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1.
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.  相似文献   

2.
Abstract The fimbriae (E4) of a virulent strain of Salmonella typhimurium were purified by ion exchange chromatography in an FPLC system. They had a channelled appearance under transmission electron microscope and showed a major structural subunit of 17-kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified fimbriae were found to agglutinate guinea pig erythrocytes, but this effect was inhibited in presence of D-mannose. Immune sera raised against the Mono-Q purified fimbriae (E4) showed cross-reactivity with the type-1 fimbriae (F1) composed of 21-kDa fimbrin subunit, purified by a different method from the same strain.  相似文献   

3.
This report describes the presence of type 1 fimbriae on Shigella boydii 5 which agglutinate guinea pig erythrocytes and feature mannose-sensitive adherence. Morphologically, the fimbriae were thin, rigid cylinders 2-5 microm in length and 35 nm in diameter, and the organella retained axial holes. This is the first study to have revealed the existence of type 1 fimbriae on S. boydii.  相似文献   

4.
Plasmid pFM205 contains the genetic determinant for the K88ab antigen and is composed of a 4.3-megadalton DNA fragment derived from wild-type K88ab plasmid pRI8801 and cloning vehicle pBR322. The K88 NA of pFM205 contains five genes, which code for polypeptides with apparent molecular weights of 17,000, 26,000 (the K88ab subunit), 27,000 27,500, and 81,000. All five polypeptides were synthesized as precursors approximately 2,000 daltons larger than the mature polypeptides, indicating that they are transported across the cytoplasmic membrane by means of a signal sequence. A set of deletion derivatives of pFM205 was constructed, each containing a deletion in one of the five genes. In strains harboring derivatives of pFM205 containing a deletion in the gene for the 17,000- or 81,000-dalton polypeptide, the K88ab subunit was synthesized and transported to the outside of the cell. However, these strains did not adhere to brushborders or guinea pig erythrocytes, suggesting that the K88ab subunits were not assembled into normal fimbriae. Strains harboring plasmids containing a deletion in the gene for the 27,500-dalton polypeptide still adhered to brush borders and guinea pig erythrocytes, although very little K88ab antigen could be detected with an immunological assay. In strains harboring plasmids containing a deletion in the gene for the 27,000-dalton polypeptide, the K88ab subunit was synthesized but was probably subsequently degraded rapidly.  相似文献   

5.
Abstract We have identified a new locus required for mediating the binding of type 1 piliated Escherichia coli cells to guinea pig erythrocytes. This locus, pilE , was discovered after restrition site mutagenesis of a cloned segment of DNA containing the chromosomal pil region from a clinical strain of E. coli. pilE Mutants failed to agglutinate guinea pig erythrocytes but expressed pili were morphologically and antigenically indistinguishable from the parental ( pilE +) strain. Construction of a chromosomal pilE mutation in E. coli K-12 was accomplished by introducing a restriction fragment containing a pilE lesion into the chromosome of a recBC sbcB host strain. Mutations in pilE could be complimented in trans by the addition of a cloned segment of DNA containing the parental pilE locus. Lesions in any of the genes required for pilus assembly also produced a hemagglutination minus phenotype suggesting that both the product(s) specified by the pilE locus and pili were required for hemagglutination. Hemagglutination experiments using partially purified pili also supported this suggestion.  相似文献   

6.
Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31 degrees C but not at 42 degrees C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42 degrees C but sensitive after growth at 31 degrees C. The ts mutant thus binds macrophages with one receptor specificity at 31 degrees C and another at 42 degrees C.  相似文献   

7.
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.  相似文献   

8.
基于禽大肠杆菌Ⅰ型菌毛黏附素fimH基因的已知序列,利用入噬菌体的Red重组系统构建禽致病性大肠杆菌国内分离株A2(血清型O2:K89)Ⅰ型菌毛黏附素fimH基因缺失突变株A2△fimH::Cat,在二次重组中利用携带能够表达FLP位点特异性重组酶的质粒pCP20(温度敏感性)以去除上述缺失突变株中抗性基因标志,结合PCR扩增和测序结果,证明fimH基因缺失株A2AfimH的正确构建。通过fimH基因互补试验使A2afimH缺失突变株恢复了与野生株具有相同的凝集活性。红细胞和酵母细胞凝集试验结果表明,野生株呈现良好的凝集效果,并能被0.5%甘露糖完全抑制,而A2afimH缺失突变株未呈现任何凝集现象。体外生长试验结果表明,在同样的培养条件下,A2afimH缺失突变株生长周期的各个阶段都要稍慢于野生株。禽致病性大肠杆菌国内分离株Ⅰ型菌毛黏附素fimH基因缺失突变株成功构建,为进一步深入研究禽大肠杆菌Ⅰ型菌毛与机体相互作用的分子机制,肠道外感染的致病机理及对国内禽大肠杆菌病的防控策略奠定了一定基础。  相似文献   

9.
Valency conversion in the type 1 fimbrial adhesin of Escherichia coli   总被引:1,自引:0,他引:1  
FimH protein is a lectin-like adhesive subunit of type 1, or mannose-sensitive, fimbriae that are found on the surface of most Escherichia coli strains. All naturally occurring FimH variants demonstrate a conserved mannotriose-specific (i.e. multivalent) binding. Here, we demonstrate that replacement of residues 185-279 within the FimH pilin domain with a corresponding segment of the type 1C fimbrial adhesin FocH leads to a loss of the multivalent mannotriose-specific binding property accompanied by the acquisition of a distinct monomannose-specific (i.e. monovalent) binding capability. Bacteria expressing the monovalent hybrid adhesins were capable of binding strongly to uroepithelial tissue culture cells and guinea pig erythrocytes. They could not, however, agglutinate yeast or bind human buccal cells -- functions readily accomplished by the E. coli-expressing mannotriose-specific FimH variants. Based on the relative potency of inhibiting compounds of different structures, the receptor binding site within monovalent FimH-FocH adhesin has an extended structure with an overall configuration similar to that within the multivalent FimH of natural origin. The monomannose-only specific phenotype could also be invoked by a single point mutation, E89K, located within the lectin domain of FimH, but distant from the receptor binding site. The structural alterations influence the receptor-binding valency of the FimH adhesin via distal effects on the combining pocket, obviously by affecting the FimH quaternary structure.  相似文献   

10.
Hemagglutinating properties of Actinobacillus pleuropneumoniae   总被引:3,自引:0,他引:3  
A total of 26 isolates of Actinobacillus pleuropneumoniae were tested for their ability to agglutinate erythrocytes of different origins. Seven different hemagglutination patterns were found. Ten (38%) isolates did not agglutinate any of the erythrocytes tested. The remaining 16 (62%) isolates agglutinated human erythrocytes, and among these, 12 also agglutinated rat, cat, dog, guinea pig, or bovine erythrocytes. No correlation was found between the seven different hemagglutination patterns observed and the serotypes. Hemagglutination activity was destroyed by heating at 100 degrees C as well as by formaldehyde treatment, but was not affected by heating at 60 degrees C, by treatment with trypsin or pronase, or by homogenization of bacterial cells. No fimbriae were observed on examination of bacterial cells negatively stained with phosphotungstate using electron microscopy. Hydrophobic surface properties of the isolates were evaluated. All the isolates appear to possess a hydrophilic cell surface. The present study provides evidence that certain isolates of A. pleuropneumoniae possess hemagglutinating properties which do not appear to be mediated by fimbriae or to involve hydrophobic interactions.  相似文献   

11.
Type 1 and F1C fimbriae are surface organelles of Escherichia coli which mediate receptor-specific binding to different host surfaces. Such fimbriae are found on strains associated with urinary tract infections. The specific receptor binding of the fimbriae is due to the presence of receptor recognition proteins present in the organelles as minor structural elements. The organization of the fim and foc gene clusters encoding these fimbriae, as well as the structures of the organelles, are very similar, although the actual sequence homology of the structural elements is not remarkable; notably, the sequence identity between the minor components of the type 1 and F1C fimbriae is only 34 to 41%. Type 1 fimbriae mediate agglutination of guinea pig erythrocytes, whereas F1C fimbriae do not confer agglutination of any types of erythrocytes tested. However, F1C fimbriae mediate specific adhesion to epithelial cells in the collecting ducts of the human kidney as well as to cells of various cell lines. This report addresses the question of fimbrial promiscuity. Our data indicate that minor fimbrial structural elements can be exchanged between the two fimbrial systems, resulting in hybrid organelles with changed receptor specificity. This is the first study on reciprocal exchange of structural components from two different fimbrial systems.  相似文献   

12.
Interaction of Actinomyces oris with salivary proline-rich proteins (PRPs), which serve as fimbrial receptors, involves type 1 fimbriae. Encoded by the gene locus fimQ-fimP-srtC1, the type 1 fimbria is comprised of the fimbrial shaft FimP and the tip fimbrillin FimQ. Fimbrial polymerization requires the fimbria-specific sortase SrtC1, which catalyzes covalent linkage of fimbrial subunits. Using genetics, biochemical methods, and electron microscopy, we provide evidence that the tip fimbrillin, FimQ, is involved in fimbrial assembly and interaction with PRPs. Specifically, while deletion of fimP completely abolished the type 1 fimbrial structures, surface display of monomeric FimQ was not affected by this mutation. Surprisingly, deletion of fimQ significantly reduced surface assembly of the type 1 fimbriae. This defect was rescued by recombinant FimQ ectopically expressed from a plasmid. In agreement with the role of type 1 fimbriae in binding to PRPs, aggregation of A. oris with PRP-coated beads was abrogated in cells lacking srtC1 or fimP. This aggregation defect of the ΔfimP mutant was mainly due to significant reduction of FimQ on the bacterial surface, as the aggregation was not observed in a strain lacking fimQ. Increasing expression of FimQ in the ΔfimP mutant enhanced aggregation, while overexpression of FimP in the ΔfimQ mutant did not. Furthermore, recombinant FimQ, not FimP, bound surface-associated PRPs in a dose-dependent manner. Thus, not only does FimQ function as the major adhesin of the type 1 fimbriae, it also plays an important role in fimbrial assembly.  相似文献   

13.
In order to develop reagents to study the immune response of guinea pigs to infection by Chlamydia psittaci guinea pig inclusion conjunctivitis strain (GPIC), we constructed a plasmid clone bank with C. psittaci DNA. One of the recombinant clones isolated produced large amounts of a 57-kilodalton (kDa) protein that was immunoreactive with sera from GPIC infected guinea pigs. While investigating this recombinant protein, we discovered that all the Gram-negative bacteria analyzed so far have immunoreactive proteins of similar size. This protein seems to be a 'common antigen' already described in various Gram-negative bacteria.  相似文献   

14.
15.
Common type 1 fimbriae were isolated from Escherichia coli and their length distribution profile was determined before and after treatment with ultrasound. As fimbriae were shortened, so their haemagglutinating capacity decreased, but their ability to bind to erythrocytes did not decrease to the same extent. Isolated fimbriae did not agglutinate inside-out vesicles prepared from horse erythrocytes or liposomes, suggesting that the binding mechanism was not based on non-specific hydrophobic interactions. The results support a lateral rather than a terminal location for the fimbrial binding site responsible for haemagglutination.  相似文献   

16.
Regulation of the gene, fimA, encoding the major fimbrial subunit of S. typhimurium S6704 was examined by using a lambda fimA-lacZ lysogen. Transformation of the lambda fimA-lacZ lysogen with various derivatives of the recombinant plasmid that encodes type 1 fimbrial expression, pISF101, indicated that two regions of this plasmid alter beta-galactosidase production. One plasmid is a deletion resulting in the loss of a 28-kDa polypeptide downstream of fimA, while the other plasmid encodes a 24- and a 27-kDa polypeptide. Northern (RNA) blot analyses indicated that the steady-state fimA mRNA levels of these transformants were high. In addition, phenotypic expression of type 1 fimbriae by agar-grown cultures is observed only in those transformants bearing plasmids which show increased beta-galactosidase and fimA mRNA levels.  相似文献   

17.
Surface haemagglutinating activity of Pseudomonas aeruginosa   总被引:2,自引:0,他引:2  
J Glick  N Garber  D Shohet 《Microbios》1987,50(203):69-80
Intact cells of several strains of Pseudomonas aeruginosa agglutinate papain-treated human erythrocytes. The agglutinating activity appears to reside in the surface layers of the bacterium-Pseudomonas surface haemagglutinin. This activity does not correlate with the existence of the internal PA-I and PA-II lectins, the presence of fimbriae or adherence to human buccal epithelial cells. Disruption of the bacterial cells by sonication abolishes their haemagglutinating activity. The intact cells of P. aeruginosa are also able to agglutinate rabbit, chicken, dog, guinea pig and sheep erythrocytes. This activity is generally higher with papain-treated erythrocytes, except those of rabbit in which lower haemagglutinating activity is observed after papain treatment. Optimal conditions for the haemagglutination are 37 degrees C and pH 6-7. Simple sugars do not inhibit, while fetuin and hydrophobic amino acids inhibit this activity. Exposure of the bacterial cells to proteolytic enzymes, EDTA or denaturating conditions abolish the haemagglutinating activity. These results indicate that the surface haemagglutinin is a protein which agglutinates red blood cells via hydrophobic interactions.  相似文献   

18.
A cosmid gene library of Actinomyces viscosus T14V was prepared in Escherichia coli to examine the expression of A. viscosus antigens and to gain insight into the structure of A. viscosus type 1 and type 2 fimbriae. Out of this library of 550 clones, 28 reacted in a colony immunoassay with antibodies against A. viscosus cells. The proteins responsible for these reactions were identified in three clones. Clones AV1209 and AV2009 displayed nonfimbrial antigens with subunits of 40 and 58 kilodaltons, respectively. Clone AV1402 showed a 59-kilodalton protein that reacted with monospecific antibody against type 2 fimbriae and that comigrated with a subunit of type 2 fimbriae during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that AV1402 expresses a gene (fimA) for a subunit of A. viscosus type 2 fimbriae.  相似文献   

19.
A full-length cDNA encoding the pig cytosolic aspartate aminotransferase (EC 2.6.1.1) (cAspAT) was constructed from two overlapping cDNA clones. One clone (Lm pcAAT-8) isolated from a lambda gt10 pig heart cDNA library contained a 3' untranslated sequence, a poly(A) segment, and a part of the coding region for amino acid positions 127-412. Another clone (Lm pcAAT-107) isolated from a lambda gt10 primer extension library contained the coding region for amino acid positions 1-148 and a 5' untranslated sequence. Rejoining of the cDNA inserts of the two clones and recloning into pUC18 gave rise to a cDNA covering an entire coding sequence for pig cAspAT mRNA. Insertion into pKK223-3 yielded an expression plasmid, ppcAAT200. Escherichia coli JM105 cells transfected with ppcAAT200 overproduced pig cAspAT to an extent of about 3% of the total cellular soluble proteins. The expressed product was indistinguishable from the alpha subform of cAspAT isolated from pig heart in terms of specific activity, absorption spectra, molecular size, crystalline form, and immunological reactivity with anti pig cAspAT antibody. Compared with the amino-terminal sequence (Ala-Pro-Pro-) reported for pig heart cAspAT, the recombinant pig cAspAT showed heterogeneity in the amino-terminal sequence: Ala 1 (26%), Pro2 (54%), and Pro3 (19%). Construction of a mutant cAspAT with deletion of residues 1-3 and its comparison with the wild-type enzyme revealed that loss of the three amino-terminal residues does not affect the catalytic activity and structural integrity of the enzyme.  相似文献   

20.
Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under the control of a strong promoter (pL, from bacteriophage lambda) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines.  相似文献   

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