Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli, and Shigella species possess an intact G4C operon.
Cell adhesion and spreading are regulated by complex interactions involving the cytoskeleton and extracellular matrix proteins. We examined the interaction of the intermediate filament protein vimentin with the actin cross-linking protein filamin A in regulation of spreading in HEK-293 and 3T3 cells. Filamin A and vimentin-expressing cells were well spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, cells treated with small interfering RNA (siRNA) to knock down filamin A or vimentin were poorly spread; both of these cell populations exhibited >50% reductions of cell adhesion, cell surface beta1 integrin expression, and beta1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions, whereas knockdown of filamin and/or vimentin inhibited the formation of cell extensions. Reduced vimentin phosphorylation, cell spreading, and beta1 integrin surface expression, and activation were phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase C-epsilon. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins, we found an association between filamin A and vimentin. Filamin A also associated with protein kinase C-epsilon, which was enriched in cell extensions. These data indicate that filamin A associates with vimentin and to protein kinase C-epsilon, thereby enabling vimentin phosphorylation, which is important for beta1 integrin activation and cell spreading on collagen.
Cells in mechanically active environments are subjected to high-amplitude exogenous forces that can lead to cell death. Filamin A (FLNa) may protect cells from mechanically induced death by mechanisms that are not yet defined. We found that mechanical forces applied through integrins enhanced Rac-mediated lamellae formation in FLNa-null but not FLNa-expressing cells. Suppression of force-induced lamella formation was mediated by repeat 23 of FLNa, which also binds FilGAP, a recently discovered Rac GTPase-activating protein (GAP). We found that FilGAP is targeted to sites of force transfer by FLNa. This force-induced redistribution of FilGAP was essential for the suppression of Rac activity and lamellae formation in cells treated with tensile forces. Depletion of FilGAP by small interfering RNA, inhibition of FilGAP activity by dominant-negative mutation or deletion of its FLNa-binding domain, all resulted in a dramatic force-induced increase of the percentage of annexin-V-positive cells. FilGAP therefore plays a role in protecting cells against force-induced apoptosis, and this function is mediated by FLNa. INTRODUCTIONMechanical forces are important physiological regulators, from the level of molecules to the whole organism. In many types of mechanically loaded tissues, supranormal force levels increase the incidence of cell death (Cheng et al., 1995;Edwards et al., 2000;Kobayashi et al., 2000) and may also contribute to pathological states such as cardiac hypertrophy (Sadoshima and Izumo, 1997) and atherosclerosis (Thubrikar and Robicsek, 1995). Cells that are subjected to highamplitude mechanical forces in vivo undergo dramatic internal structural changes (Malek and Izumo, 1996) that enable them to maintain membrane integrity, shape, and adhesion to extracellular matrix molecules (Glogauer et al., 1997;McNeil and Steinhardt, 1997). The failure to adapt to applied mechanical stimuli leads to the tissue destruction and loss of homeostasis observed in diseases including osteoarthritis (Lin et al., 2004), lung injury (Lionetti et al., 2005), and heart failure (Cheng et al., 1995).Cells may be able to sense and adapt to environmental tension, in part through cytoskeletal adaptations. One suggested mechanism of direct transfer of forces from the extracellular matrix to the actin cytoskeleton involves integrins as mechanotransducers (Huang and Ingber, 1999;Katsumi et al., 2004;. Forces applied through integrins subsequently lead to reorganization of subcortical actin and alteration of cellular gene expression (Shyy and Chien, 1997;Chien et al., 1998;Critchley, 2000). Filamin A (FLNa) is an example of a cytoskeletal gene that is induced by mechanical forces applied through 1 integrins (D'Addario et al., 2001. FLNa belongs to a family of high-molecular mass cytoskeletal proteins that cross-link actin filaments and link actin networks to cell membranes (Cunningham et al., 1992;Stossel et al., 2001;van der Flier and Sonnenberg, 2001). FLNa has been implicated in mechanoprotection (Glogauer and Ferrier, 1998) and increa...
Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains represent a major global health problem. Their virulence is mediated by the concerted activity of an array of virulence factors including toxins, a type III protein secretion system (TTSS), pili, and others. We previously showed that EPEC O127 forms a group 4 capsule (G4C), and in this report we show that EHEC O157 also produces a G4C, whose assembly is dependent on the etp, etk, and wzy genes. We further show that at early time points postinfection, these G4Cs appear to mask surface structures including intimin and the TTSS. This masking inhibited the attachment of EPEC and EHEC to tissue-cultured epithelial cells, diminished their capacity to induce the formation of actin pedestals, and attenuated TTSS-mediated protein translocation into host cells. Importantly, we found that Ler, a positive regulator of intimin and TTSS genes, represses the expression of the capsule-related genes, including etp and etk. Thus, the expression of TTSS and G4C is conversely regulated and capsule production is diminished upon TTSS expression. Indeed, at later time points postinfection, the diminishing capsule no longer interferes with the activities of intimin and the TTSS. Notably, by using the rabbit infant model, we found that the EHEC G4C is required for efficient colonization of the rabbit large intestine. Taken together, our results suggest that temporal expression of the capsule, which is coordinated with that of the TTSS, is required for optimal EHEC colonization of the host intestine.Enterohemorrhagic Escherichia coli (EHEC) is an emerging pathogen causing outbreaks of food-borne gastroenteritis manifested by bloody diarrhea, which may progress to the potentially fatal hemolytic-uremic syndrome. The latter involves severe complications, such as renal impairment, hypertension, and central nervous system manifestations mainly caused by SLT toxins (3,22). EHEC belongs to the family of the attaching and effacing (AE)-inducing pathogens, which includes the closely related species enteropathogenic E. coli (EPEC), Citrobacter rodentium, and rabbit EPEC. When colonizing the gut, these pathogens form AE lesions on the intestinal epithelial cell surface. AE lesions are characterized by localized destruction of the brush border microvilli, intimate bacterial attachment to host cells, and the formation of actin structures, termed pedestals, beneath the attached bacteria (24). This histopathology is dependent upon a type III protein secretion system (TTSS), which functions as a molecular syringe to translocate effector proteins from the bacterial cytoplasm directly into the cytoplasm of host epithelial cells (15). These effectors subvert normal host cell functions and are required for efficient host colonization (15,34,35). One of these effectors, Tir, is inserted into the host cell membrane to form a binding site for an outer membrane adhesin, intimin. Interaction of intimin with translocated Tir promotes tight bacterial attachment to the ho...
SummaryEnteropathogenic Escherichia coli (EPEC) is a human-specific pathogen that causes severe diarrhoea in young children. The disease involves intimate interaction between the pathogen and the brush border of enterocytes. During infection, EPEC uses a type III secretion system (TTSS) to inject several proteins into the infected cells, and these effector proteins modify specific processes in the host cell. We show that, upon infection, EPEC induces detachment of the infected host cells from the substratum, modification of focal adhesions (FA) in the infected cells and specific dephosphorylation of focal adhesion kinase (FAK). We also show that EPEC-induced cell detachment is dependent on FAK expression by the infected cells. Finally, we demonstrate that cell detachment, FA modification and FAK dephosphorylation are dependent on functional TTSS in the infecting EPEC. These results suggest that EPEC is using its TTSS to inject protein(s) into the infected cells, which can induce FAK dephosphorylation, as well as FAK-dependent FA modification and cell detachment. These processes are specific and probably play an important role in EPEC virulence.
Upon infection of host cells, enteropathogenic Escherichia coli (EPEC) delivers a set of effector proteins into the host cell cytoplasm via the type III secretion system (TTSS). The effectors subvert various host cell functions. We found that EPEC interferes with the spreading and ultimately with the attachment of suspended fibroblasts or epithelial cells, and we isolated mini-Tn10kan insertion mutants that failed to similarly affect host cells. In most mutants, the insertion sites were mapped to genes encoding TTSS components, including cesD, escC, escJ, escV, espD, sepL, espB, and escF. Other mutants contained insertions in micC or upstream of bfpP, yehL, or ydeP. The insertion upstream of ydeP was associated with a reduction in TTSS protein production and was studied further. To determine whether the apparent repression was due to constitutive expression of the downstream encoded genes, ydeP and ydeO expression vectors were constructed. Expression of recombinant YdeP, YdeO, or EvgA, a positive regulator of both ydeP and ydeO, repressed TTSS protein production. Our results suggest that upon activation of the EvgAS two-component system, EvgA (the response regulator) activates both ydeP and ydeO expression and that YdeP and YdeO act conjointly, directly or indirectly repressing expression of the TTSS genes.Enteropathogenic Escherichia coli (EPEC) causes infantile diarrhea, which results in the death of several hundred thousand children each year in developing countries (10). EPEC infection is characterized by intimate binding to intestinal enterocytes, localized effacement of absorptive microvilli, transient filopodium formation, and accumulation of polymerized actin beneath the bacteria (26,46,54). This histopathology is termed the attaching and effacing lesion (18, 28). The genes responsible for attaching and effacing lesion formation are clustered in the locus of enterocyte effacement (LEE) (37). The LEE consists of 41 genes encoding a type III secretion system (TTSS), as well as several effector proteins. The LEE genes are organized in five major operons, LEE1 to LEE5 (16,17). Operons LEE2 to LEE5 are positively regulated by Ler, which is encoded by the LEE1 operon (19,38). Regulation of the LEE1 operon is complex and involves many factors, including H-NS (3, 56), integration host factor (19), Fis (21), PerC (3, 38), BipA (23), GadX (48), GrlA, and GrlR (9), as well as quorum sensing (27,49). In the closely related organism enterohemorrhagic E. coli (EHEC), the LEE operons are regulated by EtrA, EivF, RpoS, and ClpX (25, 63). For efficient attachment, EPEC uses the plasmid-encoded bundle-forming pilus (BFP) (18, 46). The BFP is encoded by two operons, perABC (bfpTVW) and bfpA-L, which contains 13 genes encoding the pilus structural genes (22, 53).E. coli K-12 has 32 two-component systems (39), one of which is the EvgAS system, whose physiological role is obscure. EvgS is the sensor histidine kinase, and EvgA is the corresponding response regulator (40,43,44). The transcriptome of an E. coli K-12 evgAS mutant...
Thalassemia is among the world's most common single gene disorders, caused primarily by gene deletions. In Israel, where o-trait thalassemia is uncommon, it is of particular importance because of its phenotypic interactions with-thalassemia in het-ero-and homozygotes. In a study of 232 individuals referred for molecular evaluation of anemia, 303 chromosomes carried-globin gene abnormalities; 6 gene rearrangements and 11 point mutations were identified. This unexpected heterogeneity is in part due to the many ethnic subgroups represented by these patients. Our findings include nine unique Israeli alleles, 3 of which are described here for the first time. An equal number of point mutations was found in the 2-globin gene as compared to 1. A threonine deletion in codon 39 of the 1-globin gene, found frequently in Arabs, is unique to Israel and probably represents one of several indigenous alleles. Among Arabs, point mutations were more frequent than large deletions. Surprisingly, in Ashkenazi Jews, who resided for many centuries in a nonmalarial environment, a single-globin gene deletion − 3.7 was found in many cases. The clinical presentation of individuals carrying two or more-globin lesions was highly variable. In general, the severity correlated inversely with the number of functional-globin genes. In some cases, impairment of two-globin genes by point mutations led to a thalassemia-intermedia-like picture which could be misdiag-nosed as-thalassemia. We conclude that-thalassemia is phenotypically and geno-typically more heterogeneous than previously recognized. DNA analysis is invaluable as it provides a specific diagnosis and enables reliable genetic counseling. Am.
Cells in mechanically challenged environments cope with high-amplitude exogenous forces that can lead to cell death, but the mechanisms that mediate force-induced apoptosis and the identity of mechanoprotective cellular factors are not defined. We assessed apoptosis in NIH 3T3 and HEK (human embryonic kidney)-293 cells exposed to tensile forces applied through β1-integrins. Apoptosis was mediated by Rac-dependent activation of p38α. Depletion of Pak1 (p21-activated kinase 1), a downstream effector of Rac, prevented force-induced p38 activation and apoptosis. Rac was recruited to sites of force transfer by filamin A, which inhibited force-induced apoptosis mediated by Rac and p38α. We conclude that, in response to tensile force, filamin A regulates Rac-dependent signals, which induce apoptosis through Pak1 and p38.
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