首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
We developed quantitative fimA genotype assays and applied them in a pilot study investigating the fimbrial genotype distribution of Porphyromonas gingivalis in European subjects with or without chronic periodontitis. P. gingivalis was found in 71% and 9% of the samples from patients and healthy subjects, respectively. Enumeration of total P. gingivalis cell numbers by polymerase chain reaction and immunofluorescence showed excellent correspondence (r = 0.964). 73% of positive samples contained multiple fimA genotypes, but generally one genotype predominated by one to three orders of magnitude. Genotype II predominated in 60% of the samples. Genotype IV occurred with similar prevalence (73%) as genotype II but predominated in only 20% of the samples. Genotypes I, III and V were of much lower prevalence and cell densities of the latter two remained sparse. Our results suggest marked differences among the fimA genotypes' ability to colonize host sites with high cell numbers.  相似文献   

2.
The biosynthesis of fimbriae is a complex process requiring multiple genes which are generally found clustered on the chromosome. In Bordetella pertussis, only major fimbrial subunit genes have been identified, and no evidence has yet been found that they are located in a fimbrial gene cluster. To locate additional genes involved in the biosynthesis of B. pertussis fimbriae, we used TnphoA mutagenesis. A PhoA+ mutant (designated B176) was isolated which was affected in the production of both serotype 2 and 3 fimbriae. Cloning and sequencing of the DNA region harbouring the transposon insertion revealed the presence of at least three additional fimbrial genes, designated fimB, fimC and fimD. The transposon was found to be located in fimD. Analysis of PhoA activity indicated that the fimbrial gene cluster was positively regulated by the bvg locus. A potential binding site for BvgA was observed upstream of fimB. FimB showed homology with the so-called chaperone-like fimbrial proteins, while FimC was homologous with a class of fimbrial proteins located in the outer membrane and presumed to be involved in transport and anchorage of fimbrial subunits. An insertion mutation in fimB abolished the expression of fimbrial subunits, implicating this gene in the biosynthesis of both serotype 2 and 3 fimbriae. Upstream of fimB a pseudogene (fimA) was observed which showed homology with the three major fimbrial subunit genes, fim2, fim3 and fimX. The construction of a phylogenetic tree suggested that fimA may be the primordial major fimbrial subunit gene from which the other three were derived by gene duplication. Interestingly, the fimbrial gene cluster was found to be located directly downstream from the gene coding for the filamentous haemagglutinin, an important B. pertussis adhesin, possibly suggesting co-operation between the two loci in the pathogenesis of pertussis.  相似文献   

3.
Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.  相似文献   

4.
5.
6.
7.
Abstract Analysis of the Salmonella chromosomal region located upstream of the fimA gene (coding for the major type 1 fimbrial subunit) showed a close linkage of this gene to the folD gene (coding for the enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5, 10-methenyltetrahydrofolate cyclohydrolase), indicating that the fim gene cluster of Salmonella , unlike that of Escherichia coli , has no regulatory genes located upstream of fimA and apparently terminates with this gene. The respective locations of the fim and folD genes in the E. coli and Salmonella genetic maps suggests that the fimA-folD intergenic region of Salmonella encompasses a junctional site of a genetic rearrangement that probably originated from the different chromosomal location of the fim genes in these species.  相似文献   

8.
The gram-negative anaerobic bacterium Porphyromonas gingivalis is an etiologically important pathogen for chronic periodontal diseases in adults. Our previous study suggested that the major structural components of both Fim and Mfa fimbriae in this organism are secreted through their lipidated precursors. In this study, we constructed Escherichia coli strains expressing various fimA genes with or without the 5'-terminal DNA region encoding the signal peptide, and we determined whether lipidation of recombinant FimA proteins occurred in E. coli. Lipidation occurred for a recombinant protein from the fimA gene with the 5'-terminal DNA region encoding the signal peptide but not for a recombinant protein from the fimA gene without the signal-peptide-encoding region, as revealed by [3H]palmitic acid labeling experiments. A TLR2-dependent signaling response was induced by the recombinant protein from the fimA gene with the signal-peptide-encoding region but not by a recombinant protein from the fimA gene with the signal-peptide-encoding region that had a base substitution causing an amino acid substitution (C19A). Electron microscopic analysis revealed that recombinant FimA (A-47?- W-383) protein was autopolymerized to form filamentous structures of about 80?nm in length in vitro. The results suggest that FimA protein, a major subunit of Fim fimbriae, is transported to the outer membrane by the lipoprotein sorting system, and a mature or processed FimA protein on the outer membrane is autopolymerized to form Fim fimbriae.  相似文献   

9.
10.
Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes based on the diversity of the fimA genes encoding each fimbria subunit. It was suggested that P. gingivalis strains with type II fimbriae were more virulent than type I strains. For the present study, we generated the mutants in which fimA was substituted with different genotypes to study virulence of type II fimbriae. Using plasmid vectors, fimA of ATCC33277 (type I strain) was substituted with type II fimA, and that of OMZ314 (type II strain) with type I fimA. The substitution of type I fimA with type II enhanced bacterial adhesion/invasion to epithelial cells, whereas substitution with type I fimA resulted in diminished efficiency. Following bacterial invasion, type II clones swiftly degraded cellular paxillin and focal adhesion kinase, and inhibited cellular migration, whereas type I clones and DeltafimA mutants did not. BIAcore analysis demonstrated that type II fimbriae possess greater adhesive abilities for their receptor alpha5beta1-integrin than those of type I. In a mouse abscess model, the type II clones significantly induced serum IL-1beta and IL-6, as well as other infectious symptoms. These results suggest that type II fimbriae are a critical determinant of P. gingivalis virulence.  相似文献   

11.
The fimA gene of Xanthomonas campestris pv. vesicatoria was identified and characterized. A 20-mer degenerate oligonucleotide complementary to the N-terminal amino acid sequence of the purified 15.5-kDa fimbrillin was used to locate fimA on a 2.6-kb SalI fragment of the X. campestris pv. vesicatoria 3240 genome. The nucleotide sequence of a 1.4-kb fragment containing the fimA region revealed two open reading frames predicting highly homologous proteins FimA and FimB. FimA, which was composed of 136 amino acids and had a calculated molecular weight of 14,302, showed high sequence identity to the type IV fimbrillin precursors. fimB predicted a protein product of 135 amino acids and a molecular weight of 13,854. The open reading frame for fimB contained near the 5' end a palindromic sequence with a terminator loop potential, and the expression level of fimB in vitro and in Xanthomonas was considerably lower than that of fimA. We detected an efficiently transcribed fimA-specific mRNA of 600 bases as well as two weakly expressed, longer mRNA species that reacted with both fimA and fimB. A homolog of fimA but not of fimB was detected by Southern hybridization in strains of X. campestris pv. vesicatoria, campestris, begoniae, translucens, and graminis. A fimA::omega mutant of strain 3240 was not significantly reduced in virulence or adhesiveness to tomato leaves. However, the fimA mutant was dramatically reduced in cell aggregation in laboratory cultures and on infected tomato leaves. The fimA mutant strain also exhibited decreased tolerance to UV light.  相似文献   

12.
13.
14.
The differential host species specificities of Bordetella pertussis, B. parapertussis, and B. bronchiseptica might be explained by polymorphisms in adherence factor genes. We have found that B. parapertussis and B. bronchiseptica, unlike B. pertussis, contain a full-length gene for the fimbrial subunit FimA. B. bronchiseptica expresses fimA in a BvgAS-dependent fashion.  相似文献   

15.
16.
ABSTRACT: BACKGROUND: Salmonella enterica serotype Typhimurium produces surface-associated fimbriae that facilitate adherence of the bacteria to a variety of cells and tissues. Type 1 fimbriae with binding specificity to mannose residues are the most commonly found fimbrial type. In vitro, static-broth culture favors the growth of S. Typhimurium with type 1 fimbriae, whereas non-type 1 fimbriate bacteria are obtained by culture on solid-agar media. Previous studies demonstrated that the phenotypic expression of type 1 fimbriae is the result of the interaction and cooperation of the regulatory genes fimZ, fimY, fimW, and fimU within the fim gene cluster. Genome sequencing revealed a novel gene, stm0551, located between fimY and fimW that encodes an 11.4-kDa putative phosphodiesterase specific for the bacterial second messenger cyclic-diguanylate monophosphate (c-di-GMP). The role of stm0551 in the regulation of type 1 fimbriae in S. Typhimurium remains unclear. RESULTS: A stm0551-deleted stain constructed by allelic exchange constitutively produced type 1 fimbriae in both static-broth and solid-agar medium conditions. Quantative RT-PCR revealed that expression of the fimbrial major subunit gene, fimA, and one of the regulatory genes, fimZ, were comparably increased in the stm0551-deleted strain compared with those of the parental strain when grown on the solid-agar medium, a condition that normally inhibits expression of type 1 fimbriae. Following transformation with a plasmid possessing the coding sequence of stm0551, expression of fimA and fimZ decreased in the stm0551 mutant strain in both culture conditions, whereas transformation with the control vector pACYC184 relieved this repression. A purified STM0551 protein exhibited a phosphodiesterase activity in vitro while a point mutation in the putative EAL domain, substituting glutamic acid (E) with alanine (A), of STM0551 or a FimY protein abolished this activity. CONCLUSIONS: The finding that the stm0551 gene plays a negative regulatory role in the regulation of type 1 fimbriae in S. Typhimurium has not been reported previously. The possibility that degradation of c-di-GMP is a key step in the regulation of type 1 fimbriae warrants further investigation.  相似文献   

17.
The adhesion properties of the recombinant fimbriae (r-fimbriae) recovered from a YH522 transformant of Porphyromonas gingivalis which harbors a chimeric plasmid, pYHF2, containing the fimA gene of strain 381 were compared with those of the endogenous fimA fimbriae of strain 33277. The adhesion level of the r-fimbriae to Actinomyces viscosus was clearly lower than that of the endogenous fimbriae. In addition, the r-fimbriae were shown to lack some minor components detectable in the endogenous fimbriae. The plasmid pYHF2 prepared from the YH522 transformant was then transformed into six different P. gingivalis strains and the resultant pYHF2-containing strains were examined for their fimbrial expression. In spite of the presence of a considerable diversity in the expression level of the r-fimbriae among these transformants, it was evident that the strains expressing higher levels of the r-fimbriae exhibited a greater decrease in adhesion activity to other bacteria and to oral epithelial cells, as well as in self-aggregation.  相似文献   

18.
Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.  相似文献   

19.
Two types of adhesive fimbriae are expressed by Actinomyces; however, the architecture and the mechanism of assembly of these structures remain poorly understood. In this study we characterized two fimbrial gene clusters present in the genome of Actinomyces naeslundii strain MG-1. By using immunoelectron microscopy and biochemical analysis, we showed that the fimQ-fimP-srtC1-fimR gene cluster encodes a fimbrial structure (designated type 1) that contains a major subunit, FimP, forming the shaft and a minor subunit, FimQ, located primarily at the tip. Similarly, the fimB-fimA-srtC2 gene cluster encodes a distinct fimbrial structure (designated type 2) composed of a shaft protein, FimA, and a tip protein, FimB. By using allelic exchange, we constructed an in-frame deletion mutant that lacks the SrtC2 sortase. This mutant produces abundant type 1 fimbriae and expresses the monomeric FimA and FimB proteins, but it does not assemble type 2 fimbriae. Thus, SrtC2 is a fimbria-specific sortase that is essential for assembly of the type 2 fimbriae. Together, our experiments pave the way for several lines of molecular investigation that are necessary to elucidate the fimbrial assembly pathways in Actinomyces and their function in the pathogenesis of different biofilm-related oral diseases.  相似文献   

20.
The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号