathways during the infection of epithelial cells, as well as the host transcriptional response to the AE lesion formation induced by EPEC. We found that EspFu-mediated actin assembly promotes bacterial attachment and epithelial colonization more efficiently than Tir-Nck. Moreover, we showed that both actin polymerization mechanisms can activate inflammatory pathways and reverse the anti-inflammatory response induced by EPEC in epithelial cells. However, this activity is remarkably more evident in infections with EspFuexpressing EPEC strains. This study demonstrates the complex interactions between effector-mediated actin remodeling and inflammation. Different strains carry different combinations of these two effectors, highlighting the plasticity of pathogenic E. coli enteric infections. IMPORTANCE EPEC is among the leading causes of diarrheal disease worldwide. The colonization of the gut mucosa by EPEC results in actin pedestal formation at the site of bacterial attachment. These pedestals are referred to as attaching and effacing (AE) lesions. Here, we exploit the different molecular mechanisms used by EPEC to induce AE lesions on epithelial cells, showing that the effector EspFu is associated with increased bacterial attachment and enhanced epithelial colonization compared to the Tir-Nck pathway. Moreover, we also showed that actin pedestal formation can counterbalance the anti-inflammatory activity induced by EPEC, especially when driven by EspFu. Collectively, our findings provide new insights into virulence mechanisms employed by EPEC to colonize epithelial cells, as well as the host response to this enteric pathogen.

Interactions of Shiga toxin-producing Escherichia coli with leafy green vegetables

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens responsible for a wide spectrum of diseases including diarrhea, bloody diarrhea, and hemolytic uremic syndrome (HUS). A considerable number of outbreaks and sporadic cases of HUS have been associated with ingestion of fresh ready-to-eat products. Maintenance and persistence of STEC in the environment and foods can be related to its ability to form biofilm. A non-O157 STEC strain isolated from bovine feces was distinguished by its great ability to form biofilm in abiotic surfaces. In the present study, we aimed to investigate the ability of this strain to adhere to rocket leaves (Eruca sativa). Adherence assays were carried out for 3 h at 28 °C and analyzed by scanning electron microscopy. The non-O157 STEC strain adhered to leaf surface and inside the stomata forming several bacterial aggregates. The number of adherent bacteria per square millimeter of leaf was eightfold higher compared with an O157 STEC strain. Deletion of the STEC autotransporter protein contributing to biofilm (Sab) reduced the adherence ability of the non-O157 strain in almost 50%, and deletion of antigen 43 (Ag43) almost abolished this interaction. Very few bacteria were seen on the leaf surface, and these differences were statistically significant, suggesting the role of both proteins and especially Ag43 in the interaction of the non-O157 STEC strain with leaves. The risk posed by non-O157 STEC adherence to leaves on fresh produce contamination should not be neglected, and measures that effectively control adherence should be included in strategies to control non-O157 STEC.

EspFu-mediated actin assembly enhances enteropathogenic escherichia coli adherence and activates host cell inflammatory signaling pathways

The translocation of effectors into the host cell through type 3 secretion systems (T3SS) is a sophisticated strategy employed by pathogenic bacteria to subvert host responses and facilitate colonization. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) utilize the Tir and EspFu (also known as TccP) effectors to remodel the host cytoskeleton, culminating in the formation of attaching and effacing (AE) lesions on enterocytes. While some EPEC strains require tyrosine phosphorylation of Tir and recruitment of the host Nck to trigger actin polymerization, EHEC and certain EPEC strains, whose Tir is not phosphorylated, rely on the effector EspFu for efficient actin remodeling. Here, we investigated the role played by Tir-Nck and Tir-EspFu actin polymerization pathways during the infection of epithelial cells, as well as the host transcriptional response to the AE lesion formation induced by EPEC. We found that EspFu-mediated actin assembly promotes bacterial attachment and epithelial colonization more efficiently than Tir-Nck. Moreover, we showed that both actin polymerization mechanisms can activate inflammatory pathways and reverse the anti-inflammatory response induced by EPEC in epithelial cells. However, this activity is remarkably more evident in infections with EspFuexpressing EPEC strains. This study demonstrates the complex interactions between effector-mediated actin remodeling and inflammation. Different strains carry different combinations of these two effectors, highlighting the plasticity of pathogenic E. coli enteric infections. IMPORTANCE EPEC is among the leading causes of diarrheal disease worldwide. The colonization of the gut mucosa by EPEC results in actin pedestal formation at the site of bacterial attachment. These pedestals are referred to as attaching and effacing (AE) lesions. Here, we exploit the different molecular mechanisms used by EPEC to induce AE lesions on epithelial cells, showing that the effector EspFu is associated with increased bacterial attachment and enhanced epithelial colonization compared to the Tir-Nck pathway. Moreover, we also showed that actin pedestal formation can counterbalance the anti-inflammatory activity induced by EPEC, especially when driven by EspFu. Collectively, our findings provide new insights into virulence mechanisms employed by EPEC to colonize epithelial cells, as well as the host response to this enteric pathogen.

Interactions of Shiga toxin-producing Escherichia coli with leafy green vegetables

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens responsible for a wide spectrum of diseases including diarrhea, bloody diarrhea, and hemolytic uremic syndrome (HUS). A considerable number of outbreaks and sporadic cases of HUS have been associated with ingestion of fresh ready-to-eat products. Maintenance and persistence of STEC in the environment and foods can be related to its ability to form biofilm. A non-O157 STEC strain isolated from bovine feces was distinguished by its great ability to form biofilm in abiotic surfaces. In the present study, we aimed to investigate the ability of this strain to adhere to rocket leaves (Eruca sativa). Adherence assays were carried out for 3 h at 28 °C and analyzed by scanning electron microscopy. The non-O157 STEC strain adhered to leaf surface and inside the stomata forming several bacterial aggregates. The number of adherent bacteria per square millimeter of leaf was eightfold higher compared with an O157 STEC strain. Deletion of the STEC autotransporter protein contributing to biofilm (Sab) reduced the adherence ability of the non-O157 strain in almost 50%, and deletion of antigen 43 (Ag43) almost abolished this interaction. Very few bacteria were seen on the leaf surface, and these differences were statistically significant, suggesting the role of both proteins and especially Ag43 in the interaction of the non-O157 STEC strain with leaves. The risk posed by non-O157 STEC adherence to leaves on fresh produce contamination should not be neglected, and measures that effectively control adherence should be included in strategies to control non-O157 STEC.

Dynamic gene network analysis of Caco-2 cell response to Shiga toxin-producing Escherichia coli-associated Hemolytic–Uremic Syndrome

Shiga toxin-producing Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea and some strains may cause hemolytic–uremic syndrome (HUS). In Brazil, these strains are commonly found in cattle but, so far, were not isolated from HUS patients. Here, a system biology approach was used to investigate the differential transcriptomic and phenotypic responses of enterocyte-like Caco-2 cells to two STEC O113:H21 strains with similar virulence factor profiles (i.e., expressing stx2, ehxA, epeA, espA, iha, saa, sab, and subA): EH41 (Caco-2/EH41), isolated from a HUS patient in Australia, and Ec472/01 (Caco-2/Ec472), isolated from bovine feces in Brazil, during a 3 h period of bacteria–enterocyte interaction. Gene co-expression network analysis for Caco-2/EH41 revealed a quite abrupt pattern of topological variation along 3 h of enterocyte–bacteria interaction when compared with networks obtained for Caco-2/Ec472. Transcriptional module characterization revealed that EH41 induces inflammatory and apoptotic responses in Caco-2 cells just after the first hour of enterocyte–bacteria interaction, whereas the response to Ec472/01 is associated with cytoskeleton organization at the first hour, followed by the expression of immune response modulators. Scanning electron microscopy showed more intense microvilli destruction in Caco-2 cells exposed to EH41 when compared to those exposed to Ec472/01. Altogether, these results show that EH41 expresses virulence genes, inducing a distinctive host cell response, and is likely associated with severe pathogenicity.

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.

Dynamic gene network analysis of Caco-2 cell response to Shiga toxin-producing Escherichia coli-associated Hemolytic–Uremic Syndrome

Shiga toxin-producing Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea and some strains may cause hemolytic–uremic syndrome (HUS). In Brazil, these strains are commonly found in cattle but, so far, were not isolated from HUS patients. Here, a system biology approach was used to investigate the differential transcriptomic and phenotypic responses of enterocyte-like Caco-2 cells to two STEC O113:H21 strains with similar virulence factor profiles (i.e., expressing stx2, ehxA, epeA, espA, iha, saa, sab, and subA): EH41 (Caco-2/EH41), isolated from a HUS patient in Australia, and Ec472/01 (Caco-2/Ec472), isolated from bovine feces in Brazil, during a 3 h period of bacteria–enterocyte interaction. Gene co-expression network analysis for Caco-2/EH41 revealed a quite abrupt pattern of topological variation along 3 h of enterocyte–bacteria interaction when compared with networks obtained for Caco-2/Ec472. Transcriptional module characterization revealed that EH41 induces inflammatory and apoptotic responses in Caco-2 cells just after the first hour of enterocyte–bacteria interaction, whereas the response to Ec472/01 is associated with cytoskeleton organization at the first hour, followed by the expression of immune response modulators. Scanning electron microscopy showed more intense microvilli destruction in Caco-2 cells exposed to EH41 when compared to those exposed to Ec472/01. Altogether, these results show that EH41 expresses virulence genes, inducing a distinctive host cell response, and is likely associated with severe pathogenicity.

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.

Novel Hybrid of Typical Enteropathogenic Escherichia coli and Shiga-Toxin-Producing E. coli (tEPEC/STEC) Emerging From Pet Birds.

Exotic psittacine birds have been implicated as reservoir of diarrheagenic Escherichia coli (E. coli), including enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Here, we present a genotypic and phenotypic characterization of typical EPEC/STEC hybrid strains isolated from exotic psittacine birds. The strains were positive for eae, bfpA, and stx2f genes, belong to serotype O137:H6 and ST2678. Two strains were subject to whole genome sequencing, confirming the presence of the virulence factors of both E. coli pathotypes. Phenotypical in vitro tests confirmed their ability to adhere to HeLa cells and cause cytotoxicity to Vero cells. The rabbit ileal loop assays showed the attaching and effacing lesion, in addition to inflammatory process and overproduction of intestinal mucus. This is the first report of hybrid typical EPEC/STEC (O137:H6/ST2678) strains isolated from companion psittacine birds and the results suggest zoonotic risks.

Novel hybrid of Typical Enteropathogenic Escherichia coli and Shiga-Toxin-Producing E. coli (tEPEC/STEC) emerging from pet birds

Exotic psittacine birds have been implicated as reservoir of diarrheagenic Escherichia coli (E. coli), including enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Here, we present a genotypic and phenotypic characterization of typical EPEC/STEC hybrid strains isolated from exotic psittacine birds. The strains were positive for eae, bfpA, and stx2f genes, belong to serotype O137:H6 and ST2678. Two strains were subject to whole genome sequencing, confirming the presence of the virulence factors of both E. coli pathotypes. Phenotypical in vitro tests confirmed their ability to adhere to HeLa cells and cause cytotoxicity to Vero cells. The rabbit ileal loop assays showed the attaching and effacing lesion, in addition to inflammatory process and overproduction of intestinal mucus. This is the first report of hybrid typical EPEC/STEC (O137:H6/ST2678) strains isolated from companion psittacine birds and the results suggest zoonotic risks.

Distribution of major pilin subunit genes among atypical enteropathogenic Escherichia coli and influence of growth media on expression of the ecp operon

Atypical enteropathogenic Escherichia coil (aEPEC) strains are unable to produce the bundle-forming pilus (BFP), which is responsible for the localized adherence pattern, a characteristic of the pathogenicity of typical EPEC strains. The lack of BFP in aEPEC strains suggests that other fimbrial or non-fimbrial adhesins are involved in their adhesion to the host cells. The aim of this study was to investigate the distribution of major subunit fimbrial genes known to be important adherence factors produced by several E. coil pathotypes in a collection of 72 aEPEC strains. Our results demonstrate that a high percentage (94-100%) of aEPEC strains harbored ecpA, fimA, hcpA, and lpfA fimbrial genes. Other fimbrial genes including pilS, pilV, sfpA, daaC, papA, and sfa were detected at lower frequencies (1-8%). Genes encoding fimbrial subunits, which are characteristic of enteroaggregative E. coli or enterotoxigenic E. coli were not found. No correlation was found between fimbrial gene profiles and adherence phenotypes. Since all aEPEC strains contained ecpA, the major pilin gene of the E. coil common pilus (ECP), a subset of ecpA+ strains was analyzed for transcription of ecpRABCDE and production of ECP upon growth in three different culture conditions at 37 degrees C. Transcription of ecpRABCDE occurred in all conditions; however, ECP production was medium dependent. In all, the data suggest that aEPEC strains are highly heterogeneous in terms of their fimbrial gene profiles. Despite lacking BFP production, other mechanisms of cell adherence exist in aEPEC strains to ensure host colonization, e.g., mediated by other prevalent pili such as ECP. Moreover, the production of ECP by aEPEC strains might be influenced by yet unknown post-transcriptional factors.

Novel hybrid of Typical Enteropathogenic Escherichia coli and Shiga-Toxin-Producing E. coli (tEPEC/STEC) emerging from pet birds

Exotic psittacine birds have been implicated as reservoir of diarrheagenic Escherichia coli (E. coli), including enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Here, we present a genotypic and phenotypic characterization of typical EPEC/STEC hybrid strains isolated from exotic psittacine birds. The strains were positive for eae, bfpA, and stx2f genes, belong to serotype O137:H6 and ST2678. Two strains were subject to whole genome sequencing, confirming the presence of the virulence factors of both E. coli pathotypes. Phenotypical in vitro tests confirmed their ability to adhere to HeLa cells and cause cytotoxicity to Vero cells. The rabbit ileal loop assays showed the attaching and effacing lesion, in addition to inflammatory process and overproduction of intestinal mucus. This is the first report of hybrid typical EPEC/STEC (O137:H6/ST2678) strains isolated from companion psittacine birds and the results suggest zoonotic risks.

Distribution of major pilin subunit genes among atypical enteropathogenic Escherichia coli and influence of growth media on expression of the ecp operon

Atypical enteropathogenic Escherichia coil (aEPEC) strains are unable to produce the bundle-forming pilus (BFP), which is responsible for the localized adherence pattern, a characteristic of the pathogenicity of typical EPEC strains. The lack of BFP in aEPEC strains suggests that other fimbrial or non-fimbrial adhesins are involved in their adhesion to the host cells. The aim of this study was to investigate the distribution of major subunit fimbrial genes known to be important adherence factors produced by several E. coil pathotypes in a collection of 72 aEPEC strains. Our results demonstrate that a high percentage (94-100%) of aEPEC strains harbored ecpA, fimA, hcpA, and lpfA fimbrial genes. Other fimbrial genes including pilS, pilV, sfpA, daaC, papA, and sfa were detected at lower frequencies (1-8%). Genes encoding fimbrial subunits, which are characteristic of enteroaggregative E. coli or enterotoxigenic E. coli were not found. No correlation was found between fimbrial gene profiles and adherence phenotypes. Since all aEPEC strains contained ecpA, the major pilin gene of the E. coil common pilus (ECP), a subset of ecpA+ strains was analyzed for transcription of ecpRABCDE and production of ECP upon growth in three different culture conditions at 37 degrees C. Transcription of ecpRABCDE occurred in all conditions; however, ECP production was medium dependent. In all, the data suggest that aEPEC strains are highly heterogeneous in terms of their fimbrial gene profiles. Despite lacking BFP production, other mechanisms of cell adherence exist in aEPEC strains to ensure host colonization, e.g., mediated by other prevalent pili such as ECP. Moreover, the production of ECP by aEPEC strains might be influenced by yet unknown post-transcriptional factors.

A hemolytic-uremic syndrome-associated strain O113:H21 Shiga toxin-producing Escherichia coli specifically expresses a transcriptional module containing dicA and is related to gene network dysregulation in Caco-2 cells

Shiga toxin-producing (Stx) Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea and some of these strains may cause hemolytic uremic syndrome (HUS). The molecular mechanism underlying this capacity and the differential host cell response to HUS-causing strains are not yet completely understood. In Brazil O113:H21 strains are commonly found in cattle but, so far, were not isolated from HUS patients. Here we conducted comparative gene co-expression network (GCN) analyses of two O113:H21 STEC strains:EH41, reference strain, isolated from HUS patient in Australia, and Ec472/01, isolated from cattle feces in Brazil. These strains were cultured in fresh or in Caco-2 cell conditioned media. GCN analyses were also accomplished for cultured Caco-2 cells exposed to EH41 or Ec472/01. Differential transcriptome profiles for EH41 and Ec472/01 were not significantly changed by exposure to fresh or Caco-2 conditioned media. Conversely, global gene expression comparison of both strains cultured in conditioned medium revealed a gene set exclusively expressed in EH41, which includes the dicA putative virulence factor regulator. Network analysis showed that this set of genes constitutes an EH41 specific transcriptional module. PCR analysis in Ec472/01 and in other 10 Brazilian cattle-isolated STEC strains revealed absence of dicA in all these strains. The GCNs of Caco-2 cells exposed to EH41 or to Ec472/01 presented a major transcriptional module containing many hubs related to inflammatory response that was not found in the GCN of control cells. Moreover, EH41 seems to cause gene network dysregulation in Caco-2 as evidenced by the large number of genes with high positive and negative covariance interactions. EH41 grows slowly than Ec472/01 when cultured in Caco-2 conditioned medium and fitness-related genes are hypoexpressed in that strain. Therefore, EH41 virulence may be derived from its capacity for dysregulating enterocyte genome functioning and its enhanced enteric survival due to slow growth.

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.

Pathogenic Leptospira species acquire factor H and vitronectin via the surface protein LcpA.

Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion.