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.     

Molecular Analysis of Asymptomatic Bacteriuria Escherichia coli Strain VR50 Reveals Adaptation to the Urinary Tract by Gene Acquisition

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.     

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.     

Epithelial invasion by Escherichia coli bearing Dr fimbriae is controlled by nitric oxide-regulated expression of CD55

We previously reported that inhibition of nitric oxide (NO) increases the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr fimbria (Dr⁺). Epithelial binding and invasion by Dr⁺ E. coli has also been shown to be dependent upon the expression level of the cellular receptor decay-accelerating factor (DAF; CD55). Here, we hypothesize that NO-related severity of infection could be mediated by changes in DAF expression and in the rate of epithelial invasion. The cellular basis of NO effects on epithelial invasion with Dr⁺ E. coli was studied using Ishikawa endometrial carcinoma cells as an in vitro model of the human endometrial epithelium. Initially, we show that Ishikawa cells produce NO and express both NO synthase enzymes, NOS II and NOS III, and DAF protein. We next tested the abilities of both Dr⁺ E. coli and a Dr⁻ E. coli mutant to invade Ishikawa cells, and invasion was seen only with Dr⁺ E. coli. Invasion by Dr⁺ E. coli was decreased by elevated NO production and increased by NO inhibition. Elevated NO production significantly decreased DAF protein and mRNA expression in Ishikawa cells in a time- and dose-dependent manner. Here, we propose that in vitro invasion of an epithelial cell line is directly related to NO-regulated expression of DAF. The significance of NO-regulated receptor-ligand invasion is that it may represent a novel unrecognized phenomenon of epithelial defense against infection.     

Clonal Diversity of Chilean Isolates of Enterohemorrhagic Escherichia coli from Patients with Hemolytic-Uremic Syndrome, Asymptomatic Subjects, Animal Reservoirs, and Food Products

To determine clonal relationship among Chilean enterohemorrhagic Escherichia coli (EHEC) strains from different sources (clinical infections, animal reservoirs, and food), 54 EHEC isolates (44 of E. coli O157, 5 of E. coli O111, and 5 of E. coli O26) were characterized for virulence genes by colony blot hybridization and by pulsed-field gel electrophoresis (PFGE). By colony blotting, 12 different genotypes were identified among the 44 E. coli O157 isolates analyzed, of which the genetic profile stx₁⁺ stx₂⁺ hly⁺ eae⁺ was the most prevalent. All human O157 strains that were associated with sporadic cases of hemolytic-uremic syndrome (HUS) carried both the stx₁ and stx₂ toxin-encoding genes and were eaeA positive. Only 9 of 13 isolates from human controls were stx₁⁺ stx₂⁺, and 8 carried the eaeA gene. Comparison of profiles obtained by PFGE of XbaI-digested genomic DNA showed a great diversity among the E. coli O157 isolates, with 37 different profiles among 39 isolates analyzed. Cluster analysis of PFGE profiles showed a wide distribution of clinical isolates obtained from HUS cases and asymptomatic individuals and a clonal relationship among O157 isolates obtained from HUS cases and pigs. Analysis of virulence genes showed that a correlation exists among strains with the genotype stx₁⁺ stx₂⁺ eae⁺ and pathogenic potential. A larger difference in the PFGE restriction patterns was observed among the EHEC strains of serogroups O26 and O111. These results indicate that several different EHEC clones circulate in Chile and suggest that pigs are an important animal reservoir for human infections by EHEC. Guidelines have been proposed for better practices in the slaughter of animals in Chile.     

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.     

Immunological Characterization of Escherichia coli O157:H7 Intimin γ1

Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a(+) expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a(+). Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin typesα (O serogroups 86, 127, and 142), β1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), γ1 (O serogroups 55, 145, and 157), γ2 (O serogroups 111 and 103), and (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intγ1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin γ1-producing E. coli strains such as E. coli O157:H7.     

Pathogenicity of an Enterotoxigenic Escherichia coli Hemolysin (hlyA) Mutant in Gnotobiotic Piglets

Pigs infected with hemolytic F4⁺ strains of enterotoxigenic Escherichia coli often develop septicemia secondary to intestinal infection. We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4⁺ enterotoxigenic E. coli to cause septicemia in swine following oral inoculation. Inactivation of the hemolysin structural gene (hlyA) did not decrease the incidence of septicemia in the gnotobiotic piglet model.     

Cytotoxicity of Hemolytic, Cytotoxic Necrotizing Factor 1-Positive and -Negative Escherichia coli to Human T24 Bladder Cells

Approximately one-half of Escherichia coli isolates from patients with cystitis or pyelonephritis produce the pore-forming cytotoxin hemolysin, a molecule with the capacity to lyse erythrocytes and a range of nucleated cell types. A second toxin, cytotoxic necrotizing factor 1 (CNF1), is found in approximately 70% of hemolytic, but rarely in nonhemolytic, isolates. To evaluate the potential interplay of these two toxins, we used epidemiological and molecular biologic techniques to compare the cytotoxicity of hemolytic, CNF1⁺, and CNF1⁻ cystitis strains toward human T24 bladder epithelial cells in vitro. A total of 29 isolates from two collections of cystitis-associated E. coli were evaluated by using methylene blue staining of bladder monolayers at 1-h intervals after inoculation with each strain. Most (20 of 29) isolates damaged or destroyed the T24 monolayer (less than 50% remaining) within 4 h after inoculation. As a group, CNF1⁺ isolates from one collection (11 strains) were less cytotoxic at 4 h than the CNF1⁻ strains in that collection (P = 0.009), but this pattern was not observed among isolates from the second collection (18 strains). To directly evaluate the role of CNF1 in cytotoxicity of hemolytic E. coli without the variables present in multiple clinical isolates, we constructed mutants defective in production of CNF1. Compared to the CNF1⁺ parental isolates, no change in cytotoxicity was detected in these cnf1 mutants. Our results indicate that CNF1 does not have a detectable effect on the ability of hemolytic E. coli to damage human bladder cell monolayers in vitro.     

Hemolytically Active (Acylated) Alpha-Hemolysin Elicits Interleukin-1β (IL-1β) but Augments the Lethality of Escherichia coli by an IL-1- and Tumor Necrosis Factor-Independent Mechanism

Many pathogenic Escherichia coli produce the toxin alpha-hemolysin (Hly), and lipopolysaccharide (LPS), interleukin-1 (IL-1), and tumor necrosis factor (TNF) have all been recognized as important effector molecules during infections by gram-negative organisms. Despite the characterization of many in vitro effects of hemolysin, no direct relationship has been established between hemolysin, LPS, proinflammatory cytokine production, and E. coli-induced mortality. Previously, we have shown in vivo that hemolysin elicits a distinct IL-1α spike by 4 h into a lethal hemolytic E. coli infection. Using three transformed E. coli strains, WAF108, WAF270, and WAH540 (which produce no Hly [Hly^null], acylated Hly [Hly^active], or nonacylated Hly [Hly^inactive], respectively), we sought to determine the specific roles of hemolysin acylation, LPS, IL-1, and TNF in mediating the lethality of E. coli infection in mice. WAF270 was 100% lethal in BALB/c, C3H/HeJ, and C57BL/6 mice; in mice pretreated with antibody to the type 1 IL-1 receptor; in type 1 IL-1 receptor-deficient mice; and in dual (type 1 IL-1 receptor-type 1 TNF receptor)-deficient mice at doses which were nonlethal (0%) with both WAF108 and WAH540. At lethal doses, WAF270 killed by 6 ± 2.3 h while WAF108 and WAH540 killed at 36 ± 9.4 and 36 ± 13.8 h, respectively. These differences in mortality were not due to IL-1 or TNF release, and the enhanced expression of LPS, which corresponded to Hly expression, was not likely the primary factor causing mortality. We demonstrate that bacterial fatty acid acylation of hemolysin is required in order for it to elicit IL-1 release by monocytes and to confer its virulence on E. coli.     

Increased rate of apoptosis and diminished phagocytic ability of human neutrophils infected with Afa/Dr diffusely adhering Escherichia coli strains

The proinflammatory effect of Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains have been recently demonstrated in vitro by showing that polymorphonuclear leukocyte (PMN) transepithelial migration is induced after bacterial colonization of apical intestinal monolayers. The effect of Afa/Dr DAEC-PMN interaction on PMN behavior has been not investigated. Because of the putative virulence mechanism of PMN apoptosis during infectious diseases and taking into account the high level of expression of the decay-accelerating factor (DAF, or CD55), the receptor of Afa/Dr DAEC on PMNs, we sought to determine whether infection of PMNs by Afa/Dr DAEC strains could promote cell apoptosis. We looked at the behavior of PMNs incubated with Afa/Dr DAEC strains once they had transmigrated across polarized monolayers of intestinal (T84) cells. Infection of PMNs by Afa/Dr DAEC strains induced PMN apoptosis characterized by morphological nuclear changes, DNA fragmentation, caspase activation, and a high level of annexin V expression. However, transmigrated and nontransmigrated PMNs incubated with Afa/Dr DAEC strains showed similar elevated global caspase activities. PMN apoptosis depended on their agglutination, induced by Afa/Dr DAEC, and was still observed after preincubation of PMNs with anti-CD55 and/or anti-CD66 antibodies. Low levels of phagocytosis of Afa/Dr DAEC strains were observed both in nontransmigrated and in transmigrated PMNs compared to that observed with the control E. coli DH5alpha strain. Taken together, these data strongly suggest that interaction of Afa/Dr DAEC with PMNs may increase the bacterial virulence both by inducing PMN apoptosis through an agglutination process and by diminishing their phagocytic capacity.     

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.     

Oral Immunization with a Salmonella typhimurium Vaccine Vector Expressing Recombinant Enterotoxigenic Escherichia coli K99 Fimbriae Elicits Elevated Antibody Titers for Protective Immunity

Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mortality in piglets and newborn calves. In an effort to develop a safe and effective vaccine for the prevention of F5⁺ ETEC infections, a balanced lethal asd⁺ plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd⁺. Introduction of this plasmid into an attenuated Salmonella typhimurium Δaro Δasd strain, H683, resulted in strain AP112, which stably expresses E. coli K99 fimbriae. A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks. IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer’s patches (PP), and spleens of vaccinated and control BALB/c mice. This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen. These antibodies were important for protective immunity. One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a control Salmonella vector. These results confirm that oral Salmonella vaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity.     

Protective Effect of Immunization with Heat-Labile Enterotoxin in Gnotobiotic Rats Monocontaminated with Enterotoxigenic Escherichia coli

The protective effect of active immunization with a purified preparation of the polymyxin-release form of Escherichia coli heat-labile enterotoxin (LT), administered using a parenteral prime and peroral boosts given after ablation of gastric secretion by means of cimetidine, was assessed in gnotobiotic rats which were challenged by monocontamination with enterotoxigenic strains of E. coli. Water transport was evaluated by the in vivo marker perfusion technique at weekly intervals over a 3-week period after contamination. Water transport in unimmunized control rats was consistently in absorption in those contaminated by a nontoxigenic strain, in secretion during only week 2 in those contaminated by an LT⁺/− strain, in secretion during weeks 2 and 3 in those contaminated by an LT⁺/ST⁺ (heat-stable enterotoxin) strain, and consistently in absorption in those contaminated by an −/ST⁺ strain. Rats immunized with a booster dosage of 250 μg had a significant increase (P < 0.001) in net water absorption as compared to unimmunized rats, with values in the borderline range of absorption, when challenged with either the LT⁺/− or LT⁺/ST⁺ strains. Rats immunized with a 10-fold-higher boosting dosage had a significant increase (P < 0.001) in net water absorption as compared to those boosted at the lower dosage; water absorption was within the normal range. There was no difference between the ileal bacterial counts of unimmunized and immunized rats challenged by the various strains. These observations indicate that this immunization program provides complete protection in an animal model against challenge by intestinal contamination with enterotoxigenic strains of E. coli which produce LT, either alone or in combination with ST.     

Defects in Innate Immunity Predispose C57BL/6J-Leprdb/Leprdb Mice to Infection by Staphylococcus aureus

Foot and ankle infections are the most common cause of hospitalization among diabetic patients, and Staphylococcus aureus is a major pathogen implicated in these infections. Patients with insulin-resistant (type 2) diabetes are more susceptible to bacterial infections than nondiabetic subjects, but the pathogenesis of these infections is poorly understood. C57BL/6J-Lepr^db/Lepr^db (hereafter, db/db) mice develop type 2 diabetes due to a recessive, autosomal mutation in the leptin receptor. We established a S. aureus hind paw infection in diabetic db/db and nondiabetic Lepr^+/+ (+/+) mice to investigate host factors that predispose diabetic mice to infection. Nondiabetic +/+ mice resolved the S. aureus hind paw infection within 10 days, whereas db/db mice with persistent hyperglycemia developed a chronic infection associated with a high bacterial burden. Diabetic db/db mice showed a more robust neutrophil infiltration to the infection site and higher levels of chemokines in the infected tissue than +/+ mice. Blood from +/+ mice killed S. aureus in vitro, whereas db/db blood was defective in bacterial killing. Compared with peripheral blood neutrophils from +/+ mice, db/db neutrophils demonstrated a diminished respiratory burst when stimulated with S. aureus. However, bone marrow-derived neutrophils from +/+ and db/db mice showed comparable phagocytosis and bactericidal activity. Our results indicate that diabetic db/db mice are more susceptible to staphylococcal infection than their nondiabetic littermates and that persistent hyperglycemia modulates innate immunity in the diabetic host.Approximately 2 million of the estimated 16 million individuals with diabetes in the United States will develop chronic foot ulcers or infections during the course of their disease (38). An infection is initiated when the skin barrier is breached and bacteria, mostly skin commensals, gain access to the underlying tissues. Although limb-threatening infections are usually polymicrobial, Staphylococcus aureus is a major determinant of these infections (21). S. aureus is the predominant pathogen in non-limb-threatening infections, particularly in patients who have not received antimicrobial therapy (5, 26, 27). The emergence of S. aureus strains resistant to multiple antibiotics has made treatment of staphylococcal infections especially problematic. Methicillin-resistant S. aureus strains have become increasingly prevalent among both nosocomial and community-acquired infections within the United States (22, 36, 43). The prevalence of methicillin-resistant S. aureus is higher among diabetic patients than in the general population (11, 44, 45). Seven S. aureus strains resistant to vancomycin have been isolated in the United States, and four of these strains were isolated from patients with diabetes (42, 50). Complications of type 2 diabetes such as peripheral neuropathy and vasculopathy contribute to delayed wound healing. Although the increased susceptibility of the diabetic host to bacterial infections is well established, the chronicity of these infections is poorly understood. A consistent defect in the humoral or cell-mediated host immune system of diabetic patients has not been demonstrated. However, deficiencies in the host innate immune response are apparent since clinical investigations have indicated that phagocytes from type 2 diabetic patients are in a heightened state of oxidative stress and have impaired bactericidal activity and chemotaxis (6, 15, 41, 48). Clearly, numerous pathophysiologic perturbations contribute to the recurrence of soft tissue and bone infections in the lower extremity of patients with diabetes.C57BL/6J-Lepr^db/Lepr^db (hereafter, db/db) mice are a valuable model of type 2 diabetes since they are hyperglycemic and resistant to insulin, and they experience peripheral neuropathy, delayed wound healing, and myocardial disease. In this study we inoculated the hind paws of wild-type Lepr^+/+ (+/+) and diabetic db/db mice with S. aureus and evaluated the course of the ensuing infection in each host type, as well as the resultant host innate immune response to infection. Diabetic mice that were ≥4 months of age were more susceptible to staphylococcal infection than age-matched nondiabetic control animals. The db/db mice showed a heightened inflammatory response that was characterized by defects in phagocyte function.     

Can Results Obtained with Commercially Available MicroScan Microdilution Panels Serve as an Indicator of β-Lactamase Production among Escherichia coli and Klebsiella Isolates with Hidden Resistance to ExpandedSpectrum Cephalosporins and Aztreonam?

Among clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca, there is an ever-increasing prevalence of β-lactamases that may confer resistance to newer β-lactam antibiotics that is not detectable by conventional procedures. Therefore, 75 isolates of these species producing well-characterized β-lactamases were studied using two MicroScan conventional microdilution panels, Gram Negative Urine MIC 7 (NU7) and Gram Negative MIC Plus 2 (N+2), to determine if results could be utilized to provide an accurate indication of β-lactamase production in the absence of frank resistance to expanded-spectrum cephalosporins and aztreonam. The enzymes studied included Bush groups 1 (AmpC), 2b (TEM-1, TEM-2, and SHV-1), 2be (extended spectrum β-lactamases [ESBLs] and K1), and 2br, alone and in various combinations. In tests with E. coli and K. pneumoniae and the NU7 panel, cefpodoxime MICs of ≥2 μg/ml were obtained only for isolates producing ESBLs or AmpC β-lactamases. Cefoxitin MICs of >16 μg/ml were obtained for all strains producing AmpC β-lactamase and only 1 of 33 strains producing ESBLs. For the N+2 panel, ceftazidime MICs of ≥4 μg/ml correctly identified 90% of ESBL producers and 100% of AmpC producers among isolates of E. coli and K. pneumoniae. Cefotetan MICs of ≥ 8 μg/ml were obtained for seven of eight producers of AmpC β-lactamase and no ESBL producers. For tests performed with either panel and isolates of K. oxytoca, MICs of ceftazidime, cefotaxime, and ceftizoxime were elevated for strains producing ESBLs, while ceftriaxone and aztreonam MICs separated low-level K1 from high-level K1 producers within this species. These results suggest that microdilution panels can be used by clinical laboratories as an indicator of certain β-lactamases that may produce hidden but clinically significant resistance among isolates of E. coli, K. pneumoniae, and K. oxytoca. Although it may not always be possible to differentiate between strains that produce ESBLs and those that produce AmpC, this differentiation is not critical since therapeutic options for patients infected with such organisms are similarly limited.     

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.     

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.     

Humoral Immunity against Capsule Polysaccharide Protects the Host from magA+ Klebsiella pneumoniae-Induced Lethal Disease by Evading Toll-Like Receptor 4 Signaling

Klebsiella pneumoniae magA (for mucoviscosity-associated gene A) is linked to the pathogenesis of primary pyogenic liver abscess, but the underlying mechanism by which magA increases pathogenicity is not well elucidated. In this study, we investigated the role of the capsular polysaccharides (CPS) in the pathogenesis of magA⁺ K. pneumoniae by comparing host immunity to magA⁺ K. pneumoniae and a ΔmagA mutant. We found that Toll-like receptor 4 recognition by magA⁺ K. pneumoniae was hampered by the mucoviscosity of the magA⁺ K. pneumoniae CPS. Interestingly, monoclonal antibodies (MAbs) against magA⁺ K. pneumoniae CPS recognized all of the K1 strains tested but not the ΔmagA and non-K1 strains. Moreover, the anti-CPS MAbs protected mice from magA⁺ K. pneumoniae-induced liver abscess formation and lethality. This indicates that the K1 epitope is a promising target for vaccine development, and anti-CPS MAbs has great potential to protect host from K1 strain-induced mortality and morbidity in diabetic and other immunocompromised patients in the future.     

Relative importance of T-cell subsets in monocytotropic ehrlichiosis: a novel effector mechanism involved in Ehrlichia-induced immunopathology in murine ehrlichiosis

Infection with gram-negative monocytotropic Ehrlichia strains results in a fatal toxic shock-like syndrome characterized by a decreased number of Ehrlichia-specific CD4⁺ Th1 cells, the expansion of tumor necrosis factor alpha (TNF-α)-producing CD8⁺ T cells, and the systemic overproduction of interleukin-10 (IL-10) and TNF-α. Here, we investigated the role of CD4⁺ and CD8⁺ T cells in immunity to Ehrlichia and the pathogenesis of fatal ehrlichiosis caused by infection with low- and high-dose (10³ and 10⁵ bacterial genomes/mouse, respectively) ehrlichial inocula. The CD4⁺ T-cell-deficient mice showed exacerbated susceptibility to a lethal high- or low-dose infection and harbored higher bacterial numbers than did wild-type (WT) mice. Interestingly, the CD8⁺ T-cell-deficient mice were resistant to a low dose but succumbed to a high dose of Ehrlichia. The absence of CD8⁺ T cells abrogated TNF-α and IL-10 production, reduced tissue injury and bacterial burden, restored splenic CD4⁺ T-cell numbers, and increased the frequency of Ehrlichia-specific CD4⁺ Th1 cells in comparison to infected WT mice. Although fatal disease is perforin independent, our data suggested that perforin played a critical role in controlling bacterial burden and mediating liver injury. Similar to WT mice, mortality of infected perforin-deficient mice was associated with CD4⁺ T-cell apoptosis and a high serum concentration of IL-10. Depletion of IL-10 restored the number of CD4⁺ and CD8⁺ T cells in infected WT mice. Our data demonstrate a novel mechanism of immunopathology in which CD8⁺ T cells mediate Ehrlichia-induced toxic shock, which is associated with IL-10 overproduction and CD4⁺ T-cell apoptosis.