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。     

大肠杆菌K88ac菌毛操纵子fae全基因的克隆、表达及生物学活性初步研究

目的：在体外克隆和表达猪肠产毒性大肠杆菌（ETEC）K88ae菌毛操纵子,触结构基因,并检测重组菌毛的相关生物学活性。方法：利用长PCR技术以猪ETECK88ae株C83902基因组DNA为模板扩增编码K88菌毛操纵子触基因,克隆入表达质粒载体pBR322,构建和筛选重组质粒pBR322-fae,转化至不含任何菌毛的大肠杆菌EP株;电镜观察重组菌表面菌毛表达情况;用热抽提法提纯表达的重组菌毛;用纯化菌毛免疫小鼠制备高效价抗血清;用SDS-PAGE和Western blot检测重组菌毛的抗原性,用细胞黏附和黏附抑制试验检测其生物学活性。结果和结论：在电镜下观察到重组菌表面大量表达K88ae菌毛,该重组菌与兔抗K88ae菌毛单因子阳性血清、鼠抗K88ac菌毛单克隆抗体均产生凝集反应;纯化菌毛经SDS-PAGE,结构单位菌毛呈单一的相对分子质量约26×10^3的蛋白条带;纯化菌毛免疫小鼠后可制备出高效价的鼠抗血清,玻板凝集试验和Western blot结果表明体外表达的K88ae菌毛具有与K88ae野生菌毛相同的抗原性;猪小肠上皮细胞系黏附和黏附抑制实验结果表明重组EP菌和野生菌株一样具有较强的黏附猪小肠上皮细胞系的能力,而且提纯重组菌毛制备出的鼠抗血清能有效抑制上述重组菌或野生菌株对猪小肠上皮细胞系的黏附结合。     

Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine

Escherichia coli K12, which possess the K99 plasmid and synthesize K99 fimbriae (E. coli K99), cause severe neonatal diarrhea in piglets, calves, and lambs but not in humans. The organism binds specifically and with high affinity to only two glycolipids in piglet intestinal mucosa as demonstrated by overlaying glycolipid chromatograms with 125I-labeled bacteria. These glycolipids, which are N-glycolyl-GM3 (NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) and N-glycolylsialoparagloboside (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), occur at about 13 and 0.3 micrograms per gram wet weight of mucosa, respectively. E. coli K99 grown at 18 degrees C, a temperature at which the K99 fimbriae are not expressed, do not bind to these glycolipids. Of the standard glycolipids tested in solid phase binding assays, E. coli K99 binds with highest affinity to N-glycolylsialoparagloboside, with less affinity to N-glycolyl-GM3, and with very low affinity to N-acetylsialoparagloboside. The bacteria do not bind to GM3 (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer), GM2 (GalNAc beta 1-4[Neu-Ac alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), GM1 (Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), or several other N-acetylsialic acid-containing gangliosides and neutral glycolipids at the levels tested. N-Glycolylsialyl residues are found in the glycoproteins and glycolipids of piglets, calves, and lambs but not in the glycoproteins and glycolipids of humans. Possibly this distribution of sialyl derivatives explains the host range of infection by the organism.     

Binding specificity of Salmonella plasmid-encoded fimbriae assessed by glycomics

The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome encodes 12 intestinal colonization factors of the chaperone/usher fimbrial assembly class; however, the binding specificity is known for only one of these adhesins, known as type 1 fimbriae. Here we explored the utility of glycomics to determine the carbohydrate binding specificity of plasmid-encoded fimbriae from S. Typhimurium. A cosmid carrying the pef operon was introduced into Escherichia coli and expression of fimbrial filaments composed of PefA confirmed by flow cytometry and immune-electron microscopy. Plasmid-encoded fimbriae were purified from the surface of E. coli, and the resulting preparation was shown to contain PefA as the sole major protein component. The binding of purified plasmid-encoded fimbriae to a glycanarray suggested that this adhesin specifically binds the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc, also known as the Lewis X (Le(x)) blood group antigen. Results from the glycanarray were validated by enzyme-linked immunosorbent assay (ELISA) in which plasmid-encoded fimbriae bound Le(x)-coated wells in a concentration-dependent manner. The binding of plasmid-encoded fimbriae to Le(x)-coated wells could be inhibited by co-incubation with soluble Le(x) antigen. Our results establish glycomic analysis as a promising new approach for determining the carbohydrate binding specificity of bacterial adhesins.     

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.     

Purification and characterization of P fimbriae from an Escherichia coli strain isolated from a septicemic turkey

Abstract A pap + Escherichia coli isolate from a turkey with colisepticemia expressed P fimbriae with a major subunit of an apparent molecular mass of 18 kDa which reacted with anti-F11 serum. This fimbriae was purified and polyclonal antiserum was produced in rabbits. The N-terminal amino acid sequence of the major fimbrial subunit of the avian P fimbriae was identical to that of F11. On immunoblotting, the antiserum against the avian P fimbriae strongly reacted with the major subunit of the homologous fimbriae, with F11, and with F165₁ fimbriae. Some antigenic determinants on the major subunits of F13, F7₁, and F7₂ fimbriae, with a stronger reaction against F13 fimbriae, were also recognized. The F11 antiserum reacted similarly to the antiserum against avian P fimbriae although cross-reactions against F13, F7₁, and F7₂ fimbriae were equivalent. In a competitive enzyme-linked immunosorbent assay, serological differences were observed between the purified avian P fimbriae and F11. Thus, the avian P fimbriae is closely related but not identical to F11 fimbriae which are associated with E. coli isolated from human urinary tract infection.     

Purification and characterization of fimbriae from Salmonella enteritidis.

A human isolate of Salmonella enteritidis which displayed strong pellicle formation during static broth culture and mannose-sensitive hemagglutination produced fimbriae which were morphologically indistinguishable from type 1 fimbriae of members of the family Enterobacteriaceae. Fimbrin was purified to homogeneity, and the apparent molecular weight (Mr, 14,400) was markedly lower than that reported for the type 1 fimbrin of Salmonella typhimurium (Mr, 22,100). This fimbrin contained 40% hydrophobic amino acids and lacked cysteine. The sequence of the N-terminal 64 amino acids was determined, and sequence alignment revealed that although the 18 N-terminal residues of the S. enteritidis molecule shared considerable homology with Escherichia coli and S. typhimurium type 1 fimbrins, the S. enteritidis fimbrin lacked a 6- to 9-residue terminal sequence present in the other type 1 fimbrins and, after residue 18, shared little homology with the E. coli sequence. Antibodies raised to the purified S. enteritidis fimbrin bound to surface-exposed conformational epitopes on the native fimbriae and displayed pronounced serospecificity. These antibodies were used in the isolation of a nonfimbriated Tn10 insertion mutant which was unable to hemagglutinate.     

Type 1, P and S fimbriae, and afimbrial adhesin I are not essential for uropathogenic Escherichia coli to adhere to and invade bladder epithelial cells

Fimbrial (type 1, P, and S) and afimbrial adhesins, the unique virulence traits of uropathogenic Escherichia coli (UPEC), are well recognized for their role in the initial step of uropathogenesis. In this study, we investigated whether these adhesins are dispensable for UPEC in adherence and invasion of uroepithelial cells by using E. coli isolates (n=40) from cystitis patients and T-24 cells, the bladder carcinoma cell line. We found all isolates adherent to T-24 cells within 15 min of infection. In invasion assay, all isolates could invade T-24 cells to a variable degree; 22.5% of them were found highly invasive. About 33% of isolates that do not have any recognized adhesins were as invasive as other isolates. The amplitude of invasiveness was also independent of the adhesins. In conclusion, this study demonstrates that type 1 fimbriae, P fimbriae, S fimbriae, and afimbrial adhesin I are not required for UPEC to adhere to and invade uroepithelial cells.     

Purification and properties of cloned Salmonella typhimurium LT2 sialidase with virus-typical kinetic preference for sialyl alpha 2----3 linkages.

Subclones containing the Salmonella typhimurium LT2 sialidase gene, nanH, were expressed in Escherichia coli from multicopy derivatives of pBR329. The cloned sialidase structural gene directed overproduction of sialidase polypeptide which was detected as the major soluble protein species in cell-free extracts. Overproduced enzyme was purified to near electrophoretic homogeneity after 65-fold enrichment using conventional preparative techniques. Unlike all previously investigated sialidases, S. typhimurium sialidase was positively charged (pI greater than or equal to 9.0). Km, Vmax, and turnover number of the purified sialidase, measured using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUNeu5Ac), were 0.25 mM, 5,200 nmol min-1, and 2,700 s-1, respectively. These values are the highest yet reported for a sialidase. Sialidase was inhibited by 2-deoxy-2,3-didehydro-N-acetyl-neuraminic acid at unusually high concentrations (Ki = 0.38 mM), but not by 20 mM N-acetylneuraminic acid. Divalent cations were not required for activity. The pH optimum for hydrolysis of MUNeu5Ac was between 5.5 and 7.0 and depended on the assay buffer system. Substrate specificity measurements using natural sialoglycoconjugates showed a 260-fold kinetic preference for sialyl alpha 2----3 linkages when compared with alpha 2----6 bound sialic acids. The enzyme also efficiently cleaved residues from glycoproteins and gangliosides, but not from mucin or sialohomopolysaccharides. S. typhimurium sialidase is thus the first bacterial enzyme to be described with influenza A virus sialidase-like kinetic preference for sialyl alpha 2----3 linkages and to have a basic pI.     

Mitogenic stimulation of human B lymphocytes by the mannose-specific adhesin on Escherichia coli type 1 fimbriae

Escherichia coli type 1 fimbriae contain in association with the major structural protein a lectin-like adhesin moiety that mediates attachment of E. coli to mannose-containing receptors on the surface of host cells. We have investigated the lymphocyte mitogenic activity of this mannose-specific adhesin by comparing the ability of purified wild type type 1 fimbriae containing the adhesin and mutant type 1 fimbriae lacking the adhesin to stimulate proliferation in human lymphocytes. Both fimbriae stimulated a peak of proliferation at 8 days whereas only the wild type fimbriae stimulated an additional peak of proliferation occurring at 3 days. Proliferation at 3 days but not at 8 days could be blocked by the addition of alpha-methyl-D-mannoside. Neonatal lymphocytes from umbilical cord blood responded to both wild type and mutant fimbriae in a fashion similar to adult cells. Stimulation of separated T and non-T cell populations indicated that the proliferation seen at 3 days was solely due to non-T cells whereas the 8-day response was due to T cell proliferation. The addition of gamma-irradiated T cells did not appear to enhance the 3-day response of the non-T cells. However, the 8-day response by T cells was dependent on the presence of gamma-irradiated non-T cells. In cultures of unseparated cells, wild type fimbriae stimulated more than 75% of the B cells to enter the S and G2 phase at 3 days whereas at 8 days cycling T cells were present in both wild type and mutant fimbriae-stimulated cultures. Taken together, our observations suggest that the adhesin molecule stimulates a polyclonal mitogenic response in B cells that peaks at 3 days, and other structural components of the fimbriae are responsible for evoking an 8-day (probably immune) response in T cells.     

A novel lectin-independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin

Binding of P fimbriae of uropathogenic Escherichia coli to purified human fibronectin and human placental type IV collagen was studied. In an enzyme immunoassay, purified P fimbriae bound strongly to immobilized intact fibronectin and to the aminoterminal 30-kDa fragment and the 120-140-kDa carboxyterminal fragments of fibronectin. Binding to the gelatin-binding 40-kDa fragment of fibronectin was considerably weaker. No binding to immobilized type IV collagen was seen. The interaction between P fimbriae and immobilized fibronectin was not inhibited by alpha-D-Gal-(1-4)-beta-D-Gal-1-O-Me, a receptor analog of P fimbriae. Moreover, a mutated P fimbria lacking the lectin activity behaved similarly in the adherence assays. Recombinant strains expressing the corresponding cloned fimbriae genes bound to immobilized fibronectin, but no binding to soluble 125I-labelled fibronectin was found. The results suggest that P fimbriae interact with immobilized fibronectin and that the binding mechanism does not involve the lectin activity of the fimbriae.     

Two kinds of P-fimbrial variants of uropathogenic Escherichia coli recognizing forssman glycosphingolipid.

Various types of fimbriae on pathogenic Escherichia coli strains have been classified by their antigenicities and recognition specificities for receptors. However, the antigenicity of fimbrial proteins does not always correlate with the fimbrial recognition specificity. In this communication, the exact carbohydrate structures recognized by the fimbriae of two human uropathogenic E. coli strains, KS71 (O4) and IH11024 (O6), that have P-fimbrial antigen, were examined. Strain KS71 showed mannose-resistant (MR) hemagglutination (HA) of human blood group OP1 phenotype erythrocytes, and its HA was inhibited by blood group Pk antigen, Gal(alpha,1-4)Gal(beta,1-4)Glc-ceramide and P antigen, GalNAc(beta,1-3)Gal (alpha,1-4)Gal(beta,1-4)Glc-ceramide but not by Forssman antigen, GalNAc(alpha,1-3)GalNAc(beta,1-3)Gal(alpha,1-4)Gal (beta,1-4)Glc-ceramide, as previously described in many papers. The cells also showed MR HA of sheep erythrocytes, which was potently inhibited by Forssman, and weakly by P and Pk antigens. These phenomena could not be explained by the above P adhesin specificity. This adhesin was called Forssman-like adhesin. Strain IH11024 also caused MR HA of sheep erythrocytes but not of human erythrocytes. The HA was inhibited specifically by Forssman but neither by Pk nor P antigen. This adhesin was completely different from P adhesin and Forssman-like adhesin in recognition of the carbohydrate epitope. This adhesin, until now called a pseudotype of P fimbriae, was renamed Forssman adhesin.     

Isolation and characterization of a receptor for type 1 fimbriae of Escherichia coli from guinea pig erythrocytes

The adhesion of Escherichia coli to eukaryotic cells is mediated by proteinaceous surface appendages called fimbriae and complementary receptors on host cells. Although type 1 fimbriae, which contain a D-mannose-reactive lectin, have been well studied little is known about the binding mechanism of isolated fimbriae to individual cell receptors. This report describes the isolation and purification of a guinea pig erythrocyte receptor for type 1 fimbriae. Erythrocyte membranes were dissolved in 0.5% Triton X-100 and the receptor isolated and purified by affinity chromatography using type 1 fimbriae immobilized on Sepharose. The 65-kDa receptor, which inhibits the agglutination of guinea pig erythrocytes by type 1 fimbriated E. coli, has a pI of 8.5-8.7, and binds concanavalin A and type 1 fimbriae in a dose-dependent and saturable manner. The fimbrial binding activity of the receptor was reduced when treated with sodium metaperiodate, endoglycosidase H, trypsin, and V8 protease, suggesting the isolated receptor is a glycoprotein with N-linked carbohydrate units. Isolated type 1 fimbriae inhibited the binding of fimbriated E. coli to purified receptor in a dose- and time-related fashion. The calculated binding affinity was 6 X 10(6) M-1, a value consistent with the low binding affinity expected from previous studies of the agglutination of guinea pig erythrocytes by isolated type 1 fimbriae.     

Characterization of fimbriae produced by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC.     

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.     

Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products.

We have chemically synthesized oligopeptides corresponding to the NH2-terminal stretch of two gene products, designated FimG and FimH, of the fim gene cluster of Escherichia coli. These synthetic peptides, designated S-T1FimG(1-16) and S-T1FimH(1-25)C, evoked antibodies in rabbits that reacted with 14- and 29-kilodalton subunits, respectively, of dissociated fimbriae encoded by the recombinant plasmid pSH2 carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. Neither of these fimbrial proteins was detected in dissociated fimbrial preparations from nonadhesive E. coli cells carrying the mutant plasmid pUT2002, containing a restriction site-specific deletion of fimG and fimH. Anti-S-T1FimH(1-25)C inhibited the adherence of type 1 fimbriated E. coli to epithelial cells. Immunoelectron microscopy revealed that anti-S-T1FimH(1-25)C, but not anti-S-T1FimG(1-16), bound to intact type 1 fimbriae of E. coli at the fimbrial tips and at long intervals along the fimbrial filaments. Anti-S-T1FimG(1-16) appeared to be directed at epitopes not accessible on the intact fimbriae and consequently failed to bind to intact fimbriae or to block fimbrial attachment. Our results suggest that the fimG and fimH gene products are components of type 1 fimbriae and that FimH may be the tip adhesin mediating the binding of type 1 fimbriated E. coli to D-mannose residues on mucosal surfaces.     

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.     

Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles

Tissue-binding specificity of the type-3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild-type Klebsiella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type-3 fimbriae, as well as the purified type-3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowman's capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type-3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type-3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type-3 fimbrial gene cluster in association with the pap-encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type-3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type-3 fimbriate strains of Yersinia and Salmonella, showing that the ability to use type V collagen as tissue target is widespread among enteric bacteria.     

抗大肠杆菌987P粘着素单克隆抗体及其初步应用

共研制出ll株抗大肠杆菌987P粘着素单克隆抗体,对其部分免疫学特性作了测定。这些单抗不仅效价很高,而且特异性强,对不具有987P抗原的大肠杆菌及所试的其他肠道杆菌均无交叉反应。以FlTc或Hrpo标记的987P单抗作实验室诊断,具有简易、快速、敏感和准确的优点。酶标EPN3株单抗检测的灵敏度可达2×l03个／ml 987P+菌,对人工发病仔猪的粪样和小肠内容物的阳性检出比分别为4／4和2／2。 EP22株荧光标记单抗对病猪小肠粘膜触片的阳性检出比为6／6。 11株单抗中7株能不同程度地阻断987P+菌对仔猪小肠上皮细胞的吸附作用。EL1sA和荧光阻抑试验表明,1株单抗是针对987p粘着素上三种不同的抗原决定簇。EPN2株单抗的免疫胶体金定位还表明,987P粘着素似呈螺旋状结构,且含有许多相同的抗体结合位点。这些单抗不仅可用于ETEC菌株的987P柔毛鉴定,而且可用于987P分子结构和生物学特性的研究。     

Occurrence of p-fimbriae in enteropathogenic E coli (EPEC) and in E. coli strains isolated from patients with urinary tract infections]

The aim of this study was to gain knowledge of prevalence of P+ clones among EPEC strains isolated from children with diarrhoea and E. coli strains isolated from urine. Three hundred eighty four E. coli strains isolated from children with diarrhoea were tested. They belonged to 11 serotypes (018, 025, 026, 044, 055, 0111, 0114, 0119, 0124, 0125, and 0128). Nine hundred thirty colonies of E. coli from Mac Conkey's agar plated quantitatively with urine samples of 178 individuals suffering from urinary tract infections were also tested. All strains were assayed by mannose-resistant active haemagglutination test (MRHA) and by slide agglutination using self prepared latex reagent for detection of P fimbriae. Out of 384 E. coli strains tested 122 (31.8%) showed presence of adhesins detected by mannose-resistant active haemagglutination test (MRHA) and in 90 (23.3%) out of all tested strains the presence of P fimbriae was found. The highest percentage of P fimbriae prevalence was found in E. coli belonging to the following serotypes: 018 (in 68.9% strains), 025 (in 29.2% strains), and 0125 (in 25.0% strains). This type of fimbriae was also detected in serotypes 026 (9.1%), 044 (8.7%), 055 (5.6%), and 0119 (in 2 strains out of 5 isolated). Out of 933 colonies of E. coli, isolated from 178 urine samples, 434 (46.5%) colonies gave positive results in MRHA test, including 133 positive in latex test for P fimbriae. These studies showed that for MRHA adhesins, including P fimbriae, a parallel examination of higher number of E. coli was necessary.(ABSTRACT TRUNCATED AT 250 WORDS)