Clostridioides difficile Flagella.

Clostridioides difficile is an important pathogen for humans with a lead in nosocomial infection, but it is also more and more common in communities. Our knowledge of the pathology has historically been focused on the toxins produced by the bacteria that remain its major virulence factors. But the dysbiosis of the intestinal microbiota creating the conditions for the colonization appears to be fundamental for our understanding of the disease. Colonization implies several steps for the bacteria that do or do not use their capacity of motility with the synthesis of flagella. In this review, we focus on the current understanding of different topics on the C. difficile flagellum, ranging from its genetic organization to the vaccinal interest in it.

Clostridioides difficile Flagellin Activates the Intracellular NLRC4 Inflammasome.

Clostridioides difficile (C. difficile), is a major cause of nosocomial diarrhea and colitis. C. difficile flagellin FliC contributes toxins to gut inflammation by interacting with the immune Toll-like receptor 5 (TLR5) to activate nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) signaling pathways. Flagella of intracellular pathogens can activate the NLR family CARD domain-containing protein 4 (NLRC4) inflammasome pathway. In this study, we assessed whether flagellin of the extracellular bacterium C. difficile internalizes into epithelial cells and activates the NLRC4 inflammasome. Confocal microscopy showed internalization of recombinant green fluorescent protein (GFP)-FliC into intestinal Caco-2/TC7 cell line. Full-length GFP-FliC activates NLRC4 in Caco-2/TC7 cells in contrast to truncated GFP-FliC lacking the C-terminal region recognized by the inflammasome. FliC induced cleavage of pro-caspase-1 into two subunits, p20 and p10 as well as gasdermin D (GSDMD), suggesting the caspase-1 and NLRC4 inflammasome activation. In addition, colocalization of GFP-FliC and pro-caspase-1 was observed, indicating the FliC-dependent NLRC4 inflammasome activation. Overexpression of the inflammasome-related interleukin (interleukin (IL)-1ß, IL-18, and IL-33) encoding genes as well as increasing of the IL-18 synthesis was detected after cell stimulation. Inhibition of I-kappa-B kinase alpha (IKK-α) decreased the FliC-dependent inflammasome interleukin gene expression suggesting a role of the NF-κB pathway in regulating inflammasome. Altogether, these results suggest that FliC internalizes into the Caco-2/TC7 cells and activates the intracellular NLRC4 inflammasome thus contributing to the inflammatory process of C. difficile infection.

Respective roles of migration and social deprivation for virological non-suppression in HIV-infected adults on antiretroviral therapy in France.

Barriers to achieve sustained HIV virological suppression on antiretroviral therapy (ART) jeopardize the success of the 90:90:90 UNAIDS initiative which aims to end the HIV/AIDS epidemic. In France, where access to ART is free and universally available, we analyze the way in which social determinants of health (i.e. cultural, environmental) and economic factors might influence virological outcomes. A cross-sectional study was performed in two hospitals located in Paris area. All consecutive people living with HIV (PLHIV) on ART for at least 6 months attending the outpatient clinics between 01/05/2013 and 31/10/2014 answered an individual score of deprivation, EPICES, retrieving information on health insurance status, economic status, family support and leisure activity. This score varies from 0 to 100 with deprivation state defined above 30.17. Factors associated with HIV viral load >50 copies/ml were assessed by logistic regression modeling with a backward stepwise selection to select the final multivariable model. Sensitivity analyses were performed using two other thresholds for virological non-suppression (100 or 200 copies/ml). Overall, 475 PLHIV were included (53% male, median age 47 years, 66% not born in France mainly in a sub-Saharan African country). Half of French natives and 85% of migrants were classified as deprived. Median duration on ART was 9.7 years with virological suppression in 95.2% of non-deprived participants and in 83.5% of deprived ones (p = 0.001). The final multivariable model retained ART tiredness, younger age, a previous AIDS event and social deprivation (adjusted Odds Ratio, 2.9; 95%CI, 1.2-7.0) as determinants of virological non-suppression but not migration in itself. When using separate components of EPICES score, reporting economic difficulties and non-homeownership were associated with virological non-suppression. In addition to interventions focusing on cultural aspects of migration, social interventions are needed to help people with social vulnerability to obtain sustained responses on ART.

Clostridium difficile flagella induce a pro-inflammatory response in intestinal epithelium of mice in cooperation with toxins.

Clostridium difficile is the most important enteropathogen involved in gut nosocomial post-antibiotic infections. The emergence of hypervirulent strains has contributed to increased mortality and morbidity of CDI. The C. difficile toxins contribute directly to CDI-associated lesions of the gut, but other bacterial factors are needed for the bacteria to adhere and colonize the intestinal epithelium. The C. difficile flagella, which confer motility and chemotaxis for successful intestinal colonization, could play an additional role in bacterial pathogenesis by contributing to the inflammatory response of the host and mucosal injury. Indeed, by activating the TLR5, flagella can elicit activation of the MAPK and NF-κB cascades of cell signaling, leading to the secretion of pro-inflammatory cytokines. In the current study, we demonstrate, by using an animal model of CDI, a synergic effect of flagella and toxins in eliciting an inflammatory mucosal response. In this model, the absence of flagella dramatically decreases the degree of mucosal inflammation in mice and the sole presence of toxins without flagella was not enough to elicit epithelial lesions. These results highlight the important role of C. difficile flagella in eliciting mucosal lesions as long as the toxins exert their action on the epithelium.

Intestinal Epithelial Cell Response to Clostridium difficile Flagella.

Clostridium difficile is the bacterium responsible for most antibiotic-associated diarrhea in North America and Europe. This bacterium, which colonizes the gut of humans and animals, produces toxins that are known to contribute directly to damage of the gut. It is known that bacterial flagella are involved in intestinal lesions through the inflammatory host response. The C. difficile flagellin recognizes TLR5 and consequently activates the NF-κB and the MAPK signaling pathways which elicit the synthesis of pro-inflammatory cytokines. Increasing interest on the role of C. difficile flagella in eliciting this cell response was recently developed and the development of tools to study cell response triggered by C. difficile flagella will improve our understanding of the pathogenesis of C. difficile.

Deciphering Adaptation Strategies of the Epidemic Clostridium difficile 027 Strain during Infection through In Vivo Transcriptional Analysis.

Clostridium difficile is responsible for a wide spectrum of infection from asymptomatic carriage to severe, relapsing colitis. Since 2003, C. difficile infections have increased with a higher morbidity and mortality due to the emergence of epidemic and hypervirulent C. difficile strains such as those of the epidemic lineage 027/BI/NAP1. To decipher the hypervirulence and epidemicity of 027 strains, we analyzed gene expression profiles of the R20291 027 strain using a monoxenic mouse model during the first 38h of infection. A total of 741 genes were differentially expressed during the course of infection. They are mainly distributed in functional categories involved in host adaptation. Several genes of PTS and ABC transporters were significantly regulated during the infection, underlying the ability of strain R20291 to adapt its metabolism according to nutrient availability in the digestive tract. In this animal model, despite the early sporulation process, sporulation efficiency seems to indicate that growth of R20291 vegetative cells versus spores were favored during infection. The bacterial mechanisms associated to adaptability and flexibility within the gut environment, in addition to the virulence factor expression and antibiotic resistance, should contribute to the epidemicity and hypervirulence of the C. difficile 027 strains.

Clostridium difficile flagella predominantly activate TLR5-linked NF-κB pathway in epithelial cells.

Clostridium difficile has become the most common enteropathogen responsible for intestinal nosocomial post-antibiotic infections. This has coincided with the appearance of serious cases related to the emergence of hypervirulent strains. The toxins are the main virulence factors and elicit an inflammatory response during C. difficile infection. However, other bacterial components appear to be involved in the inflammatory process. In some pathogens, flagella play a role in pathogenesis through abnormal stimulation of the TLR5-mediated host immune response. To date, few studies have addressed this role for C. difficile flagella. In the current study, we confirm in two different epithelial cell models that C. difficile thanks to its FliC flagellin interacts with TLR5. In addition, thanks to inhibition and transcriptomic studies we demonstrate that the interaction of flagellin and TLR5 predominantly activates the NF-κB and, in a lesser degree, the MAPK pathways, via TLR5, leading to up-regulation of pro-inflammatory gene expression and synthesis of pro-inflammatory mediators. These results suggest a role for C. difficile flagella in contributing to inflammatory response in host intestinal cells.

The flagellin FliC of Clostridium difficile is responsible for pleiotropic gene regulation during in vivo infection.

Clostridium difficile is the main agent responsible for hospital acquired antibiotic associated diarrhoea. In recent years, epidemic strains have emerged causing more severe infections. Whilst C. difficile has two major virulence factors, toxins TcdA and TcdB, it is generally accepted that other virulence components of the bacterium contribute to disease. Previously, it has been suggested that flagella expression from pathogenic bacteria might be implicated in virulence. In a recent study, we observed an increased mortality in a gnotobiotic mouse model when animals were colonized with an isogenic fliC mutant constructed in the PCR-ribotype 027 (B1/NAP1) strain R20291, while animals survived when colonized by the parental strain or after colonization by other high-toxin-producing C. difficile strains. To understand the reasons for this increased virulence, we compared the global gene expression profiles between the fliC-R20291 mutant and its parental strain using an in vitro and in vivo transcriptomic approach. The latter made use of the gnotobiotic mouse model. Interestingly, in the fliC mutant, we observed considerable up-regulation of genes involved in mobility, membrane transport systems (PTS, ABC transporters), carbon metabolism, known virulence factors and sporulation. A smaller but significant up-regulation of genes involved in cell growth, fermentation, metabolism, stress and antibiotic resistance was also apparent. All of these genes may be associated with the increased virulence of the fliC-R20921 mutant. We confirmed that the fliC mutation is solely responsible for the observed changes in gene expression in the mutant strain since expression profiles were restored to that of the wild-type strain in the fliC-complemented strain. Thus, the absence of FliC is directly or indirectly involved in the high mortality observed in the fliC mutant infected animals. Therefore, we provide the first evidence that when the major structural component of the flagellum is neutralized, deregulation of gene expression can occur during infection.

The role of flagella in Clostridium difficile pathogenesis: comparison between a non-epidemic and an epidemic strain.

Clostridium difficile is a major cause of healthcare-associated infection and inflicts a considerable financial burden on healthcare systems worldwide. Disease symptoms range from self-limiting diarrhoea to fatal pseudomembranous colitis. Whilst C. difficile has two major virulence factors, toxin A and B, it is generally accepted that other virulence components of the bacterium contribute to disease. C. difficile colonises the gut of humans and animals and hence the processes of adherence and colonisation are essential for disease onset. Previously it has been suggested that flagella might be implicated in colonisation. Here we tested this hypothesis by comparing flagellated parental strains to strains in which flagella genes were inactivated using ClosTron technology. Our focus was on a UK-outbreak, PCR-ribotype 027 (B1/NAP1) strain, R20291. We compared the flagellated wild-type to a mutant with a paralyzed flagellum and also to mutants (fliC, fliD and flgE) that no longer produce flagella in vitro and in vivo. Our results with R20291 provide the first strong evidence that by disabling the motor of the flagellum, the structural components of the flagellum rather than active motility, is needed for adherence and colonisation of the intestinal epithelium during infection. Comparison to published data on 630Δerm and our own data on that strain revealed major differences between the strains: the R20291 flagellar mutants adhered less than the parental strain in vitro, whereas we saw the opposite in 630Δerm. We also showed that flagella and motility are not needed for successful colonisation in vivo using strain 630Δerm. Finally we demonstrated that in strain R20291, flagella do play a role in colonisation and adherence and that there are striking differences between C. difficile strains. The latter emphasises the overriding need to characterize more than just one strain before drawing general conclusions concerning specific mechanisms of pathogenesis.

Surface-layer (S-layer) of human and animal Clostridium difficile strains and their behaviour in adherence to epithelial cells and intestinal colonization.

Clostridium difficile is a frequent cause of severe, recurrent post-antibiotic diarrhoea and pseudomembranous colitis. The surface layer (S-layer) is the predominant outer surface component of C. difficile which is involved in pathogen-host interactions critical to pathogenesis. In this study, we characterized the S-layer protein A (SlpA) of animal and human strains belonging to different PCR-ribotypes (PR) and compared the in vitro adherence and in vivo colonization properties of strains showing different SlpA variants. Since each SlpA variant has been recently associated with an S-layer cassette, we were able to deduce the cassette for each of our strains. In this study, an identity of 99-100 % was found among the SlpA of isolates belonging to PR 012, 014/020, 045 and 078. One exception was the SlpA of a poultry isolate, PR 014/020, which showed 99 % identity with that of strain 0160, another PR 014/020 which contains an S-layer cassette 6. Interestingly, this cassette has also been found in a PR 018 strain, an emerging virulent type currently predominant in Italy. Five other SlpA variants (v014/020a-e) were identified in strains PR 014/020. In vitro adherence assays and in vivo colonization experiments were performed on five PR 014/020 strains: human 1064 (v014/020e), human 4684/08 (v014/020b), human IT1106 (v078a), poultry P30 (v014/020d) and poultry PB90 (v014/020b) strains. Adhesion assays indicate that C. difficile strains vary in their capacity to adhere to cells in culture and that adhesion seems to be independent of the SlpA variant. Colonization properties were assessed in vivo using a dixenic mouse model of colonization. The kinetics of faecal shedding and caecal colonization were similar when human 4684/08 (v014/020b) strain was compared with human 1064 (v014/020e) and poultry PB90 (v014/020b) strain. In contrast, poultry P30 (v014/020d) strain outcompeted both human 4684/08 (v014/020b) and IT1106 (v078a) strains and its adherence to caeca at day 7 was significantly higher. The peculiar characteristics of C. difficile P30 seem to advantage it in colonizing the intestinal mice niche, increasing its ability to compete and adapt. The results obtained underline the need of an increased attention to the genetic evolution of C. difficile to prevent and limit the consequences of the emergence of increasingly virulent strains.

Apical expression of human full-length hCEACAM1-4L protein renders the Madin Darby Canine Kidney cells responsive to lipopolysaccharide leading to TLR4-dependent Erk1/2 and p38 MAPK signalling.

CEACAM1 expressed by granulocytes and epithelial cells is recognized as a membrane-associated receptor by some Gram-negative pathogens. Here we report a previously unsuspected role of human CEACAM1-4L (hCEACAM1-4L) in polarized epithelial cells. We find that in contrast with non-transfected cells, Madin Darby Canine Kidney strain II (MDCK) engineered for the apical expression of the long cytoplasmic chain protein hCEACAM1-4L showed a serum-independent increase in the phosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinases (MAPKs) after treatment with lipopolysaccharide (LPS) of wild-type, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strain IH11128. Aggregates of FITC-LPS bind the apical domain of MDCK-hCEACAM1-4L cells colocalizing with the apically expressed hCEACAM1-4L protein and do not bind MDCK-pCEP cells, and surface plasmon resonance analysis shows that LPS binds to the extracellular domain of the CEACAM1-4L protein. We showed that cell polarization and lipid rafts positively control the LPS-IH11128-induced phosphorylation of Erk1/2 in MDCK-hCEACAM1-4L cells. Structure-function analysis using mutated hCEACAM1-4L protein shows that the cytoplasmic domain of the protein is needed for LPS-induced MAPK signalling, and that phosphorylation of Tyr-residues is not increased in association with MAPK signalling. The hCEACAM1-4L-dependent Erk1/2 phosphorylation develops in the presence of lipid A and does not develop in the presence of penta-acylated LPS. Finally, small interfering RNA (siRNA) silencing of canine TLR4 abolishes the hCEACAM1-4L-dependent, LPS-induced phosphorylation of Erk1/2. Collectively, our results support the notion that the apically expressed, full-length hCEACAM1-4L protein functions as a novel LPS-conveying molecule at the mucosal surface of polarized epithelial cells for subsequent MD-2/TLR4 receptor-dependent MAPK Erk1/2 and p38 signalling.

Role of fibronectin-binding protein A in Clostridium difficile intestinal colonization.

Clostridium difficile is a frequent cause of severe, recurrent, post-antibiotic diarrhoea and pseudomembranous colitis. Its pathogenicity is mediated mainly by two toxins, TcdA and TcdB. However, different adhesins have also been described as important colonization factors which are implicated in the first step of the intestinal infection. In this study, we focused our interest on one of these adhesins, fibronectin-binding protein A (FbpA), and on its role in the intestinal colonization process. A mutant of FbpA (CDΔFbpA) was constructed in C. difficile strain 630Δerm by using ClosTron technology. This mutant was characterized in vitro and in vivo and compared to the isogenic wild-type strain. Adhesion of the CDΔFbpA mutant to the human colonic epithelial cell line Caco-2 and to mucus-secreting HT29-MTX cells was examined. Surprisingly, the CDΔFbpA mutant adhered more than the wild-type parental strain. The CDΔFbpA mutant was also analysed in three different mouse models by following the intestinal implantation kinetics (faecal shedding) and caecal colonization (7 days post-challenge). We showed that in monoxenic mice, CDΔFbpA shed C. difficile in faeces at the same rate as that of the isogenic wild-type strain but its colonization of the caecal wall was significantly reduced. In dixenic mice, the shedding rate was slower for the CDΔFbpA mutant than for the isogenic wild-type strain during the first days of infection, but no significant difference was observed in caecal colonization. Similar rates of intestinal implantation and caecal colonization were observed for both strains in assays performed in human microbiota-associated mice. Taken together, our data suggest that FbpA plays a role in intestinal colonization by C. difficile.

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 alpha5 or beta1 integrin subunits nor alpha5beta1 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.

Escherichia coli type 1 pili trigger late IL-8 production by neutrophil-like differentiated PLB-985 cells through a Src family kinase- and MAPK-dependent mechanism.

The innate immune response to enteropathogenic bacteria includes chemokine-induced polymorphonuclear neutrophil (PMN) migration across mucosal epithelia leading to bacterial clearance and resolution of infection. Among these bacteria, diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), causing childhood diarrhea, can promote IL-8-dependent PMN transmigration across cultured intestinal epithelial cell monolayers via MAPK pathway activation. However, interactions between PMN and Afa/Dr DAEC are poorly documented and constitute the aim of the present study. Using the human PLB-985 cell line differentiated into fully mature PMN, we described the coordinated response to various E. coli. The rapid and strong release of reactive oxygen species and preformed intragranular mediators (myeloperoxidase and IL-8) is followed by a later TNF-alpha, IL-1beta, and IL-8 synthesis. The use of wild-type (IH11128, C1845, LF82), control (AAEC185), and recombinant (AAEC185 bearing Dr or F1845 fimbriae, AdLF82, or type 1 pili) bacterial strains allowed us to demonstrate that late IL-8 hyperproduction is triggered by type 1 pili but not by Dr or F1845 fimbriae; MAPKs (p38, ERK, Src) and NF-kappaB activations are implicated in this response. Thus, in the course of Afa/Dr DAEC intestinal infection, epithelium- and neutrophil-derived IL-8 could, at least in part, control the flow of neutrophils through the lamina propria. Afa/Dr DAEC-induced IL-8 hyperproduction by PMN might thus be important for inducing and perpetuating local inflammation, and this self-amplifying loop might play a role in the pathogenesis of inflammatory bowel diseases such as Crohn's disease.

Environmental signals implicated in Dr fimbriae release by pathogenic Escherichia coli.

Afa/Dr diffusely adhering Escherichia coli have been shown to cause urinary tract infections and enteric infections. Virulence of Dr-positive IH11128 bacteria is associated with the presence of Dr fimbriae. In this report, we show for the first time that the Dr fimbriae are released in the extracellular medium in response to multiple environmental signals. Production and secretion of Dr fimbriae are clearly thermoregulated. A comparison of the amounts of secreted fimbriae showed that the secretion is drastically increased during anaerobic growth in minimal medium. The effect of anaerobiosis on secretion seemed to depend on both the growth phase and the culture medium. The secretion was maximal during the logarithmic-phase growth and corresponded to 27 and 57% of total Dr fimbriae produced by bacteria grown in mineral medium+glucose and LB broth, respectively. Thus, the anaerobic environment of the colon would favour the secretion of Dr fimbriae during bacterial multiplication. The controlled release of the Dr fimbriae, which is carried out in the absence of cellular lysis, appears independent of the action of proteases or a process of maturation. The mechanism employed in the liberation of Dr fimbriae thus seems different from that described for the adhesins FHA and Hap of Bordetella pertussis and Haemophilus influenzae.

Zipper-like internalization of Dr-positive Escherichia coli by epithelial cells is preceded by an adhesin-induced mobilization of raft-associated molecules in the initial step of adhesion.

We undertook a study of the mechanism by which Dr-positive bacteria invade epithelial cells. Our findings show that Dr-positive bacteria enter via a zipper-like mechanism that is independent of the Dr-induced mobilization of F-actin and of the signaling molecules that control Dr-induced F-actin rearrangements. We also observed that Dr-positive IH11128 bacteria entered cells that were positive for the caveola marker VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) to the same extent as those that were not (parental Caco-2 cells). Using fluorescence labeling and confocal laser scanning microscopy, we provide evidence that during the adhesion step, the alpha5beta1 integrin, which plays a pivotal role in Afa/Dr diffusely adhering Escherichia coli bacterial entry, is mobilized around adhering Dr-positive bacteria. We show that the receptor for Afa/Dr adhesins, glycosylphosphatidylinositol-anchored CD55; the raft marker, ganglioside GM1; and VIP21/caveolin are all recruited around adhering Dr-positive bacteria. We also observed that extracting membrane cholesterol with methyl-beta-cyclodextrin (MBCD) did not affect the recruitment of CD55, GM1, or beta1 integrin to adhering Dr-positive bacteria. In contrast, extracting or changing membrane-bound cholesterol by means of drugs that modify lipid rafts (MBCD, filipin III, or mevalonate plus lovastatin plus MBCD) inhibited the entry of Dr-positive IH11128 both into cells that expressed VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) and into those that did not (parental Caco-2 cells). Finally, restoring cholesterol within the cell membrane of MBCD-treated cells restored Dr-positive IH11128 internalization.

Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC).

Little is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins. These adhesins recognize as receptors the GPI-anchored proteins CD55 (decay-accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA). CD66e is a member of the CEA-related cell adhesion molecules (CEACAM) family, comprising seven members. We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM-expressing CHO and HeLa cells. The results demonstrate that only E. coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr-I, including Dr, F1845 and AfaE-III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6. Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr-I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6. In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recruited around bacteria in HeLa cells. The recruited CEACAM1, CEA and CEACAM6 around adhering bacteria resist totally or in part a detergent extraction, whereas the recruited CEACAM3 does not. Finally, the results show that recognition of CEA and CEACAM6, but not CEACAM1, is accompanied by tight attachment to bacteria of cell surface microvilli-like extensions, which are elongated. Moreover, recognition of CEA is accompanied by an activation of the Rho GTPase Cdc42 and by a phosphorylation of ERM, which in turn elicit the observed cell surface microvilli-like extensions.

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.

Pathogenesis of cerebral Cryptococcus neoformans infection after fungemia.

The pathogenesis of cerebral infection after Cryptococcus neoformans fungemia in outbred mice was investigated. Confocal microscopy and cultures on ficoll-hypaque gradient-separated blood cells were used to detect yeasts in the cytoplasms of monocytes. In semithin brain sections, poorly capsulated yeasts were seen in macrophages in the leptomeningeal space, in monocytes circulating in leptomeningeal capillaries, or in the endothelial cells themselves, strengthening the hypothesis that monocytes and endothelial cells play key roles in the pathogenesis of cryptococcal meningitis. Similar fungal loads and cellular reactions were seen in mice and in 1 patient with acquired immune deficiency syndrome (AIDS), all with acute cryptococcal meningoencephalitis, and in mice and in 1 patient with AIDS, all with cured cryptococcal infection. Immunostaining revealed both the presence of cryptococcal polysaccharide in various brain cells and antigenic variability both from yeast cell to yeast cell and over time. Thus, our data established the relevance and interest that this experimental model has for investigation of the pathogenesis of human cryptococcal meningitis.