Use of the coagglutination reaction of yeast-like Candida maltosa fungi for detecting fimbriae in intestinal bacteria

The capacity of nonpathogenic yeast-like C. maltosa strains to coagglutinate Escherichia coli has been studied. C. maltosa cells have also been shown to coagglutinate E. coli possessing mannose-sensitive adhesins in a wide range of their concentrations (5-140 bacterial cells per C. maltosa cell). Strains belonging to types CFA/I and CFA/II with fimbriae, similarly to their corresponding paired genetically related strains without these adhesins, are practically incapable of agglutinating C. maltosa cells, while strains K88 and B41 react with them. The reaction occurs at a concentration of 9.5-37.0 and 38.0-55.5 bacteria respectively per C. maltosa cell and is not inhibited by 1% d-mannose. The suggestion that C. maltosa cell surface glycoproteins contain not only receptors for E. coli fimbriae, type I, but also components similar in their structure to receptors specific to the mannose-resistant adhesins of strains K88, K99 and 41, has been confirmed by hemagglutination inhibition with C. maltosa surface antigens as inhibiting agents.     

The production of F41 fimbriae by piglet strains of enterotoxigenic Escherichia coli that lack K88, K99 and 987P fimbriae

The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen. The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin. Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Positive regulation of colonization factor antigen I (CFA/I) production by enterotoxigenic Escherichia coli producing the colonization factors CS5, CS6, CS7, CS17, PCFO9, PCFO159:H4 and PCFO166

Entertoxigenic Escherichia coli (ETEC) strains of nineteen serogroups which produced colonization factors (coli-surface-associated antigens CS5, CS6, CS7 and CS17, colonization factor antigen CFA/III and putative colonization factors PCFO159:H4, PCFO166 and PCFO9) were tested for hybridization with a DNA probe containing the cfaD sequence that regulates expression of CFA/I. Strong colony hybridization, similar to that with the CFA/I-positive control strain H10407, occurred with ETEC strains of serogroups O27, O159 and O169 which produced CS6 antigen, and with all the strains which produced PCFO166 fimbriae. Weak colony hybridization, compared to the control strain, was found with ETEC producing CS5 fimbriae with CS6 antigen, CFA/III fimbriae with CS6 antigen, CS7 fimbriae or PCFO159:H4 fimbriae. CS6-antigen-positive strains of serogroups O79, O89 and O148 and all the CS17-antigen-positive and PCFO9-fimbriae-positive strains were negative in colony hybridization tests with the cfaD probe. Plasmid DNA of nine ETEC strains and their colonization-factor-negative derivatives was tested for hybridization with the cfaD probe and with ST and LT oligonucleotide probes. The sequences that hybridized with the cfaD probe were on the plasmids which coded for enterotoxin production. Fifteen strains were transformed with NTP513, a recombinant plasmid which contains the CFA/I region 1 fimbrial subunit operon but lacks a functional cfaD sequence, in order to determine whether DNA in any of these strains could substitute for the cfaD sequence in the regulation of production of CFA/I fimbriae.(ABSTRACT TRUNCATED AT 250 WORDS)     

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。     

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。     

Characterization of a putative colonization factor (PCFO166) of enterotoxigenic Escherichia coli of serogroup O166

Enterotoxigenic Escherichia coli (ETEC) of serogroup O166 gave mannose-resistant haemagglutination (MRHA) with bovine and human erythrocytes. The strains did not react with antisera prepared against the known colonization factors CFA/I, CFA/II, CFA/III, CFA/IV and PCFO159:H4. Strain E7476 of serotype O166:H27, which produced heat-stable enterotoxin (ST), was examined initially. It produced fimbriae about 7 nm in diameter. On SDS-PAGE two possible fimbrial polypeptides of molecular mass 15.5 and 17.0 kDa were seen. When variants of strain E7476 were isolated, loss of ST and MRHA together was associated with loss of a 98 MDa plasmid, while loss of ST alone correlated with plasmid deletion. An absorbed anti-strain E7476 antiserum reacted specifically with the 15.5 and 17.0 kDa polypeptides in Western immunoblotting and bound to the intact fimbriae by immuno-electron microscopy. When this antiserum was used in an ELISA to examine other strains of serogroup O166, a positive reaction was obtained with all the ST- and MRHA-positive strains. One strain of serotype O71:H27 and two strains of serotype O98:H- also reacted with the absorbed anti-strain E7476 antiserum. The antiserum did not react with ETEC carrying known colonization factors. E. coli K12 and a number of E. coli of different serotypes carrying a plasmid coding for ST transferred from strain E7476, all gave MRHA and reacted with the absorbed anti-strain E7476 antiserum. The term putative colonization factor O166 (PCFO166) is proposed to describe the adhesive factor(s) on ETEC of serogroup O166 because of the similarity of properties with those of known colonization factors.     

Role of fimbrial adhesins in the pathogenesis of Escherichia coli infections.

Escherichia coli strains are able to cause intestinal (enteritis, diarrhoeal diseases) and extraintestinal (urinary tract infections, sepsis, meningitis) infections. Most pathogenic E. coli strains produce specific fimbrial adhesins, which represent essential colonization factors: intestinal E. coli strains very often carry transferable plasmids with gene clusters specific for fimbrial adhesins, like K88 and K99, or colonization factor antigens (CFA) I and II. In contrast, the fimbrial gene clusters of extraintestinal E. coli strains, such as P, S, or F1C fimbriae, are located on the chromosomes. The fimbrial adhesin complexes consist of major and minor subunit proteins. Their binding specificity can generally be assayed in hemagglutination tests. In the case of fimbrial adhesins of intestinal E. coli strains, the major subunit proteins preferentially represent the hemagglutinating adhesins, whereas minor subunit proteins are the hemagglutinins of extraintestinal E. coli strains. Recently "alternative" adhesin proteins were identified, which have the capacity to bind to eukaryotic structures different from the receptors of the erythrocytes. Fimbrial adhesins are not constitutively expressed but are stringently regulated on the molecular level. Extraintestinal E. coli wild-type strains normally carry three or more fimbrial adhesin determinants, which have the capacity to influence the expression of one another (cross talk). Furthermore the fimbrial gene clusters undergo phase variation, which seems to be important for their contribution to pathogenesis of E. coli.     

Comparative study of inhibition of mannose-resistant haemagglutination caused by CFA/I, CFA/II, K88 and K99-positive Escherichia coli strains

Abstract We have studied the inhibition of mannose-resistant haemagglutination (MRHA) caused by Escherichia coli strains with CFA/I, CFA/II, K88, K99 and by other faecal E. coli lacking these colonisation antigens, by means of 30 sugar compounds and by enzymatic treatment of erythrocytes with neuraminidase, α-mannosidase, β-galactosidase, trypsin and pronase, and with formaldehyde. Inhibition of MRHA by sugars was effective only in K88-positive strains with d (+)glucosamine, mucic acid and bovine submaxillary mucin. Enzymatic treatment and the formolisation of erythrocytes gave different results on MRHA activity in strains possessing each colonisation antigen type. Results suggest that the erythrocyte receptor for CFA/I and CFA/II may possibly be sialoglycoprotein in which N -acetylneuraminic acid (NANA) plays an important role, because MRHA activity in these strains was inhibited by treatment of erythrocytes with neuraminidase and pronase. On the other hand, erythrocyte receptors for K88 and K99, like receptors for haemagglutinins of faecal E. coli lacking these colonization antigens, may have other glycoconjugate structures in which proteins and NANA are not essential. Our observations also suggest that the nature (or structure) of the receptor for a specific colonisation antigen on diverse erythrocyte types may be different.     

Occurrence of type-1C fimbriae on Escherichia coli strains isolated from human extraintestinal infections

Two monoclonal antibodies specific for type-1C fimbriae of Escherichia coli were produced. In enzyme-linked immunosorbent assay and immunoblotting the antibodies, which were of the IgG1 isotype, reacted with type-1C, but not with P or type-1 fimbriae of E. coli strain KS71. Immunoblotting and immunoprecipitation of crude fimbrial extracts from 25 strains invariably gave an apparent molecular weight of 17 000 for the type-1C fimbrillin. A total of 313 E. coli strains, isolated from patients with extraintestinal infection or from faeces of healthy children, were screened for the presence of type-1C fimbriae using both the monoclonal and polyclonal antibodies. Of these, 45 (14%) strains had type-1C fimbriae, with the highest frequency (27%) on strains isolated from patients with pyelonephritis. No faecal strain had type-1C fimbriae, and the frequency on the other diagnostic groups ranged from 11 to 15%. Thus, no direct correlation between type-1C fimbriae and bacterial virulence in human extraintestinal infections was found. Type-1C fimbriae were detected on only a few E. coli serotypes, notably on all O6:K2:H1 and O22:K13:H1 strains tested.     

Control of temperature-dependent synthesis of K99 fimbriae

The influence of temperature on the production of K99 fimbriae by Escherichia coli was determined in cultures growing at constant specific growth rate in continuous cultures. In a wild type strain, in which the K99 operon is present on a low copy number plasmid, low cultivation temperature repressed the K99 production. This temperature-dependent production was not observed after introduction of multicopies of the regulatory region of the K99 operon into this strain, nor in E. coli K12 harbouring a recombinant, multicopy plasmid encoding the K99 operon. These results are in agreement with a regulation model in which a regulatory factor, most likely a repressor, inhibits expression of the K99 operon at low temperatures.     

Expression of cloned plasmid regions encoding colonization factor antigen I (CFA/I) in Escherichia coli

Molecular cloning from a plasmid encoding colonization factor antigen I (CFA/I) and heat-stable enterotoxin isolated two regions, 1 and 2, that are required for the production of CFA/I fimbriae. The level of CFA/I synthesis measured by ELISA was similar in an Escherichia coli K12 strain carrying regions 1 and 2 cloned separately on compatible plasmid vectors to that in the same strain containing the parental plasmid. The structural gene for the CFA/I fimbrial subunit was within region 1. This region directed production in E. coli minicells of at least six independent polypeptides, of which the fimbrial subunit and at least three others appeared to be synthesized as precursor molecules that underwent processing. Cloned DNA containing CFA/I region 2 specified three polypeptides in minicells. Attempts to reduce the size of the cloned region 1 resulted in a derivative plasmid that carried the CFA/I structural gene but did not complement a region-2 recombinant plasmid to restore production of CFA/I fimbriae.     

Expression of CS fimbriae in Escherichia coli strains of human and animal origin belonging to O-serovar 6, K-serovar 15 or H-serovar 16

Abstract A non-conjugative CS-fimbriae-associated plasmid pCS001 was mobilized into rifampicin-resistant mutants of strains of Escherichia coli of O-serovar 6 or K-serovar 15 or H-serovar 16. By a variety of serological procedures only the production of CS3 fimbriae was detected in transoconjugants. The finding extend previous observations that only strains of E. coli of serotype O6:K15:H16 or H− and of appropriate biotype are able to express either CS1 or CS2 fimbriae, as even a recipient of serotype O6:K15:H31 possessing a rhamnose-positive fermentation phenotype did not express either of these two fimbriae. The results indicate that expression of CS1 or CS2 fimbriae probably involves chromosomal determinants only found in strains of serotype O6:K15:H16 or H−.     

Incidence and some characteristics of fimbriae FY and 31A of Escherichia coli isolates from calves with diarrhea in Japan

Escherichia coli isolates from calves with diarrhea (1 day to 8 weeks old, 140 individuals) were surveyed for the three immunologically distinct fimbrial adhesins FY, 31A, and K99. Of a total of 1,370 strains isolated, 96 (7.0%), 34 (2.5%), 75 (5.5%), and 13 (0.9%) were identified as FY+, 31A+, FY+.31A+, and K99+, respectively. The K99+ strains also manifested heat-stable enterotoxin production (ST+), while FY+, 31A+, and FY+.31A+ strains were ST-. Expression of FY and 31A was repressed at lower temperatures or poor aeration. The FY+ and 31A+ E. coli showed mannose-resistant hemagglutinating activity with bovine erythrocytes. Electron microscopy revealed that FY is a gently curled fimbria with a mean diameter of 4.2 nm, and 31A is a fimbria with a mean diameter of 5.1 nm. The molecular mass of protein subunits was found to be approximately 20 kilodaltons (Kd) and 19 Kd for FY and 31A, respectively. Lethal diarrhea of neonatal calves was induced by challenge with the combination of a K99+.ST+ E. coli strain and either a 31A+ E. coli strain or a 31A+ E. coli strain plus an FY+ E. coli strain under the experimental conditions in which lethal diarrhea was not induced by challenge with a K99+.ST+ E. coli strain alone.     

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.     

Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88.

The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning. The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon. Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88. Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E. coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain. This observation was extended to other F41-producing animal isolates. A large number of animal E. coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production. All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes. Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production. In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal. The K88 antigen-producing strains showed no homology with the K99 probe. A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens. This suggests that there are adhesins among animal isolates of E. coli which are genetically related to but antigenically distinct from K88 and F41.     

CFA/I-ST plasmids: comparison of enterotoxigenic Escherichia coli (ETEC) of serogroups O25, O63, O78, and O128 and mobilization from an R factor-containing epidemic ETEC isolate

Colonization factor antigen I (CFA/I) plays an important role in the pathogenesis of diarrhea due to enterotoxigenic Escherichia coli. In this study, we examined 11 CFA/I+ enterotoxigenic E. coli from serogroups O25, O63, O78, and O128 and found that with all strains, spontaneous loss of CFA/I was associated with the loss of heat-stable toxin (ST) and with the loss of a single plasmid ranging in size from 54 to 60 megadaltons; when heat-labile toxin was lost, this was associated with the loss of another plasmid. The R factor of one strain, TX432 (O78:H12:CFA/I+; ST+), was found to mobilize the CFA/I-ST plasmid into E. coli K-12 at a frequency of 20%. These studies provide further evidence that CFA/I production is plasmid mediated in enterotoxigenic E. coli belonging to serogroups O25, O63, O78, and O128.     

Stimulation of mucosal immune response following oral administration of enterotoxigenic Escherichia coli fimbriae (CFA/I) entrapped in liposomes in conjunction with inactivated whole-cell Vibrio cholerae vaccine

In this study, we have searched for an effective mucosal vaccine. An oral enterotoxigenic E. coli vaccine containing colonization factor antigen (CFA/I) associated with inactivated whole-cell V. cholerae vaccine (WCV) has been tested for safety and immunogenicity in animals. Five groups of animals were used. The results showed the following: (a) vaccine containing CFA/I antigen entrapped in liposomes and associated with WCV (batch C) had increased titers of specific antibodies to CFA/I antigen in 15 to 18 (83.3%) animals; (b) specific Peyer's patches (PP), lymph nodes (LN) and spleen (SPL) lymphocytes proliferation was detected following in vitro restimulation with CFA/I antigen or WCV. This response gradually increased to the highest value by the 35th postimmunization day. Moreover, lower PP, LN and spleen (SPL) proliferation was observed in rabbits receiving soluble CFA/I antigen (S-CFA/I) or free liposomes (F-L) alone; (c) adhesion of E. coli H10407 strain labelled with 3H-leucine in immunized and control animals revealed the following local effects: (i) protection of rabbit intestinal mucosa against virulent E. coli cells; (ii) inhibition of adhesion of ETEC bacteria to intestinal mucosa and (iii) significantly faster release of E. coli H 10407 strain labelled with 3H-leucine from the intestinal tract of immunized animals. The histopathological and electron microscope findings confirmed the above results. The experimental results point out an efficient protection against infection with E. coli strains (ETEC), after mucosal vaccination with CFA/I antigen entrapped in liposomes associated with inactivated whole-cell Vibrio cholerae as immunological adjuvant.     

Lactobacillus acidophilus expressing recombinant K99 adhesive fimbriae has an inhibitory effect on adhesion of enterotoxigenic Escherichia coli

The most common enteric colibacillosis in neonatal and newborns is caused by enterotoxigenic Escherichia coli(ETEC). Colonization of ETEC in the small intestine is associated with adhesions using fimbriae, which is known as a specific adhesion factor and provides highly specific means for anchoring and prerequisite for an infectious agent. In the present study we have engineered Lactobacillus acidophilus to produce recombinant K99 fimbriae, which is used for the colonization to the intestine of pigs. The expression of K99 fimbrial protein was confirmed using SDS-PAGE, immunoblot and agglutination analyses. To evaluate a function of the K99 fimbrial protein, inhibition and competition tests were performed on pre-screened intestinal brush border from pigs. The tests showed that recombinant L. acidophilus, not control L. acidophilus, had a significant inhibitory effect to and competition against K99+ E. coli in a dose dependent manner. In conclusion, we demonstrated that recombinant K99 fimbriae producing L. acidophilus was able to prevent E. coli binding to intestinal brush border.     

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.