Dra/AfaE adhesin of uropathogenic Dr/Afa+ Escherichia coli mediates mortality in pregnant rats.

Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa+ E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE+ afaD and afaE afaD+ mutants. The clinical E. coli strain (afaE+ afaD+) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE+ afaD+ strain, followed by the group infected with the afaE+ afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE+ strains were the most invasive (afaE+ afaD strain > afaE+ afaD+ strain), while the afaE mutant strains (afaE afaD+ and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE+ E. coli strains in vitro.

Dr operon-associated invasiveness of Escherichia coli from pregnant patients with pyelonephritis.

We used a gentamicin protection assay to assess the ability of gestational pyelonephritis isolates of Escherichia coli to invade HeLa cells. The ability to enter HeLa cells was strongly associated with the presence of Dr operons coding for Dr adhesins. In contrast, the nonivasive isolates predominantly expressed papG, coding for P fimbriae.

Rapid receptor-clustering assay to detect uropathogenic and diarrheal Escherichia coli isolates bearing adhesins of the Dr family.

Infections caused by Escherichia coli isolates expressing adhesins of the Dr family are associated with diarrhea and urinary tract infections, and these E. coli strains recognize the complement regulatory protein decay-accelerating factor (DAF) as their receptor. Clustering of the DAF receptor at the sites of bacterial adherence to epithelial cells is proposed as an alternative to PCR assay for rapid detection of Dr-positive E. coli.

Ampicillin-resistant Escherichia coli in gestational pyelonephritis: increased occurrence and association with the colonization factor Dr adhesin.

The pattern of ampicillin resistance and possible association with virulence factors of 78 Escherichia coli isolates taken from 78 pregnant women with pyelonephritis were evaluated. The current incidence of ampicillin resistance among pyelonephritis isolates (46%) was significantly higher than that reported in 1985 (22%). Resistance was found more frequently during the first (60%) and third (53%) trimesters than during the second trimester (33%). Of all dra(+) E. coli isolates, 75% were ampicillin resistant, whereas dra(+) isolates of O75 serotype E. coli accounted for 87% of ampicillin-resistant strains. The significant increase of ampicillin resistance among gestational pyelonephritis E. coli and the association with the dra gene cluster encoding colonization and invasive capacity may warrant further study involving obstetric and neonate wards, with the latter being at the higher risk for potential problems.

Role of decay-accelerating factor domains and anchorage in internalization of Dr-fimbriated Escherichia coli.

Dr-fimbriated Escherichia coli capable of invading epithelial cells recognizes human decay-accelerating factor (DAF) as its cellular receptor. The role of extracellular domains and the glycosylphosphatidylinositol anchor of DAF in the process of internalization of Dr(+) E. coli was characterized in a cell-cell interaction model. Binding of Dr(+) E. coli to the short consensus repeat 3 domain of DAF expressed by Chinese hamster ovary cells was critical for internalization to occur. Deletion of short consensus repeat 3 domain or replacement of Ser(165) by Leu in this domain, or the use of a monoclonal antibody to this region abolished internalization. Replacing the glycosylphosphatidylinositol anchor of DAF with the transmembrane anchor of membrane cofactor protein or HLA-B44 resulted in abolition or reduction of internalization respectively. Cells expressing glycosylphosphatidylinositol-anchored DAF but not the transmembrane-anchored DAF internalized Dr(+) E. coli through a glycolipid pathway, since the former cells were more sensitive to inhibition by methyl-beta-cyclodextrin, a sterol-chelating agent. Electron microscopic studies revealed that the intracellular vacuoles containing the internalized Dr(+) E. coli were morphologically distinct between the anchor variants of DAF. The cells expressing glycosylphosphatidylinositol-anchored DAF contained a single bacterium in tight-fitting vacuoles, while the cells expressing transmembrane-anchored DAF contained multiple (two or three) bacteria in spacious phagosomes. This finding suggests that distinct postendocytic events operate in the cells expressing anchor variants of DAF. We provide direct evidence for the DAF-mediated internalization of Dr(+) E. coli and demonstrate the significance of the glycosylphosphatidylinositol anchor, which determines the ability and efficiency of the internalization event.

Decay-accelerating factor and cytoskeleton redistribution pattern in HeLa cells infected with recombinant Escherichia coli strains expressing Dr family of adhesins.

Escherichia coli strains expressing Dr fimbriae are able to enter epithelial cells by interacting with a complement-regulatory protein, decay-accelerating factor. This model of bacterial internalization, with a well-characterized bacterial ligand and host receptor, provides a unique opportunity to investigate the early stages of invasion. We used immunofluorescence staining techniques to examine the distribution of receptor and cytoskeletal proteins in HeLa cells infected with E. coli recombinant strains that expressed Dr family of adhesins: Dr, Dr-II, F1845, AFA-I, and AFA-III. A major rearrangement of decay-accelerating factor was found at the adherence sites of recombinant strains expressing Dr, Dr-II, and F1845 adhesins. The changes in the distribution of receptor were significantly smaller on HeLa cells infected with E. coli bearing AFA-I or AFA-III afimbrial adhesins. Receptor aggregation was associated with the redistribution of cytoskeleton-associated proteins such as actin, alpha-actinin, ezrin, and occasionally tropomyosin. Purified Dr fimbriae coated on polystyrene beads were capable of triggering clustering of receptor and accumulating actin at the adhesion sites of beads to HeLa cells. Using scanning and transmission electron microscopic techniques, we have shown that beads coated with Dr fimbriae, as opposed to beads coated with bovine serum albumin, were enwrapped by cellular microvilli and ultimately internalized into HeLa cells. This indicates that interaction of Dr fimbriae with decay-accelerating factor is associated with redistribution of receptor and is sufficient to promote bacterial internalization.

Molecular cloning and characterization of Dr-II, a nonfimbrial adhesin-I-like adhesin isolated from gestational pyelonephritis-associated Escherichia coli that binds to decay-accelerating factor.

Bacterial adhesins play an important role in the colonization of the human urogenital tract. Escherichia coli Dr family adhesins have been found to be frequently expressed in strains associated with pyelonephritis in pregnant females. The tissue receptor for known Dr adhesins has been localized to the short consensus repeat-3 (SCR-3) domain of decay accelerating factor (DAF), a complement regulatory protein. In this report, we identified and cloned draE2, a gene encoding a novel 17-kDa DAF-binding adhesin, Dr-II, from a strain of E. coli associated with acute gestational pyelonephritis. Despite the significant sequence diversity between Dr-II and Dr family adhesins, the receptor of Dr-II was found to be the SCR-3 domain of DAF. Sequence analysis of the 186-amino-acid Dr-II open reading frame revealed significant diversity from other members of the Dr adhesin family, including Dr, AFA-I, AFA-III, and F1845, but only an 8-amino-acid difference in sequence from that of the 17-kDa nonfimbrial adhesin NFA-I of unknown receptor specificity. N-terminal peptide sequencing of the purified adhesin confirmed the identity of the open reading frame and indicated cleavage of a 28-amino-acid signal peptide. Antibodies raised against purified Dr-II adhesin exhibited little or no cross-reactivity to Dr adhesin. Characterization of the biological properties demonstrated that like the Dr adhesins, Dr-II was associated with the ability of E. coli to bind to tubular basement membranes and Bowman's capsule and to be internalized into HeLa cells.

Dr fimbriae operon of uropathogenic Escherichia coli mediate microtubule-dependent invasion to the HeLa epithelial cell line.

Escherichia coli Dr adhesin and decay-accelerating factor (DAF) receptor-mediated interaction was proposed as the mechanism of ascending urinary tract infection (UTI) and chronic interstitial nephritis. This report provides novel evidence for Dr fimbriae operon-mediated invasive capacity of Dr+ E. coli. Insertional mutants draE, draC, and draB, and adherent draD and UV-inactivated BN406 were unable to enter HeLa cells. Complementation of the dra mutation restored invasiveness. Internalization was inhibited by anti-Dr fimbriae IgG (100%), anti-SCR-3 domain of DAF (75%), and nocodazole (95%). Increased receptor-ligand density occurred at the site of internalization. Internalized Dr+ E. coli did not significantly multiply in the HeLa cell line. Accordingly, the dra operon and DAF were required for microtubule-dependent internalization of E. coli to HeLa cells. The relatively low invasion and multiplication rates of Dr+ E. coli may hypothetically contribute to the postattachment steps of ascending UTI and chronic renal infection.

Pelvic inflammatory disease isolates of Neisseria gonorrhoeae are distinguished by C1q-dependent virulence for newborn rats and by the sac-4 region.

The virulence mechanism of Neisseria gonorrhoeae in pelvic inflammatory disease (PID) is not well understood, and an objective diagnostic method to identify patients with PID is lacking. We investigated the hypothesis that development of PID was associated with a C1q-dependent virulence property of gonococcal strains. Recent development of a C1q-dependent experimental model of gonococcal infection (S. Nowicki, M. Martens, and B. Nowicki, Infect. Immun. 63:4790-4794, 1995) created an opportunity to evaluate this hypothesis in vivo. Therefore, the virulence of 32 clinical isolates (18 PID isolates and 14 local infection [LI] isolates) was evaluated in experimental rat pups. A serum bactericidal assay was used to characterize a gonococcal serum-resistant (ser(r)) phenotype. PCR primers designed to amplify a suitable-size gonococcal sac-4 DNA fragment (unique for serum-resistant donor JC1) were used to evaluate the association of serum-resistant genotype sac-4 with two phenotypes: C1q-dependent virulence expressed in vivo and resistance to bactericidal activity of human serum expressed in vitro. Strains were also characterized by auxotyping and serotyping. Of 32 gonococcal strains, 15 (46.7%) caused C1q-dependent bacteremia in rat pups and were sac-4 positive and ser(r). However, of the 15 isolates, 13 (87%) represented strains associated with human PID and 2 (13%) were associated with LI. None of the strains that were completely serum-sensitive (ser(s)) and sac-4 negative produced C1q-dependent bacteremia in rat pups, suggesting that both ser(r) and sac-4 were required for infection. The serum-resistant recombinant recipient of sac-4 produced C1q-dependent bacteremia in the rat model similarly to the serum-resistant donor of sac-4; the serum-sensitive parent strain did not produce bacteremia. These data suggest that sac-4-mediated serum resistance conferred C1q-dependent virulence and is a unique characteristic associated with PID. These newly identified features may contribute to the understanding of the pathogenic mechanism of PID-associated strains and open perspectives for establishing novel diagnostic methods.

Development of experimental model of chronic pyelonephritis with Escherichia coli O75:K5:H-bearing Dr fimbriae: mutation in the dra region prevented tubulointerstitial nephritis.

Escherichia coli that express Dr fimbriae and related adhesins recognize the common receptor decay accelerating factor. E. coli strains that express adhesins of the Dr family were postulated to be associated with cystitis (30-50%), pregnancy-associated pyelonephritis (30%), and chronic diarrhea (50%). In this study, we investigated the hypothesis that E. coli renal interstitial binding mediated by the Dr adhesin may be important for the development of chronic pyelonephritis. An insertional dra mutant, E. coli DR14, of the clinical E. coli isolate IH11128 bearing Dr fimbriae, was constructed and used to characterize persistence of infection and interstitial tropism in an experimental model of ascending pyelonephritis. Quantitative cultures of kidney homogenates indicated that Dr hemagglutinin positive (Dr+) E. coli IH11128 established a 1-yr colonization of renal tissue. In the Dr hemagglutinin negative (Dr-) group, 50% of animals cleared infection within 20 wk and 100% between 32 to 52 wk. Dr+ E. coli colonized the renal interstitium. Significant histological changes corresponding to tubulointerstitial nephritis including interstitial inflammation, fibrosis, and tubular atrophy were found in the kidney tissue of the Dr+ but not the Dr- group. A substantial amount of fimbrial antigen was detected in the parenchymal regions affected by interstitial inflammation and fibrosis. The obtained results are consistent with the hypothesis that mutation within the dra region, affecting E. coli binding to tubular basement membranes, prevented renal interstitial tropism and the development of the changes characteristically seen in tubulointerstitial nephritis.

Rapid cyclic changes in density and accessibility of endometrial ligands for Escherichia coli Dr fimbriae.

The mechanisms of developing infection in young, noncompromised individuals are well understood. Colonization is prerequisite for the development of infection. In human, ligands serving bacterial colonization belong to common antigens. Consequently, a majority of individuals should be sensitive to infection at all times. We hypothesize that the temporal patterns of some infections and sensitivity to them are associated with sudden changes in the density and accessibility of common receptors. Endometrial samples from women having normal menstrual cycles were examined for histological location, receptor density, and in situ hybridization of Dr (decaying-accelerating factor) ligands for Escherichia coli Dr fimbriae. Significant up-regulation and luminal expression of Dr ligands occurred during the secretory phase, whereas receptors were expressed in the basement membrane and in smaller quantities during the proliferative phase. This observation agrees with our hypotheses that some ligands recognized by bacterial adhesins change their compartmentalization and, most importantly, that they up-regulate expression at specific times.

Association of colony variation in Serratia marcescens with the differential expression of protease and type 1 fimbriae.

Several clinical isolates of Serratia marcescens were found to dissociate on peptone glycerol agar into colonies with red and pink or white and gray phenotypes that differ in the expression of proteolytic activity and mannose-sensitive type of hemagglutination. Colonies of red and white type were proteolytically active but did not express hemagglutination, whereas pink and gray colonies were protease-deficient but agglutinated guinea pig erythrocytes. Site-directed mutagenesis of a red laboratory strain S. marcescens SM6 resulted in selection of protease negative derivative prt::G7 which expressed the pink phenotype with hemagglutinating activity. It is suggested that a DNA-regulatory element may be involved in this type of colony variation.

Dr fimbriae coding region associated hemolytic activity of Escherichia coli.

We investigated the hemolytic activity of Escherichia coli strain EC901 carrying plasmid pBJN406 containing genes draA-E involved in expression of the mannose-resistant Dr hemagglutinin, and in its isogenic insertion mutants devised with Tn5, Tn3, and TnphoA. While E. coli BN406 displayed rapid hemolytic activity against equine erythrocytes, insertion mutations in draD and draE, but not in draA, draB, and draC, abolished all hemolytic activity. These data suggest a role for draD and draE in the expression of hemolysis.

dra-related X adhesins of gestational pyelonephritis-associated Escherichia coli recognize SCR-3 and SCR-4 domains of recombinant decay-accelerating factor.

Bacterial adhesins are important virulence factors that allow colonization of the human urogenital tract by Escherichia coli. Adhesins of the Dr family have been found to be more frequently expressed in strains associated with symptomatic urinary tract infections. Because of the high frequency of symptomatic urinary tract infections during pregnancy, we screened E. coli isolates from 64 gestational pyelonephritis patients for the expression of Dr and X adhesins to address their potential virulence roles in this population. Using PCR and primers for the afaB gene, we detected dra-related operons in 17 isolates (27%). On the basis of the lack of hemagglutination of Dr(a-) erythrocytes containing a point mutation in the decay-accelerating factor (DAF) short consensus repeat-3 (SCR-3) domain, 12 of these strains were categorized as classical Dr adhesins. The hemagglutination of O erythrocytes by Dr+ strains was blocked or reduced by a monoclonal antibody to the DAF SCR-3 domain. The remaining five dra-positive strains agglutinated Dr(a-) erythrocytes. Monoclonal antibody to the DAF SCR-3 domain failed to block O-erythrocyte hemagglutination. Adhesins in these strains did not fulfill criteria for Dr hemagglutinins because of the undefined receptor specificities and were categorized as X. E. coli strains bearing dra-related X adhesins bound to DAF cDNA-transfected Chinese hamster ovary cells. Three of these dra-related X-adhesin-bearing E. coli strains failed to attach to the SCR-3 delta deletion transfectant, which suggested that binding sites were located in the SCR-3 domain but outside the region blocked by the monoclonal anti-SCR-3 immunoglobulin G. The binding sites of the remaining two dra-related X adhesin strains were localized to the SCR-4 domain, as the attachment was shown to be abolished on an SCR-4 delta mutant but unaffected by an SCR-3 delta deletion. The heterogeneity in the binding sites of E. coli DAF (Dr) family adhesins from gestational pyelonephritis isolates may reflect the ability of the adhesins to evolve to recognize alternate peptide epitopes for efficient colonization.

Decay-accelerating factor is expressed in the human endometrium and may serve as the attachment ligand for Dr pili of Escherichia coli.

PROBLEM: We evaluated the hypothesis that different tissue substructures in uteri may express decay accelerating factor (DAF), a complement regulatory protein that also may serve as ligand for bacterial attachment. METHOD: Purified Dr pili, anti-Dr pili IgG, anti-DAF (SCR-3) IgG, and fluorescein-isothiocyanate-conjugated secondary IgG were used for binding and inhibition experiments. RESULT: We observed staining of endometrial glands, spiral arterioles, and myometrial arteries with Dr adhesin (pili) and anti-DAF (SCR-3) IgG, and found variation in distribution and amount of Dr ligands in different individuals. Anti-DAF (SCR-3) IgG blocked the binding of Dr pili to the endometrium. CONCLUSION: Presence of DAF in endometrium may protect tissues from complement-induced damage. Differences between individuals in DAF density in the endometrium may affect sensitivity to attachment of Dr-bearing E. coli and/or complement activation.

Extracellular haemolytic activity of Serratia marcescens.

Blood agar medium with dialysis membrane mounted between two layers of agar was applied to study the haemolytic activity of 28 strains of Serratia marcescens. Two kinds of lytic substances differing with their ability to pass through dialysis membrane were found. Haemolytic activity was not detected in cell-free filtrates from liquid cultures. The discrepancies between haemolytic activity in blood agar media and activity of liquid cultures were observed. Stable attachment of bacterial cells to the erythrocytes was not necessary to lysis. The possibility of extracellular haemolysin is discussed.

Haemolytic activity of Morganella morganii strains.

Haemolytic activity on solid and liquid media of 103 Morganella morganii strains isolated from clinical sources was investigated. The ability to produce haemolysin was found in 42.7% of strains. All strains capable to produce haemolysin on blood agar media also revealed haemolytic activity in some liquid media. Haemolysins were found in the supernatants and filtrates of the cultures in peptone water but not in Brain Heart Infusion and Trypticase Soy Broth. The maximal titer of haemolysin was observed in the logarithmic phase of growth. Heating and incubation with trypsin led to complete loss of haemolytic activity.