The Afa/Dr adhesins of diffusely adhering Escherichia coli stimulate interleukin-8 secretion,activate mitogen-activated protein kinases,and promote polymorphonuclear transepithelial migration in T84 polarized epithelial cells

Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and the E. coli laboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody that recognized the short consensus repeat 3 domain of DAF (monoclonal antibody 1H4). Moreover, Afa/Dr DAEC strains induced tyrosine phosphorylation of several T84 proteins and activated the mitogen-activated protein kinases (ERK1/2 mitogen-activated protein, P38, and Jun-C kinases). These data demonstrated for the first time that, in vitro, Afa/Dr DAEC strains exert a proinflammatory signal in intestinal epithelial cells.     

Pathogenesis of Afa/Dr diffusely adhering Escherichia coli

Over the last few years, dramatic increases in our knowledge about diffusely adhering Escherichia coli (DAEC) pathogenesis have taken place. The typical class of DAEC includes E. coli strains harboring AfaE-I, AfaE-II, AfaE-III, AfaE-V, Dr, Dr-II, F1845, and NFA-I adhesins (Afa/Dr DAEC); these strains (i) have an identical genetic organization and (ii) allow binding to human decay-accelerating factor (DAF) (Afa/Dr(DAF) subclass) or carcinoembryonic antigen (CEA) (Afa/Dr(CEA) subclass). The atypical class of DAEC includes two subclasses of strains; the atypical subclass 1 includes E. coli strains that express AfaE-VII, AfaE-VIII, AAF-I, AAF-II, and AAF-III adhesins, which (i) have an identical genetic organization and (ii) do not bind to human DAF, and the atypical subclass 2 includes E. coli strains that harbor Afa/Dr adhesins or others adhesins promoting diffuse adhesion, together with pathogenicity islands such as the LEE pathogenicity island (DA-EPEC). In this review, the focus is on Afa/Dr DAEC strains that have been found to be associated with urinary tract infections and with enteric infection. The review aims to provide a broad overview and update of the virulence aspects of these intriguing pathogens. Epidemiological studies, diagnostic techniques, characteristic molecular features of Afa/Dr operons, and the respective role of Afa/Dr adhesins and invasins in pathogenesis are described. Following the recognition of membrane-bound receptors, including type IV collagen, DAF, CEACAM1, CEA, and CEACAM6, by Afa/Dr adhesins, activation of signal transduction pathways leads to structural and functional injuries at brush border and junctional domains and to proinflammatory responses in polarized intestinal cells. In addition, uropathogenic Afa/Dr DAEC strains, following recognition of beta(1) integrin as a receptor, enter epithelial cells by a zipper-like, raft- and microtubule-dependent mechanism. Finally, the presence of other, unknown virulence factors and the way that an Afa/Dr DAEC strain emerges from the human intestinal microbiota as a "silent pathogen" are discussed.     

Recruitment of CD55 and CD66e brush border-associated glycosylphosphatidylinositol-anchored proteins by members of the Afa/Dr diffusely adhering family of Escherichia coli that infect the human polarized intestinal Caco-2/TC7 cells

The Afa/Dr family of diffusely adhering Escherichia coli (Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structural draE gene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.     

Afa/Dr diffusely adhering Escherichia coli infection in T84 cell monolayers induces increased neutrophil transepithelial migration,which in turn promotes cytokine-dependent upregulation of decay-accelerating factor (CD55), the receptor for Afa/Dr adhesins

Ulcerative colitis and Crohn's disease are inflammatory bowel diseases thought to involve strains of Escherichia coli. We report here that two wild-type Afa/Dr diffusely adhering E. coli (DAEC) strains, C1845 and IH11128, which harbor the fimbrial F1845 adhesin and the Dr hemagglutinin, respectively, and the E. coli laboratory strain HB101, transformed with the pSSS1 plasmid to produce Afa/Dr F1845 adhesin, all induced interleukin-8 (IL-8) production and transepithelial migration of polymorphonuclear leukocytes (PMNL) in polarized monolayers of the human intestinal cell line T84 grown on semipermeable filters. We observed that after PMNL migration, expression of decay-accelerating factor (DAF, or CD55), the brush border-associated receptor for Afa/Dr adhesins, was strongly enhanced, increasing the adhesion of Afa/Dr DAEC bacteria. When examining the mechanism by which DAF expression was enhanced, we observed that the PMNL transepithelial migration induced epithelial synthesis of tumor necrosis factor alpha and IL-1beta, which in turn promoted the upregulation of DAF.     

Pyelonephritogenic diffusely adhering Escherichia coli EC7372 harboring Dr-II adhesin carries classical uropathogenic virulence genes and promotes cell lysis and apoptosis in polarized epithelial caco-2/TC7 cells

Diffusely adhering Escherichia coli (DAEC) strains expressing adhesins of the Afa/Dr family bind to epithelial cells in a diffuse adherence pattern by recognizing a common receptor, the decay-accelerating factor (CD55). Recently, a novel CD55-binding adhesin, named Dr-II, was identified from the pyelonephritogenic strain EC7372. In this report, we show that despite the low level of sequence identity between Dr-II and other members of the Afa/Dr family, EC7372 induces pathophysiological effects similar to those induced by other Afa/Dr DAEC strains on the polarized epithelial cell line Caco-2/TC7. Specifically, the Dr-II adhesin was sufficient to promote CD55 and CD66e clustering around adhering bacteria and apical cytoskeleton rearrangements. Unlike other Afa/Dr DAEC strains, EC7372 expresses a functional hemolysin that promotes a rapid cellular lysis. In addition, cell death by apoptosis or necrosis was observed in EC7372-infected Caco-2/TC7 cells, depending on infection time. Our results indicate that EC7372 harbors a pathogenicity island (PAI) similar to the one described for the pyelonephritogenic strain CFT073, which carries both hly and pap operons. Cumulatively, our findings indicate that strain EC7372 can be considered a prototype of a subclass of Afa/Dr DAEC isolates that have acquired a PAI harboring several classical uropathogenic virulence genes.     

Representational difference analysis between Afa/Dr diffusely adhering Escherichia coli and nonpathogenic E. coli K-12

Diffusely adhering Escherichia coli strains harboring Afa/Dr adhesins (Afa/Dr DAEC) have been associated with diarrhea and urinary tract infections (UTIs). The present work is the first extensive molecular study of a Afa/Dr DAEC strain using the representational difference analysis technique. We have searched for DNA sequences present in strain C1845, recovered from a diarrheagenic child, but absent from a nonpathogenic K-12 strain. Strain C1845 harbors part of a pathogenicity island (PAI(CFT073)) and several iron transport systems found in other E. coli pathovars. We did not find genes encoding factors known to subvert host cell proteins, such as type III secretion system or effector proteins. Several C1845-specific sequences are homologous to putative virulence genes or show no homology with known sequences, and we have analyzed their distribution among Afa/Dr and non-Afa/Dr clinical isolates and among strains from the E. coli Reference Collection. Three C1845-specific sequences (MO30, S109, and S111) have a high prevalence (77 to 80%) among Afa/Dr strains and a low prevalence (12 to 23%) among non-Afa/Dr strains. In addition, our results indicate that strain IH11128, an Afa/Dr DAEC strain recovered from a patient with a UTI, is genetically closely related to strain C1845.     

Afa/Dr-Expressing,Diffusely Adhering Escherichia coli Strain C1845 Triggers F1845 Fimbria-Dependent Phosphatidylserine Externalization on Neutrophil-Like Differentiated PLB-985 Cells through an Apoptosis-Independent Mechanism

The enterovirulent Escherichia coli strains potentially involved in inflammatory bowel diseases include diffusely adherent strains expressing Afa/Dr fimbriae (Afa/Dr DAEC). We have previously observed type 1 pilus-mediated interleukin-8 (IL-8) hyperproduction in infected neutrophils. As pathogen induction of host cell death programs and clearance of apoptotic infected cells are crucial for innate immune system homeostasis and host integrity, we examined modulation of neutrophil cell death by Afa/Dr DAEC. Using the human PLB-985 cell line differentiated into fully mature neutrophils, we found that the wild-type enterovirulent E. coli strain C1845 and the recombinant strain DH5α/pF1845 (expressing the fimbrial adhesin F1845) similarly induced time-dependent phosphatidylserine (PS) externalization, suggesting a major specific role of this virulence factor. Using small interfering RNA (siRNA) decay-accelerating factor (DAF)-transfected PLB-985 cells, we then showed that this PS externalization was triggered in part by glycosylphosphatidylinositol (GPI)-anchored DAF receptor engagement (leading to tyrosine kinase and protein kinase C activation) and that it required cytoskeleton and lipid raft architectural integrity. PS externalization under these conditions was not dependent on caspases, mitochondria, lysosomes, or reactive oxygen or nitrogen species. F1845-mediated PS externalization was sufficient to enable macrophage engulfment of infected differentiated PLB-985 cells. These findings provide new insights into the neutrophil response to Afa/Dr DAEC infection and highlight a new role for F1845 fimbriae. Interestingly, although apoptosis pathways were not engaged, C1845-infected PLB-985 cells displayed enhanced removal by macrophages, a process that may participate in the resolution of Afa/Dr DAEC infection and related inflammation.Strains of diffusely adhering Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC) belong to group 6 of the pathogenic E. coli (35). They can cause childhood diarrhea and are responsible for one-third of recurrent urinary tract infections in adults (53). In vitro, enteric wild-type Afa/Dr DAEC strain C1845, which bears F1845 fimbriae, triggers decay-accelerating factor (DAF)-dependent and mitogen-activated protein kinase (MAPK)-dependent interleukin-8 (IL-8) synthesis by polarized colonic epithelial T84 cell monolayers. This leads to transepithelial migration of human polymorphonuclear neutrophils (PMN), which in turn induce epithelial synthesis of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β (4, 5). These interactions between PMN and apical enterovirulent E. coli colonizing the intestinal brush border were documented only recently. First, Brest et al. obtained evidence that Afa/Dr DAEC could modulate PMN apoptosis and was inefficiently engulfed by PMN (8). Then, using the human myeloid cell line PLB-985 differentiated into fully mature PMN, our group found that Afa/Dr DAEC could activate PMN, triggering an oxidative burst and rapid release of preformed myeloperoxidase and IL-8, followed by IL-1α, TNF-α, and IL-8 synthesis (52); type 1 pili were identified as the promoting bacterial virulence factor, and DAF was identified as the PMN membrane-bound receptor that triggers cell signaling via Erk1/2 and p38 MAPKs, Src tyrosine kinase, and NF-κB.Proinflammatory responses might contribute to inducing and perpetuating local gut inflammation. Indeed, delayed death and clearance of infected PMN in tissues can cause exaggerated inflammation and prolonged infection (15); in particular, enzymes and reactive oxygen species (ROS) produced by PMN can damage surrounding tissues. Alternatively, a decrease in the PMN life span due to rapid apoptosis can be a contributing factor in severe and recurrent infections (39). PMN become apoptotic and are then recognized, engulfed, and cleared by professional phagocytes, such as tissue macrophages, which prevents them from releasing their toxic contents (22). The detection, recognition, and ingestion of apoptotic cells involve at least three “eat me” molecules, namely, phosphatidylserine (PS), endocytic receptors, and soluble molecules bridging apoptotic PMN and macrophages (33). A nonapoptotic PS externalization mechanism has also been described, which allows PMN engulfment by macrophages in certain conditions (34, 54, 58). Many microbial pathogens have evolved to circumvent PMN attack through six main strategies: activation of survival and stress responses, contact avoidance, phagocytosis prevention, intracellular survival, PMN death induction, and evasion of PMN extracellular traps (32, 60). Pathogen-induced stimulation of host cell death pathways may eliminate key immune cells or be involved in evasion of other host defenses, while, on the other hand, suppression of death pathways may facilitate the proliferation of intracellular pathogens (20, 36).Here, we report that E. coli wild-type strain C1845 and its recombinant counterpart DH5α/pF1845, which harbors a plasmid encoding F1845 fimbriae, similarly induce time-dependent PS externalization on differentiated PLB-985 cells, suggesting a role for the F1845 adhesin. Further investigation showed that PS externalization followed interaction between F1845 fimbriae and PLB-985 cell membrane-bound DAF. F1845-induced DAF-dependent PS externalization involved tyrosine kinase and protein kinase C (PKC) activation and required cytoskeleton and lipid raft integrity. We also found that PS externalization was not related to three of the main apoptosis pathways (caspase activation and the mitochondrial and lysosomal pathways) or to release of reactive oxygen or nitrogen species. Finally, we showed that the nonapoptotic PS externalization enabled macrophage engulfment of infected PLB-985 cells. Together, these results suggest that this PMN response could participate in resolution of Afa/Dr DAEC infection and the related inflammation.     

Human diffusely adhering Escherichia coli expressing Afa/Dr adhesins that use human CD55 (decay-accelerating factor) as a receptor does not bind the rodent and pig analogues of CD55

Afa/Dr diffusely adhering Escherichia coli (DAEC) bacteria that are responsible for recurrent urinary tract and gastrointestinal infections recognized as a receptor the glycosylphosphatidylinositol (GPI)-anchored protein decay-accelerating factor (DAF; CD55) at the brush border of cultured human intestinal cells. Results show that Afa/Dr DAEC C1845 bacteria were poorly associated with the mucosa of the gastrointestinal tract of infected mice. We conducted experiments with Chinese hamster ovary (CHO) cells stably transfected with mouse (GPI or transmembrane forms), pig, or human CD55 or mouse Crry cDNAs or transfected with empty vector pDR2EF1 alpha. Recombinant E. coli AAEC185 bacteria expressing Dr or F1845 adhesins bound strongly to CHO cells expressing human CD55 but not to the CHO cells expressing mouse (transmembrane and GPI anchored), rat, or pig CD55 or mouse Crry. Positive clustering of CD55 around Dr-positive bacteria was observed in human CD55-expressing CHO cells but not around the rarely adhering Dr-positive bacteria randomly distributed at the cell surface of CHO cells expressing mouse, rat, or pig CD55.     

Structural and functional lesions in brush border of human polarized intestinal Caco-2/TC7 cells infected by members of the Afa/Dr diffusely adhering family of Escherichia coli

Diffusely adhering Escherichia coli (DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush border-associated decay-accelerating factor (DAF; CD55) as a receptor. The wild-type Afa/Dr DAEC strain C1845 has been shown to induce brush border lesions by an adhesin-dependent mechanism triggering apical F-actin rearrangements. In the present study, we undertook to further characterize cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and Dr hemagglutinin, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin, proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, actin-depolymerizing factor, and tubulin was not modified. Using draE mutants, we found that a mutant in which cysteine replaces aspartic acid at position 54 conserved binding capacity but failed to induce F-actin disassembly. Accompanying the cytoskeleton injuries, we found that the distribution of brush border-associated functional proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV), glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, SI and DPPIV enzyme activity decreased.     

Afa/Dr diffusely adhering Escherichia coli C1845 infection promotes selective injuries in the junctional domain of polarized human intestinal Caco-2/TC7 cells

The Afa/Dr diffusely adhering Escherichia coli (DAEC) C1845 strain harboring the F1845 fimbrial adhesin interacts with the brush border-associated CD55 molecule and promotes elongation of brush border microvilli resulting from rearrangement of the F-actin network. This phenomenon involves the activation of a cascade of signaling coupled to the glycosylphosphatidylinositol-anchored receptor of the F1845 adhesin. We provide evidence that infection of the polarized human intestinal cell line Caco-2/TC7 by strain C1845 is followed by an increase in the paracellular permeability for [(3)H]mannitol without a decrease of the transepithelial resistance of the monolayers. Alterations in the distribution of tight-junction (TJ)-associated occludin and ZO-1 protein are observed, whereas the distribution of the zonula adherens-associated E-cadherin is not affected. Using the recombinant E. coli strains HB101(pSSS1) and -(pSSS1C) expressing the F1845 fimbrial adhesin, we demonstrate that the adhesin-CD55 interaction is not sufficient for the induction of structural and functional TJ lesions. Moreover, using the actin filament-stabilizing agent Jasplakinolide, we demonstrate that the C1845-induced functional alterations in TJs are independent of the C1845-induced apical cytoskeleton rearrangements. The results indicated that pathogenic factor(s) other than F1845 adhesin may be operant in Afa/Dr DAEC C1845.     

Increased Escherichia coli phagocytosis in neutrophils that have transmigrated across a cultured intestinal epithelium

The functionality of polymorphonuclear leukocytes (PMNs) once they migrate into the digestive lumen is still ill defined. More specifically, phagocytic function and bactericidal action of PMNs after transepithelial migration have not received much attention. The aim of the present study is to compare PMN behavior before and after transepithelial migration, in particular (i) phagocytosis and bactericidal activity; (ii) expression of surface molecules, particularly those involved in phagocytosis; and (iii) apoptosis. Cultured human intestinal epithelial T84 cell monolayers were used. The effect of transepithelial migration on phagocytosis was evaluated by immunofluorescence and electron microscopy and by flow cytometric assessment of the engulfment of a strain of Escherichia coli transfected with the green fluorescent protein. Superoxide production by PMNs was investigated by luminol-mediated chemiluminescence. Expression of various surface molecules on PMNs was evaluated by flow cytometry, while PMN apoptosis was assayed by morphologic changes and DNA fragmentation. E. coli phagocytosis by the PMNs was markedly increased after transepithelial migration without modification of superoxide production. CD11b/CD18 and CD47 expression was increased upon PMN transmigration, whereas CD16 expression was decreased and CD29, CD46, CD49e, CD49f, CD55, CD59, CD61, CD95 levels remained unchanged. Apoptosis in transmigrated PMNs was slightly advanced and was observed after 12 h compared to 16 h for nontransmigrated PMNs. In conclusion, the phagocytic capacity of the PMNs is augmented after transepithelial migration, with a dramatic increase in the level of CD11b/CD18 and preservation of the superoxide production. These results suggest a higher bactericidal activity of the PMNs once they have translocated into the digestive lumen.     

Nonlethal adherence to human neutrophils mediated by Dr antigen-specific adhesins of Escherichia coli.

Uropathogenic Escherichia coli strains express a variety of adhesins, including members of the Dr adhesin family such as the Dr hemagglutinin, AFAI, and AFAIII. Certain E. coli adhesins (e.g., type 1 and S fimbriae) are known to mediate adherence to human polymorphonuclear leukocytes (PMNs). The receptor on erythrocytes for Dr family adhesins, decay accelerating factor, is also present on PMNs. To determine whether Dr family adhesins mediate adherence to PMNs and to characterize the specificity and consequences of such adherence, we studied agglutination of PMNs and adherence to PMNs by recombinant E. coli strains expressing various mannose-resistant or mannose-sensitive adhesins, in the presence or absence of inhibitors of adherence. Dr family adhesins, like type 1 fimbriae, mediated concentration-dependent adherence to PMNs. Adherence to PMNs was mannose sensitive for type 1 fimbriae but mannose resistant for Dr family adhesins. Chloramphenicol inhibited PMN adherence for the Dr hemagglutinin with the same potency as that with which it inhibited hemagglutination, but it was inactive against PMN adherence and hemagglutination mediated by other members of the Dr adhesin family. In contrast to PMN adherence mediated by type 1 fimbriae, adherence mediated by the Dr hemagglutinin did not lead to significantly increased bacterial killing. These data suggest that Dr family adhesins mediate a novel pattern of adherence to PMNs, probably by recognizing decay accelerating factor, with minimal consequent bacterial killing.     

Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells

We recently documented the neutrophil response to enterovirulent diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), using the human myeloid cell line PLB-985 differentiated into fully mature neutrophils. Upon activation, particularly during infections, neutrophils release neutrophil extracellular traps (NETs), composed of a nuclear DNA backbone associated with antimicrobial peptides, histones, and proteases, which entrap and kill pathogens. Here, using fluorescence microscopy and field emission scanning electron microscopy, we observed NET production by PLB-985 cells infected with the Afa/Dr wild-type (WT) E. coli strain C1845. We found that these NETs were able to capture, immobilize, and kill WT C1845 bacteria. We also developed a coculture model of human enterocyte-like Caco-2/TC7 cells and PLB-985 cells previously treated with WT C1845 and found, for the first time, that the F-actin cytoskeleton of enterocyte-like cells is damaged in the presence of bacterium-induced NETs and that this deleterious effect is prevented by inhibition of protease release. These findings provide new insights into the neutrophil response to bacterial infection via the production of bactericidal NETs and suggest that NETs may damage the intestinal epithelium, particularly in situations such as inflammatory bowel diseases.     

Role of Src kinases in mobilization of glycosylphosphatidylinositol-anchored decay-accelerating factor by Dr fimbria-positive adhering bacteria

Afa/Dr fimbriae constitute the major virulence factor of diffusely adhering Escherichia coli (Afa/Dr DAEC). After recognizing membrane-bound signaling receptors, they trigger cell responses. One of these receptors is the human decay-accelerating factor (hDAF). It has previously been reported that the binding of Afa/Dr fimbriae to hDAF quickly induces recruitment of hDAF around adhering bacteria. The aim of our study is to analyze the role of Src kinases in the Dr fimbria-induced recruitment of hDAF. Using biochemical methods and confocal microscopy followed by 3-dimensional (3D) analysis, we have shown that the activation and cell membrane targeting of Src kinases are necessary for the recruitment and organization of hDAF around adhering bacteria. We identified c-Src to be the specific kinase involved in this process. Using a set of Src-green fluorescent protein mutants, we showed that the catalytic activity and the Src homology 2 (SH2) and SH3 domains of the Src kinases are necessary for Dr fimbria-induced hDAF mobilization to occur. In addition, using mutated Dr fimbriae and a set of mutated hDAFs in which each of the complement control protein (CCP) domains had successively been deleted, we found that the aspartic acids at position 54 in the Dr fimbriae and in CCP domain 4 of hDAF played pivotal roles in the mobilization of the Src kinases and hDAF, respectively.     

Human Decay-Accelerating Factor and CEACAM Receptor-Mediated Internalization and Intracellular Lifestyle of Afa/Dr Diffusely Adhering Escherichia coli in Epithelial Cells

We used transfected epithelial CHO-B2 cells as a model to identify the mechanism mediating internalization of Afa/Dr diffusely adhering Escherichia coli. We provide evidence that neither the α5 or β1 integrin subunits nor α5β1 integrin functioned as a receptor mediating the adhesion and/or internalization of Dr or Afa-III fimbria-positive bacteria. We also demonstrated that (i) whether or not the AfaD or DraD invasin subunits were present, there was no difference in the cell association and entry of bacteria and that (ii) DraE or AfaE-III adhesin subunits are necessary and sufficient to promote the receptor-mediated bacterial internalization into epithelial cells expressing human decay-accelerating factor (DAF), CEACAM1, CEA, or CEACAM6. Internalization of Dr fimbria-positive E. coli within CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells occurs through a microfilament-independent, microtubule-dependent, and lipid raft-dependent mechanism. Wild-type Dr fimbria-positive bacteria survived better within cells expressing DAF than bacteria internalized within CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells. In DAF-positive cells, internalized Dr fimbria-positive bacteria were located in vacuoles that contained more than one bacterium, displaying some of the features of late endosomes, including the presence of Lamp-1 and Lamp-2, and some of the features of CD63 proteins, but not of cathepsin D, and were acidic. No interaction between Dr fimbria-positive-bacterium-containing vacuoles and the autophagic pathway was observed.Diffusely adhering Escherichia coli (DAEC) organisms comprise two classes of strains, the typical DAEC and the atypical DAEC strains, each subdivided into two subclasses of strains (67). These pathogenic E. coli strains belong to group six of enterovirulent E. coli (38). Typical Afa/Dr DAEC strains have been shown to be involved in age-dependent diarrhea in infants (48, 63). Typical Afa/Dr DAEC strains have been shown to belong to the recently reported type IV pathotype of uropathogenic E. coli (UPEC) (46). Typical Afa/Dr DAEC strains are involved in urinary tract infections (UTIs), since 25 to 50% of children with cystitis and 30% of pregnant women with pyelonephritis are infected with E. coli bearing Afa/Dr fimbriae (30, 57). Moreover, typical Afa/Dr DAEC strains are involved in recurrent UTIs, and the vast majority of the typical Afa/Dr DAEC isolates (90%) are multiantibiotic resistant (29). The typical Afa/Dr DAEC strain expresses a family of genes that is organized to form a family of afa-, dra-, daa-, and/or nfa-related operons encoding Afa-I, Afa-II, Afa-III, Afa-V, Dr, Dr-II, F1845, and Nfa-I fimbriae (67). The genes are organized in similar ways, with at least five genes (A to E), of which the last, the E gene, encodes a major structural adhesin subunit (70). The D gene encodes the invasin subunit (21, 35, 72). Importantly, DraE and AfaE-III proteins display 98% sequence identity, whereas DraD and AfaD-III share 100% sequence identity. Atomic resolution models of Dr and Afa-III fibrils have revealed that the structural basis for assembly occurs by donor strand complementation and that the architecture of capped surface fibers results from the assembly of several DraE or AfaE subunits, with one invasin subunit, DraD or AfaD-III, at their distal ends (1, 14).Typical Afa/Dr fimbriae govern the adhesion of the bacteria to host epithelial cells and the cells'' responses to their presence. All these fimbriae (Afa/Dr_DAF) are able to bind specifically to the complement control protein repeats 2 and 3 (CCP2 and CCP3), domains of human decay-accelerating factor (DAF; CD55) (56). The DAF binding domain into the Afa/Dr adhesin subunit is located in its central part, localized on strands B and E of DraE (1, 31). Moreover, our group has recently reported that a subclass of Afa/Dr fimbriae, including Afa-III, Dr, and F1845 (Afa/Dr_CEACAM), recognized members of the human CEACAM family (3), which includes CEACAM1 (biliary glycoprotein; CD66a), CEA (carcinoembryonic antigen; CD66e), and CEACAM6 (nonspecific cross-reacting antigen; CD66c) (4, 27). AfaE-I, AfaE-III, AfaE-V, DraE, and DaaE adhesin subunits of Afa/Dr DAEC targeted the N-terminal domains of CEACAMs (40). The CEACAM binding site is located primarily in the A, B, E, and D strands of the Dr adhesin opposite the beta-sheet encompassing the previously determined binding site for DAF (40). Typical Afa/Dr DAEC strains have been described as invasive in unpolarized epithelial cells expressing several Afa/Dr fimbriae receptors but with a low level of efficiency (24, 26, 35). In cultured human polarized intestinal cells forming a cell monolayer that mimics an epithelium, these bacteria are apically uninvasive and enter the cells via the basolateral domain (26). The process of internalization into unpolarized epithelial cells involves lipid rafts (26, 37, 66) and dynamic, unstable microtubules (24, 26). Previous studies conducted with Dr and Afa-III fimbriae have not elucidated the processes by which typical Afa/Dr DAEC strains enter epithelial cells, which remain controversial. Two different working hypotheses have been proposed. According to one possible mechanism, after the Dr fimbriae have recognized the membrane-bound human DAF, the entire dra operon is necessary to trigger a receptor-mediated internalization of the bacteria (24). Selvarangan et al. (66) have shown that a transposon mutant with a mutation in the DraE adhesin subunit lacks adhesiveness and consequently fails to enter the cells. More convincingly, Das et al. (15) have demonstrated that the DraE subunit is both an adhesin and an invasin, since by mutagenesis to replace selected amino acids in hydrophilic domain II of the DraE protein, bacterial internalization was reduced or abolished without modifying the bacterial cell association. The second possible mechanism would imply that DraD and AfaD-III invasin subunits recognize the membrane-bound α5β1 integrin, and this is sufficient to trigger the entry of bacteria (26, 61) via a zipper-like mechanism (37). For this second mechanism, it has not been clearly established whether there is an initial step of recognition of the membrane-bound receptor DAF or CEACAM members by the AfaE/DraE adhesin subunits.We decided to conduct a series of experiments to analyze the role of DAF and/or α5β1 integrin in the receptor-mediated internalization of Afa/Dr DAEC. We extended our investigation by analyzing the role of the CEACAMs that act as receptors for Afa/Dr_CEACAM adhesins during the receptor-mediated internalization of Afa/Dr DAEC. We also investigated the role of AfaE/DraE adhesins and/or AfaD/DraD invasin subunits in bacterial internalization. We also examined the intracellular lifestyle of Dr fimbria-positive bacteria by comparing the survival rates of the intracellular bacteria in a series of cell lines, each of which expresses one of the membrane-bound epithelial receptors of Afa/Dr fimbriae, and by characterizing the late vacuole-containing internalized bacteria in a DAF-positive epithelial cell line.     

Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens

Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048-5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr(+) adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr(-) adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr(-) adhesin) was found to predominate in afa-positive isolates from sepsis patients (75%); it was frequent in afa-positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr(+) strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa-positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection caused by afa-positive strains.     

Interaction of Escherichia coli with Different Fimbriae and Polymorphonuclear Leukocytes

The effects of Escherichia coli strains with various fimbriae on bacteria-polymorphonuclear leukocyte (PMN) interactions were studied. Strains of E. coli were cultivated at 37 degrees C to express and at 18 degrees C to suppress the formation of fimbriae. The presence of fimbriae was confirmed by electron microscopic studies and hemagglutination and salt aggregation tests. Fimbriated E. coli strains were more readily PMN associated than the nonfimbriated strains in the absence of opsonins, confirming the results of previous studies. However, the PMN chemiluminescence (CL) induced by the various strains in the absence of serum opsonins depended on the type of fimbriae they expressed. Strains with type 1 fimbriae expressing mannose-sensitive hemagglutination induced 5 to 15 times more CL than the same strains grown at 18 degrees C, i.e., not expressing this type of fimbriae. For strains showing mannose-resistant hemagglutination, the differences between fimbriated and nonfimbriated variants of the same strains grown at 37 and 18 degrees C, respectively, were less pronounced. Analysis of enterotoxigenic strains expressing colonization factor antigen I (CFA/I) fimbriae showed that these induced only 25 to 33% of the CL induced by the same E. coli strains not expressing CFA/I, whereas enterotoxigenic strains expressing CFA/II fimbriae induced 100 to 200% of the CL induced by the nonfimbriated variants. Although less CL was induced by bacteria with CFA/I fimbriae than by nonfimbriated variants, this situation was reversed when the microorganisms were opsonized. Thus, CFA/I fimbriae, while enhancing adhesion to cells, induce less activation of PMN-killing mechanisms in a serum-free environment. These findings may be relevant for the virulence in certain body sites, since CFA/I fimbriae, while facilitating adhesiveness, may protect the bacteria from PMN killing. Our findings indicate that PMN interactions with fimbriated E. coli in the host defense may be complex. Certain fimbriae may indeed be advantageous to the bacteria, enabling them to interact with PMNs without activating the bactericidal oxidative metabolism.     

Escherichia coli Cytotoxic Necrotizing Factor 1 Effaces Microvilli and Decreases Transmigration of Polymorphonuclear Leukocytes in Intestinal T84 Epithelial Cell Monolayers

Cytotoxic necrotizing factor type 1 (CNF1), a 110-kDa toxin-like protein from pathogenic Escherichia coli strains, induces an actin cytoskeleton reorganization consisting of the formation of prominent stress fibers by permanent activation of the small GTP-binding protein Rho. Since p21Rho regulates tight-junction permeability and perijunctional actin reorganization in epithelial intestinal cells (A. Nusrat, M. Giry, J. R. Turner, S. P. Colgan, C. A. Parkos, E. Lemichez, P. Boquet, and J. L. Madara, Proc. Natl. Acad. Sci. USA 92:10629–10633, 1995), we used polarized T84 epithelial intestinal cell monolayers to examine whether CNF1 could affect microvillus structure, transepithelial resistance, and polymorphonuclear leukocyte (PMN) transmigration. Incubation of T84 cells with CNF1 did not influence transepithelial resistance, suggesting that barrier function and surface polarity were not affected by the toxin. However, CNF1 effaced intestinal cell microvilli and induced a strong decrease of PMN transepithelial migration in either the luminal-to-basolateral or the basolateral-to-luminal direction. CNF1 could thus be a virulence factor exhibiting a new type of combined activity consisting of effacing of microvilli and occlusion of the epithelial barrier to PMNs. Attenuated transepithelial migration of PMNs could result in the enhanced growth and protection of luminal bacteria.     

Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli

O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.     

Diminished neutrophil oxidative metabolism after phagocytosis of virulent Salmonella typhi.

The interactions of human polymorphonuclear neutrophils (PMNs) with virulent and avirulent strains of Salmonella typhi were examined. Ingestion of the S. typhi strains by PMNs was evaluated with three techniques: visual examination of PMN monolayers (phagocytic index); uptake of radiolabeled S. typhi by PMNs; and removal of S. typhi from the supernatant of suspensions of PMNs and bacteria. All three techniques indicated equivalent phagocytosis of the strains. Postphagocytic PMN oxidative metabolism was quantitated with measurements of oxygen consumption, protein iodination, and chemiluminescence. We found that although PMNs ingested equal numbers of virulent and avirulent S. typhi, those PMNs ingesting the virulent organisms exhibited a significantly smaller increase in postphagocytic oxidative metabolism than PMNs ingesting avirulent S. typhi. Despite this muted oxidative burst the virulent bacteria were killed as well as the avirulent strains. Virulent S. typhi either fail to stimulate receptors that trigger PMN oxidative metabolism or inhibit PMN oxidative metabolism. Our data support the former hypothesis.