Expression of a cloned 987P adhesion-antigen fimbrial determinant in Escherichia coli K-12 strain HB101

The properties of three independent enterotoxigenic Escherichia coli isolates known to express 987P adhesion fimbriae in a manner subject to phase variation were examined. Phase variation could not be correlated with any major changes in the plasmid DNA content of these strains or with readily detectable changes in any other tested phenotypic markers. The 987P genetic determinant from one of these strains, E. coli 987, was cloned into the non-fimbriated E. coli K-12 strains HB101, and expressed, using the cosmid vector system. 987P fimbriae produced by cells harbouring these recombinant plasmids (987P+ phenotype) could not be distinguished from 987P fimbriae produced by strain 987. Expression of 987P fimbriae from some recombinant plasmids was unstable but none of the recombinants exhibited the phase variation phenotype displayed by the parental strain. One recombinant plasmid, pPM200, contained an insert of strain 987 DNA of ca. 33 kb. The HB101[pPM200] displayed a rather stable 987P+ phenotype, but this was not true for several hosts, since pPM200 acquired approx. 20-kb deletions following transformations of E. coli K-12 strains other than HB101. The deletions mapped to the same region of pPM200 irrespective of the host strain transformed. Cells harbouring the deleted plasmids did not express 987P fimbriae (987P- phenotype).     

Quinolone/fluoroquinolone susceptibility in Escherichia coli correlates with human polymicrobial bacteriuria and with in vitro interleukine-8 suppression

Urinary tract infections (UTIs) are frequently polymicrobial diseases mainly sustained by Escherichia coli in association with other opportunistic pathogens. Cystitis and pyelonephritis are usually accompanied by an inflammatory response, which includes neutrophil recruitment. Uropathogenic E. coli possess the ability to evade host defenses, modulating the innate immune response. The aim of this study was to determine whether particular E. coli strains correlate with polymicrobial bacteriuria and whether escape from the early host defenses and microbial synergy could lead to mixed UTIs. We evaluated 188 E. coli-positive urine samples and assessed the relationships among polymicrobism, neutrophil presence and several traits of E. coli isolates (virulence factors such as hlyA, fimA, papC and their relative products, i.e. hemolysin, type 1 and P fimbriae, and cnf1, their phylogenetic group) and their ability to suppress cytokine response in 5637 bladder epithelial cells. Escherichia coli susceptibility toward quinolones and fluoroquinolones, known to be linked to the pathogenicity of this species, was also considered. We found significant correlations among polymicrobial bacteriuria, absence of pyuria and quinolone/fluoroquinolone susceptibility of E. coli isolates and their enhanced capability to suppress interleukin-8 urothelial production when compared with the patterns induced by the resistant strains.     

Transepithelial neutrophil migration is CXCR1 dependent in vitro and is defective in IL-8 receptor knockout mice

Neutrophil migration across infected mucosal surfaces is chemokine dependent, but the role of chemokine receptors has not been investigated. In this study, chemokine receptors were shown to be expressed by epithelial cells lining the urinary tract, and to play an essential role for neutrophil migration across the mucosal barrier. Uroepithelial CXCR1 and CXCR2 expression was detected in human urinary tract biopsies, and in vitro infection of human uroepithelial cell lines caused a dramatic increase in both receptors. As a consequence, there was higher binding of IL-8 to the cells and the IL-8-dependent neutrophil migration across the infected epithelial cell layers was enhanced. Abs to IL-8 or to the CXCR1 receptor inhibited this increase by 60% (p<0.004), but anti-CXCR2 Abs had no effect, suggesting that CXCR1 was the more essential receptor in this process. Similar observations were made in the mouse urinary tract, where experimental infection stimulated epithelial expression of the murine IL-8 receptor, followed by a rapid flux of neutrophils into the lumen. IL-8 receptor knockout mice, in contrast, failed to express the receptor, their neutrophils were unable to cross the epithelial barrier, and accumulated in massive numbers in the tissues. These results demonstrate that epithelial cells express CXC receptors and that infection increases receptor expression. Furthermore, we show that CXCR1 is required for neutrophil migration across infected epithelial cell layers in vitro, and that the murine IL-8 receptor is needed for neutrophils to cross the infected mucosa of the urinary tract in vivo.     

Fractionation of a bacterial cell population by adsorption to erythrocytes and yeast cells

Abstract Fluorochrome-labeled antibodies specific for either S or type-1 fimbriae of Escherichia coli were used to show that in broth culture the two fimbrial types of strain 3040 mostly occurred on different cells. 12% of the cells were nonfimbriated. A fractionation procedure that involved adsorption of bacterial cells onto erythrocytes and yeast cells was developed to isolate homogeneous subpopulations (S-fimbriated, type-1-fimbriated, and non-fimbriated) of. E. coli. The level of contamination in each isolated subpopulation was 4% at the highest. The method is useful in obtaining homogeneous bacterial populations for adherence studies and for purification of specific fimbrial antigens.     

Fimbriae in Escherichia Coli Isolated from the Small Intestine of Piglets

Ninety E. coli strains, isolated from piglets which had died from neonatal diarrhea, were tested for the presence of K88, K99, 987P and type 1 fimbriae. Two or more types of fimbriae were demonstrated in 14 of the strains, a single fimbria! type in 44 strains while in 32 strains no fimbriae were detected. Of the 14 E. coli strains with more than 1 type of fimbriae, 12;, 10, 8 and 4 strains showed K88, K99, 987P and type 1, respectively. Twelve E. coli strains were isolated from piglets which had died in the neonatal period without showing signs of neonatal diarrhea at necropsy. One strain showed 987P and 3 strains showed type 1 fimbriae, while the remaining 8 strains were unfimbriated. Sixteen fimbriated E. coli strains were subcultured in order to examine the stability of fimbrial expression in the strains. The K88 and the type 1 fimbriae were regularly expressed, while the K99 and 987P were inconsistently demonstrated.     

Precise and rapid assessment of Escherichia coli adherence to vaginal epithelial cells by flow cytometry

BACKGROUND: In the pathogenesis of Escherichia coli urinary tract infections (UTIs) in women, infecting bacteria adhere to vaginal and periurethral epithelial cells prior to ascending to the bladder and causing infection. Complex interactions among specific bacterial adhesins and various host factors appear to influence adherence of E. coli to mucosal surfaces such as the urogenital epithelium. To conduct population-based studies assessing host epithelial cell determinants that influence bacterial attachment, a method of measuring bacterial adherence utilizing clinically derived epithelial cell samples is needed. METHODS: We developed and standardized an efficient, accurate, high-throughput method for analyzing the adherence of uropathogenic E. coli to clinical samples containing a large number of exfoliated vaginal epithelial cells (VEC). Three wild-type E. coli strains isolated from women with UTI (IA2 expressing pap-encoded, class II fimbriae only; F24 expressing pap-encoded, class II and type 1 fimbriae; and F20, without pap-encoded or type I fimbriae) were transformed with gfpmut3, encoding green fluorescent protein, incubated with VECs, and analyzed by flow cytometry. RESULTS: Enumeration of the binding of each E. coli strain to 10,000 VECs showed reproducible, highly significant strain-dependent differences in adherence to VECs. Differential analysis of the relative contributions of type 1 pili and P fimbrial-mediated binding to the adherence phenotype was performed. It demonstrated that IA2 binding was dependent entirely on P fimbriae, whereas F24 binding was dependent on both P and type 1 fimbriae. CONCLUSIONS: This method has great potential for use in high-throughput analyses of clinically derived epithelial cell samples and will be valuable in population-based investigations of host-parasite interactions in UTI utilizing VECs collected from specific patient groups.     

Binding of K99 fimbriae of enterotoxigenic Escherichia coli to pig small intestinal mucin glycopeptides

Binding of purified K99 fimbriae to cryostat sections of pig small intestine was detected. Binding sites were located in the mucus layer, but not in the submucosal connective tissue. High-Mr mucin glycopeptides from pig small intestine were found to bind to K99-fimbriated enterotoxigenic Escherichia coli, in contrast to non-fimbriated cells. Sialic acid specificity of K99 fimbriae was demonstrated by the significant reduction in binding upon desialylation of mucin glycopeptides. The binding was saturable and the dissociation constant was estimated to be 6 x 10(-7) M. Fimbriated bacteria were calculated to possess 2.3 x 10(3) binding sites per cell.     

Analysis of the variability of S-fimbriae expression in an Escherichia coli pathogen

The uropathogenic Escherichia coli wild-type strain 536 produces S-fimbriae, P-related fimbriae and type I fimbriae. Using immuno-colony dot and ELISA techniques, variants were detected showing an increased degree of S-fimbrial production. It was demonstrated by immunofluorescence microscopy that in normal (wild-type) and hyper-S-fimbriated E. coli populations non-fimbriated cells also exist, and that the percentage of S-fimbriated and non-fimbriated bacteria was roughly identical in either population. Hyper-S-fimbriated variants could be stably maintained. The transition from wild-type to hyper-S-fimbriation, which occurs spontaneously, is markedly higher than vice versa. Southern blot analysis of the S fimbrial adhesin (sfa) determinants of normal and hyper-fimbriated strains revealed no marked difference in the gene structure.     

Establishment of a mouse model of cystitis and roles of type 1 fimbriated Escherichia coli in its pathogenesis.

The role of type 1 fimbriae in promoting bladder colonization and the course of Escherichia coli cystitis were examined with type 1 fimbriated strains of clinically isolated E. coli. In the experiments of mice in vivo, intact bladder epithelium showed natural resistance to the adherence of type 1 fimbriated and non-fimbriated E. coli. However, the exfoliation of bladder superficial cells by trypsinization before the bacterial inoculation promoted the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium. After colonization of E. coli, maximum numbers of E. coli and leukocytes were observed 3 days after inoculation. Nine days after inoculation, both of E. coli and leukocytes disappeared and the regeneration of superficial cells was observed. On the other hand, superficial cells in mice injected with phosphate-buffered saline or non-fimbriated E. coli regenerated 5 days after trypsinization. The present study demonstrated that the removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium in vivo and a new model of E. coli cystitis in mice was established. The model which we established is valuable for histopathological, immunological, and therapeutic studies.     

Type 1 fimbria-mediated adhesion of enteric bacteria to grass roots

Type 1 fimbriae of Klebsiella pneumoniae and Enterobacter agglomerans mediated bacterial adhesion to the roots of bluegrass, Poa pratensis. Purified, radiolabeled fimbriae bound to grass roots in vitro; binding was inhibited by alpha-methyl-d-mannoside or Fab fragments to the fimbriae. Anti-type 1 fimbriae Fab fragments and alpha-methyl-d-mannoside also inhibited adhesion of type 1-fimbriated bacteria to P. pratensis roots. It is proposed that associative nitrogen fixation by Klebsiella and Enterobacter strains also involves type 1 fimbriae, in addition to the type 3 fimbriae of Klebsiella spp. (T. K. Korhonen, E. Tarkka, H. Ranta, and K. Haahtela, J. Bacteriol. 155:860-865, 1983).     

Characterization of the second long polar (LP) fimbriae of Escherichia coli O157:H7 and distribution of LP fimbriae in other pathogenic E. coli strains

A second region containing five genes homologous to the long polar fimbrial operon of Salmonella enterica serovar Typhimurium is located in the chromosome of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A non-fimbriated E. coli K-12 strain carrying the cloned EHEC lpf (lpf2) genes expressed thin fibrillae-like structures on its surface and displayed reduced adherence to tissue culture cells. Neither mutation in the lpfA2 gene in either the parent or lpfA1 mutant strains showed an effect in adherence or in the formation of A/E lesions on HeLa cells. lpfA2 isogenic mutant strains adhere to Caco-2 cells almost as well as the wild-type at 5 h, but they were deficient in adherence at early time points. A collection of diarrheagenic E. coli strains were investigated for the presence of lpfA1 and lpfA2 and results showed that these genes are present in specific serogroups which are phylogenetically related. Our results suggest that LP fimbriae 2 may contribute to the early stages of EHEC adhesion and that genes encoding the major LP fimbrial subunits are present in a small group of EHEC and EPEC serotypes.     

大肠杆菌F18菌毛操纵子全基因克隆、表达及生物学活性

利用PCR技术以猪产肠毒素大肠杆菌F18标准菌株107/86和2134P基因组DNA为模板成功地扩增出编码F18ab和F18ac完整菌毛操纵子fed基因。将它们分别克隆入表达质粒载体pET-22b( ),结合酶切和核苷酸序列分析证明了PCR预期扩增产物的正确性。然后将克隆的重组载体DNA转化至大肠杆菌BL21(DE3),构建和筛选出分别含F18ab和F18ac完整fed基因的重组菌,经过IPTG诱导表达,在电镜下观察到上述两种重组菌能分别大量表达F18ab和F18ac菌毛。用热抽提法提纯其诱导表达的F18ab和F18ac菌毛,经SDS-PAGE电泳和考马斯亮蓝染色发现提纯后菌毛获单一分子量约为15kDa蛋白条带,免疫家兔后制备出高效价的兔抗血清,玻板凝集试验和Western blot结果表明:体外诱导表达的F18ab和F18ac菌毛具有和野生F18菌毛相同的抗原性。用表达F18ab和F18ac菌毛的上述2株重组菌分别进行小肠上皮细胞体外吸附试验和吸附抑制试验,结果表明:2株重组菌和野生菌株一样具有较强的粘附易感仔猪小肠上皮细胞的能力,而用表达F18ab和F18ac重组菌提纯的菌毛制备出兔抗血清都能有效地抑制上述重组菌或野生菌株对易感仔猪小肠上皮细胞的吸附结合。     

Macrophage inflammatory protein-2 is required for neutrophil passage across the epithelial barrier of the infected urinary tract

IL-8 is a major human neutrophil chemoattractant at mucosal infection sites. This study examined the C-X-C chemokine response to mucosal infection, and, specifically, the role of macrophage inflammatory protein (MIP)-2, one of the mouse IL-8 equivalents, for neutrophil-epithelial interactions. Following intravesical Escherichia coli infection, several C-X-C chemokines were secreted into the urine, but only MIP-2 concentrations correlated to neutrophil numbers. Tissue quantitation demonstrated that kidney MIP-2 production was triggered by infection, and immunohistochemistry identified the kidney epithelium as a main source of MIP-2. Treatment with anti-MIP-2 Ab reduced the urine neutrophil numbers, but the mice had normal tissue neutrophil levels. By immunohistochemistry, the neutrophils were found in aggregates under the pelvic epithelium, but in control mice the neutrophils crossed the urothelium into the urine. The results demonstrate that different chemokines direct neutrophil migration from the bloodstream to the lamina propria and across the epithelium and that MIP-2 serves the latter function. These findings suggest that neutrophils cross epithelial cell barriers in a highly regulated manner in response to chemokines elaborated at this site. This is yet another mechanism that defines the mucosal compartment and differentiates the local from the systemic host response.     

In vitro adhesion of n(2)-fixing enteric bacteria to roots of grasses and cereals

Nitrogen-fixing Klebsiella and Enterobacter strains isolated from several plants were assayed for fimbriae and for adhesion to plant roots in vitro. All eight Klebsiella strains formed type 3 fimbriae, and five strains also formed type 1 fimbriae; all 21 Enterobacter strains had type 1 fimbriae. Three strains of Klebsiella carrying either type 1, type 3, or no fimbriae were used as model organisms in developing an in vitro adhesion test. Adhesion was assayed with bacterial cells labeled with [H]leucine. Fifteen N(2)-fixing strains and the three model strains were compared for adhesion to the roots of seven grasses and five cereals. Type 3-fimbriated Klebsiella strains adhered better than the other strains, and type 3 fimbriae appeared to be major adhesins for the Klebsiella strains. Although variations between plants were observed, no host specificity for bacterial adhesion was found.     

Characterization of the type 3 fimbriae with different MrkD adhesins: possible role of the MrkD containing an RGD motif

Four novel mrkD alleles namely mrkD(V1), mrkD(V2), mrkD(V3), and mrkD(V4) were identified in seventeen Klebsiella pneumoniae meningitis strains using PCR-RFLP and sequence determination. Comparative analysis revealed a most variable region containing an RGD motif in the receptor domain of MrkD(V3). In order to determine if the sequence confers the K. pneumoniae mrkD(V3) the highest level of the fimbrial activity, a type 3 fimbriae display system was constructed in Escherichia coli. The E. coli JM109[pmrkABCD(V3)F] displaying meshwork-like fimbriae also had the most fimbrial activity, supporting a possible role of the varied sequences. In a dose-dependent manner, the GRGDSP hexapeptide appeared to inhibit the adhesion of the E. coli JM109[pmrkABCD(V3)F] to HCT-8, an ileocecal epithelial cell line. In addition, the adhesion activity was reduced by the addition of anti-alpha5beta1 integrin monoclonal antibody, indicating that the RGD containing region in MrkD(V3) is responsible for the binding of type 3 fimbriae to integrin.     

Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-alpha/CXC chemokine ligand 1 (GRO-alpha/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-alpha/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-alpha/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.     

The role of E. coli adhesiveness in the pathogenesis and clinical course of urinary tract infections]

An infection with E. coli is the most frequent cause of the urinary infections in childhood. Virulence depends on several factors out of which a principal role is played by the adhesion of bacteria to the urinary tract epithelium. Such a property have E. coli strains with adherence mannose-positive fimbriae of type P with antigens recognizing and binding glycolipid receptors on epithelial cells in the urinary tract. Children with such infections owe their "sensitivity+" (10% of the population) to genetically determined large number o receptors binding E. coli strains. Incidence and clinical course of the urinary tract infections have been analysed in the group of 184 children. Moreover, sequelae of the urinary tract infections with E. coli have been analysed in dependence on E. coli strain characteristics, i.e. presence or absence of adherent fimbriae from cases of cystitis and significant asymptomatic bacteriuria. Considering pathogenesis of the urinary tract infections as the result of interactions between bacteria and host, antigenic properties of adherent fimbriae might be used for preparation of a vaccine preventing such infections.