Renal allograft injury is associated with urinary tract infection caused by Escherichia coli bearing adherence factors.

Urinary tract infections are the most common infection in renal transplant patients and Escherichia coli (E. coli) is the most common clinical isolate. Although acute allograft injury (AAI) secondary to urinary tract infection (UTI) has been reported, the incidence of AAI associated with UTI, the virulence factors express by uropathic E. coli and whether virulence factors are associated with renal allograft outcome have not been described. We collected E. coli from our renal transplant patients with UTI, determined O:H serotypes, P and Dr fimbriae expression and the clinical presentation and allograft function during the UTI and post-UTI period. Pyelonephritis occurred in 40% of our patients, 82% of which had AAI (>20% increase in SCr). Sixty-two percent of E. coli isolates that expressed P fimbriae were associated with AAI, whereas only 29% that did not express P fimbriae had AAI (p = 0.03). The pattern of P fimbriae and O serotypes differed from reported isolates, as the P fimbriae PapG class II and the O25 serotype were the most common. Dr adhesin was expressed on 7 isolates, including 2 of 3 with urosepsis. We propose a unique pattern of uropathogenic serotypes and adherence factors contribute to acute allograft injury in renal transplant patients with UTI.

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.

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.

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.

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.

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.

Localized increase in nitric oxide production and the expression of nitric oxide synthase isoforms in rat uterus with experimental intrauterine infection.

OBJECTIVE: We recently reported that nitric oxide was associated with increased mortality among pregnant rats with intrauterine infection. In our current study we investigated the expression of different isoforms of nitric oxide synthases and nitric oxide in the nonpregnant rat uterus with experimental intrauterine infection. STUDY DESIGN: Pathogenic Escherichia coli was inoculated into the uterine lumen of ovariectomized rats. Animals were killed after inoculation, and uterine horns were collected for assessing nitric oxide production with high-performance liquid chromatography and nitric oxide synthase (type II and type III) protein expression with Western immunoblotting and immunofluorescence methods. RESULTS: (1) Nitric oxide production increased in the infected uterine horn in a time-dependent manner after intrauterine infection but did not increase in the uninfected horn. (2) Nitric oxide synthase type III protein contents did not show a difference between infected and uninfected horns, and type III nitric oxide synthase was expressed by the epithelial cells and smooth muscle cells. (3) Type II nitric oxide synthase was abundantly expressed in infected horns but was not expressed in uninfected horns. Immunofluorescence data indicated that macrophages and natural killer cells, located in the endometrial layer clustering around epithelial cells, expressed type II protein. CONCLUSION: We suggest that localized increase in type II nitric oxide synthase expression and nitric oxide production occurs in response to intrauterine infection and that the nitric oxide system may play a role in host response to restrict the infection.

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.

Gestational pyelonephritis--associated Escherichia coli isolates represent a nonrandom, closely related population.

OBJECTIVE: A select group of Escherichia coli strains known as uropathogenic cause pyelonephritis in nonpregnant individuals. We investigated whether Escherichia coli from gestational pyelonephritis represent a random population or possess common uropathogenic characteristics. STUDY DESIGN: Repetitive element sequence-based polymerase chain reaction, plasmid profiles, hemolysin, and O serotypes were assayed from Escherichia coli isolates of 57 pregnant patients with acute pyelonephritis at different gestational ages. RESULTS: The majority of the first trimester isolates fell primarily into repetitive element sequence-based patterns 1 and 3 and O6, O15, and O75 serotypes. Second-trimester isolates had multiple patterns with high-frequency repetitive element sequence-based polymerase chain reaction 1 and 5 and an unknown (OX) serotype. Pattern 3, predominantly O75 serotype, was found primarily among third-trimester isolates. CONCLUSION: It is likely that Escherichia coli associated with acute pyelonephritis during different trimesters of pregnancy represents nonrandom closely related isolates, and some of these strains may be characteristic in pregnant patients only.

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.

Gonococcal infection in a nonhuman host is determined by human complement C1q.

Human C1q displayed a dose-dependent protection of gonococcal cells (GC) from the bactericidal effect of newborn rat serum. All rat pups injected with C1q-preincubated GC developed bacteremia, while none of the animals injected with GC only were infected. After clearance of bacteremia at day 6, live GC could still be recovered from tested organs, including the liver. Preincubation of GC with higher concentrations of C1q was associated with increased morbidity. In contrast to human serum as a source of C1q, rat, rabbit, and mouse sera did not increase the in vivo virulence of Neisseria gonorrhoeae. C1q-deficient human serum, heat-inactivated C1q or human serum, type IV collagen, and complement C3 were inefficient in inducing infection. Experimental infection by C1q-preincubated GC was inhibited by anti-C1q antibodies in a dose-dependent fashion, demonstrating a causal effect of C1q function. This report demonstrates the novel finding that human C1q, a component of the human immune system with a general function for elimination of infection, may increase GC virulence and result in the development of disseminated infection in a nonhuman host.

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.

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.

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.

Human cultured intestinal cells express attachment sites for uropathogenic Escherichia coli bearing adhesins of the Dr adhesin family.

We have recently demonstrated that cultured human intestinal HT-29 and Caco-2 cell lines express receptors for the F1845 fimbrial adhesin harbored by the diarrheagenic C1845 Escherichia coli (Kernéis et al., Infect. Immun. 59 (1991) 4013-4018). This adhesin belongs to a family of adhesins including the Dr hemagglutinin and the afimbrial adhesin AFA-I harbored by uropathogenic E. coli. Here we investigated the cell association of laboratory E. coli strains expressing the Dr hemagglutinin and the afimbrial adhesin AFA-I with human cultured enterocyte-like or mucosecreting cells. We observed that the E. coli strains bearing these adhesins adhere both to human intestinal undifferentiated and differentiated fluid-transporting cells, and to mucus-secreting cells. This result strongly suggests a high capacity of intestinal colonization for the uropathogenic E. coli harboring adhesive factors belonging to the Dr adhesin family. These results further corroborate the intestinal colonization by uropathogenic E. coli of the Dr family related to the fecal-perineal-urethral hypothesis of urinary tract infection pathogenesis.

pap-2-encoded fimbriae adhere to the P blood group-related glycosphingolipid stage-specific embryonic antigen 4 in the human kidney.

A subtype of P fimbriae, encoded by the pap-2 gene cluster, has been analyzed for agglutination of erythrocytes and for binding to cryostat sections of the human kidney. We have demonstrated that pap-2-encoded fimbriae are capable of binding to erythrocytes from some animal species and to human erythrocytes which express globoside and the LKE (stage-specific embryonic antigen 4 [SSEA-4]) antigen. The pap-2 fimbriae bind to Bowman's capsule in the human kidney. Monoclonal antibodies directed against glycosphingolipids were used for the detection of specific P blood group-related antigens in the human kidney and on erythrocytes. Preincubation of kidney sections with monoclonal antibody MC813-70, which binds to the SSEA-4 antigen, inhibited adherence of purified pap-2-encoded fimbriae to Bowman's capsule. We suggest that one receptor for pap-2-encoded fimbriae is the antigen known as LKE (Luke) on human erythrocytes or SSEA-4 in the tissues.