Pic Protein From Enteroaggregative E. coli Induces Different Mechanisms for Its Dual Activity as a Mucus Secretagogue and a Mucinase.

A hallmark of enteroaggregative Escherichia coli (EAEC) infection is the formation of an intestinal biofilm, which comprises a mucus layer with immersed bacteria. Pic is an autotransporter secreted by EAEC, and other E. coli pathotypes, and has been involved in two apparently contradictory phenotypes, as a mucus secretagogue and as a mucinase. Here, we investigated this Pic dual activity, mucus secretagogue capability and mucinolytic activity, in human goblet cells that secrete MUC2 and MUC5AC. Pic induced mucus hypersecretion directly in the goblet cells, without other intestinal cell types involved. At the same time, Pic exhibited strong proteolytic activity on the secreted mucins. These activities were independent since a mutation in the serine protease motif (PicS258I) abolished mucin degradation while maintaining the mucus secretagogue activity intact. Furthermore, deoxycholic acid (DCA)-induced mucins were proteolytically degraded when goblet cells were co-incubated with DCA/Pic, while co-incubation with DCA/PicS258I induced a synergistic effect on mucus hypersecretion. Pic was more efficient degrading MUC5AC than MUC2, but no degradation was detected with Pic inactivated at the active site by mutation or pharmacological inhibition. Remarkably, Pic cleaved MUC2 and MUC5AC in the C-terminal domain, leaving N-terminal subproducts, impacting the feature of gel-forming mucins and allowing mucus layer penetration by EAEC. Astonishingly, Pic stimulated rapid mucin secretion in goblet-like cells by activating the intracellular calcium pathway resulting from the PLC signal transduction pathway, leading to the production of DAG and releasing IP3, a second messenger of calcium signaling. Therefore, the dual activity of Pic, as a mucus secretagogue and a mucinase, is relevant in the context of carbon source generation and mucus layer penetration, allowing EAEC to live within the layer of mucus but also access epithelial cells.

Cytolethal distending toxin-producing Escherichia coli strains causing severe diarrhoea in young Mexican children.

Introduction. Cytolethal distending toxins (CDTs), encoded by cdt genes, have DNase activity leading to cellular and nuclear distension, resulting in irreversible cell cycle arrest and apoptosis of target cells. cdt-positive Escherichia coli strains have been isolated from children with diarrhoea. There is, however, scant information on the prevalence and clinical presentation of diarrhoeal disease caused by these strains. Furthermore, toxin production of cdt-positive strains is rarely confirmed. We report five young children with diarrhoea caused by CDT-producing E. coli in whom stools were negative for other bacterial or enteric pathogens. Case presentation. On admission to hospital, all children presented watery diarrhoea with high stool output (range 7-20 stools/24 h); five had fever of 38 °C or more and four presented vomiting. Dehydration was present in four patients, one of whom had hypovolaemic shock; one child also presented hyponatraemia and hypokalaemia. In two children, cdt-positive strains were classified as typical and atypical enteropathogenic E. coli, and the remaining three harboured cdt-positive strains that did not belong to any diarrhoeagenic pathogroup. One cdt-positive strain from each case was characterized by a CDT cytotoxic assay and a cdt type-specific PCR. All strains produced the characteristic cellular intoxication due to CDT. Two strains carried the cdt-I, one cdt-III, one cdt-IV, and one concurrently had cdt-I, cdt-II and cdt-III genes. Conclusion. Our results suggest that CDT-producing E. coli strains are an infrequent, albeit significant, cause of severe diarrhoeal illness in children. Future research should measure the true burden of cdt-positive E. coli diarrhoea among children.

A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins.

The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia, require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens.IMPORTANCE The type 3 secretion system is essential for injection of virulence factors, which are delivered directly into the cytosol of the host cells for usurping and subverting host processes. Recent studies have shown that these effectors proteins indeed travel inside an "injectisome" conduit through a single step of translocation by connecting the bacterium and host cell cytoplasms. However, all findings are not compatible with this model. For example, both YopH, a protein detected on the surface of Yersinia, and EspC, an autotransporter protein secreted by enteropathogenic E. coli, require a functional T3SS for host cell translocation. Both proteins have an intermediate extracellular step before their T3SS-dependent translocation. Here, we show an alternative delivery mechanism for these extracellularly secreted virulence factors that are then incorporated into the T3SS to enter the cells; this novel mechanism coexists with but diverges from the canonical injection model that involves the passage of the protein inside the injectisome.

Subdomain 2 of the Autotransporter Pet Is the Ligand Site for Recognizing the Pet Receptor on the Epithelial Cell Surface.

Most autotransporter passenger domains, regardless of their diversity in function, fold or are predicted to fold as right-handed ß-helices carrying various loops that are presumed to confer functionality. Our goal here was to identify the subdomain (loop) or amino acid sequence of the Pet passenger domain involved in the receptor binding site on the host cell for Pet endocytosis. Here, we show that d1 and d2 subdomains, as well as the amino acid sequence linking the subdomain d2 and the adjacent ß-helix (PDWET), are not required for Pet secretion through the autotransporter system and that none of our deletion mutants altered the predicted long right-handed ß-helical structure. Interestingly, Pet lacking the d2 domain (PetΔd2) was unable to bind on the epithelial cell surface, in contrast to Pet lacking d1 (PetΔd1) subdomain or PDWET sequences. Moreover, the purified d1 subdomain, the biggest subdomain (29.8 kDa) containing the serine protease domain, was also unable to bind the cell surface. Thus, d2 sequence (54 residues without the PDWET sequence) was required for Pet binding to eukaryotic cells. In addition, this d2 sequence was also needed for Pet internalization but not for inducing cell damage. In contrast, PetΔd1, which was able to bind and internalize inside the cell, was unable to cause cell damage. Furthermore, unlike Pet, PetΔd2 was unable to bind cytokeratin 8, a Pet receptor. These data indicate that the surface d2 subdomain is essential for the ligand-receptor (Pet-Ck8) interaction for Pet uptake and to start the epithelial cell damage by this toxin.

The serine protease Pic as a virulence factor of atypical enteropathogenic Escherichia coli.

Autotransporter proteins (AT) are associated with bacterial virulence attributes. Originally identified in enteroaggregative Escherichia coli (EAEC), Shigella flexneri 2a and uropathogenic E. coli, the serine protease Pic is one of these AT. We have previously detected one atypical enteropathogenic E. coli strain (BA589) carrying the pic gene. In the present study, we characterized the biological activities of Pic produced by BA589 both in vitro and in vivo. Contrarily to other Pic-producers bacteria, pic in BA589 is located on a high molecular weight plasmid. PicBA589 was able to agglutinate rabbit erythrocytes, cleave mucin and degrade complement system molecules. BA589 was able to colonize mice intestines, and an intense mucus production was observed. The BA589Δpic mutant lost the capacity to colonize as well as the above-mentioned in vitro activities. Thus, Pic represents an additional virulence factor in aEPEC strain BA589, associated with adherence, colonization and evasion from the innate immune system.

Actin cytoskeleton manipulation by effector proteins secreted by diarrheagenic Escherichia coli pathotypes.

The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology.

VirK is a periplasmic protein required for efficient secretion of plasmid-encoded toxin from enteroaggregative Escherichia coli.

Despite the autotransporter (AT) moniker, AT secretion appears to involve the function of periplasmic chaperones. We identified four periplasmic proteins that specifically bound to plasmid-encoded toxin (Pet), an AT produced by enteroaggregative Escherichia coli (EAEC). These proteins include the 17-kDa Skp chaperone and the 37-kDa VirK protein. We found that the virK gene is present in different Enterobacteriaceae. VirK bound to misfolded conformations of the Pet passenger domain, but it did not bind to the folded passenger domain or to the ß domain of Pet. Assays with an EAECΔvirK mutant and its complemented version showed that, in the absence of VirK, Pet was not secreted but was instead retained in the periplasm as proteolytic fragments. In contrast, Pet was secreted from a Δskp mutant. VirK was not required for the insertion of porin proteins into the outer membrane but assisted with insertion of the Pet ß domain into the outer membrane. Loss of VirK function blocked the EAEC-mediated cytotoxic effect against HEp-2 cells. Thus, VirK facilitates the secretion of the AT Pet by maintaining the passenger domain in a conformation that both avoids periplasmic proteolysis and facilitates ß-domain insertion into the outer membrane.

Progressive paralysis associated with diffuse astrocyte anaplasia in delta 202 mice homozygous for a transgene encoding the SV40 T antigen.

A convenient transgenic astrocytoma model in delta202 mice, homozygous for a construct encoding the early region of the SV40 virus genome, is described. In the offspring of crosses between delta202 mice heterozygous for the transgene nearly 60% were transgenic; one third of these developed progressive paralysis starting in the hindlimbs at approximately 35 days of age and died at 90 +/- 30 days of age. In affected mice proliferating-non-neuronal cells immunostained with antibodies to the GFAP, an astrocyte marker, whose number increased with age were found in the white matter of the brain, cerebellum and spinal cord, and progressive degeneration and necrosis of spinal motoneurons was observed that-may explain the paralysis. The early onset and reproducible time course of the neurological disease suggest that homozygous delta202 mice, whose proliferating astrocytes appear to damage spinal motoneurons, are a useful model to study astrocyte differentiation, function and tumorigenesis.