Cloning and sequencing of the gene encoding Vibrio cholerae O1 fimbrial subunit (fimbrillin)

Abstract The gene encoding an 18 kDa fimbrial subunit of Vibrio cholerae O1 was identified in a fimbriate strain Bgd17. Mixed oligoprimers were prepared based on the amino acid sequence of the N-terminus and that from a cyanogen bromide-cleaved fragment of the fimbrillin. A PCR-amplified 185 bp DNA fragment was sequenced. This 185 bp fragment was further extended to 540 bp to 3' and 5' termini by RNA-PCR using a primer containing a random hexamer at its 3' end. This fragment did not contain the stop codons. It was further extended by a gene walking method using Eco RI cassette and its primers. Finally a 660 bp fragment was obtained and sequenced. This fragment contained the complete open reading frame of the structural subunit of the fimbriae, composed of 169 amino acids with a molecular mass of 17435.65 and a leader sequence of 6 or 9 amino acids. The deduced amino acid sequence of the polypeptide encoded by the gene, designated fim A, displayed a highly conserved sequence of MKXXXGFTLI EL of type 4 fimbriae.     

Characterization of type 1 and mannose-resistant fimbriae of Erwinia spp.

Type 1 fimbriae from Erwinia carotovora subsp. carotovora and mannose-resistant fimbriae from Erwinia rhapontici were purified and characterized. The type 1 fimbrillin had an apparent molecular weight of 16,500; that of the mannose-resistant fimbrillin was 18,000. The amino-terminal amino acid sequences of the two fimbrillins were related, but tryptic peptide maps showed significant differences between the proteins. No serological cross-reaction was found between the two fimbrial filaments, nor did they cross-react with type 1 or type 3 fimbriae purified from other enterobacterial species. Immunofluorescent staining of bacterial populations revealed that they were heterogeneous with respect to fimbriation.     

Genomic heterogeneity in Chlorobium limicola: chromosomic and plasmidic differences among strains

Abstract The purpose of this study is to certify the importance of the fimbriae as an attachment factor of Actinobacillus actinomycetemcomitans , a human periodontopathic bacterium, and the significance of anti-fimbrial antibody function as an attachment inhibitor. Fimbrial antigen was prepared from the A. actinomycetemcomitans 310-a strain. Oligopeptides were synthesized according to the amino acid sequence of the fimbrial protein. The peptide antigen was conjugated with branched lysine polymer resin beads. The peptide antigen was suspended in PBS emulsified with incomplete Freund's adjuvant and used to immunize rabbits. A rabbit antiserum reacted with an approximately 54 kDa protein of the fimbriae protein from A. actinomycetemcomitans 310-a and with those of other fimbriated strains. This antiserum strongly inhibited the attachment of fimbriated A. actinomycetemcomitans strains to saliva-coated hydroxyapatite beads, buccal epithelial cells, and a fibroblast cell line, Gin-1. Such a synthetic fimbrial peptide antigen may be effective in inducing antibodies which inhibit adhesion and subsequent colonization by A. actinomycetemcomitans .     

Purification and Characterization of Type 1 Fimbriae of Salmonella typhi

Type 1 fimbriae have been purified from a Salmonella typhi strain of clinical origin. Purified fimbriae retained their ability to bind to erythrocytes in a mannose-inhibitable fashion and, in doing so, behaved preferentially as a monovalent adhesin. SDS-PAGE analysis of the fimbrial preparation showed the presence of a 20-kDa major polypeptide component (fimbrillin) and of additional larger polypeptides present in smaller amounts. The amino-terminal sequence of fimbrillin was determined and turned out to be very similar but not identical to that of type 1 fimbrillins of other Salmonella serovars. A Western blot analysis of the purified fimbrial preparation using an antiserum raised against native fimbriae suggested that fimbrial proteins did not carry any major sequential epitope and that, in native fimbriae, conformational epitopes, possibly generated between different subunits, might provide for the major immunogenic epitopes. Analysis of different S. typhi clinical isolates using the anti-fimbrial antiserum showed an overall immunological similarity of these structures within this serovar.     

Porphyromonas gingivalis fimbriae and their synthetic peptides induce proinflammatory cytokines in human peripheral blood monocyte cultures

Abstract Porphyromonas gingivalis fimbriae as well as synthetic peptides that mimic the fimbrial subunit protein, which includes the amino acid sequence XLTXXLTXXNXX, induced high production of proinflammatory cytokines such as interleukin-1β, interleukin-6, interleukin-8, tumor necrosis factor-α in human peripheral blood monocyte/macrophage cultures. Responses induced by some peptide segments were comparable to those induced by Escherichia coli lipopolysaccharides. A chemically modified peptide analogous to an active peptide segment was found to be antagonistic with regard to interleukin-6 production induced by the native fimbriae. It may be suggested that P. gingivalis fimbriae and their degraded peptides function as proinflammatory agents in vivo, while certain analog peptides inhibited the process.     

Streptococcus salivarius fimbriae are composed of a glycoprotein containing a repeated motif assembled into a filamentous nondissociable structure

Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivarius fimbriae did not dissociate when they were incubated at 100 degrees C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 x 10(6) to 40 x 10(6) Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 microm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/phi, where X represents a modified amino acid residue and phi represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis and Streptococcus constellatus.     

Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae

Binding of Porphyromonas gingivalis to the host cells is an essential step in the pathogenesis of periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are thought to be involved in this process. In our earlier studies, two major epithelial cell components of 40 and 50 kDa were identified as potential fimbrial receptors. Sequencing of a cyanogen bromide digestion fragment of the 50-kDa component resulted in an internal sequence identical to keratin I molecules, and hence this cytokeratin represents one of the epithelial cell receptors for P. gingivalis fimbriae. In this study, the 40-kDa component of KB cells was isolated and its amino-terminal sequence determined. The N-terminal amino sequence was found to be GKVKVGVNGF and showed perfect homology with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, purified P. gingivalis fimbriae were found to bind to rabbit muscle GAPDH. Antibodies directed against internal peptide 49-68 and 69-90 of fimbrillin were shown to inhibit the binding of P. gingivalis and of fimbriae to epithelial cells. Antibodies against these peptides also inhibited the binding of fimbriae to GAPDH. Our results confirmed that the amino-terminal domain corresponding to amino residues 49-68 of the fimbrillin protein is the major GAPDH binding domain. These studies point to GAPDH as a major receptor for P. gingivalis major fimbriae and, as such, GAPDH likely plays a role in P. gingivalis adherence and colonization of the oral cavity, as well as triggering host cell processes involved in the pathogenesis of P. gingivalis infections.     

Proteus mirabilis MR/P fimbriae: molecular cloning, expression, and nucleotide sequence of the major fimbrial subunit gene.

Proteus mirabilis, a cause of serious urinary tract infection and acute pyelonephritis, produces several putative virulence determinants, among them, fimbriae. Principally, two fimbrial types are produced by this species: mannose-resistant/Proteus-like (MR/P) fimbriae and mannose-resistant/Klebsiella-like (MR/K) fimbriae. To isolate MR/P fimbrial gene sequences, a P. mirabilis cosmid library was screened by immunoblotting and by hybridization with an oligonucleotide probe based on the N-terminal amino acid sequence of the isolated fimbrial polypeptide, ADQGHGTVKFVGSIIDAPCS. One clone, pMRP101, reacted strongly with a monoclonal antibody specific for MR/P fimbriae and with the DNA probe. This clone hemagglutinated both tannic acid-treated and untreated chicken erythrocytes with or without 50 mM D-mannose and was shown to be fimbriated by transmission electron microscopy. A 525-bp open reading frame, designated mrpA, predicted a 175-amino-acid polypeptide including a 23-amino-acid hydrophobic leader peptide. The unprocessed and processed polypeptides are predicted to be 17,909 and 15,689 Da, respectively. The N-terminal amino acid sequence of the processed fimbrial subunit exactly matched amino acid residues 24 to 43 predicted by the mrpA nucleotide sequence. The MrpA polypeptide shares 57% amino acid sequence identity with SmfA, the major fimbrial subunit of Serratia marcescens mannose-resistant fimbriae.     

Purification and characterization of a novel secondary fimbrial protein from Porphyromonas gingivalis strain 381

We previously reported the existence of two different kinds of fimbriae expressed by Porphyromonas gingivalis ATCC 33277. In this study, we isolated and characterized a secondary fimbrial protein from strain FPG41, a fimA-inactivated mutant of P. gingivalis 381. FPG41 was constructed by a homologous recombination technique using a mobilizable suicide vector, and failed to express the long fimbriae (41-kDa fimbriae) that were produced on the cell surface of P. gingivalis 381. However, short fimbrial structures were observed on the cell surface of FPG41 by electron microscopy. The fimbrial protein was purified from FPG41 by DEAE-Sepharose CL-6B column chromatography. The secondary fimbrial protein was eluted at 0.15 M NaCl, and the molecular mass of this protein was approximately 53 kDa as estimated by SDS-PAGE. An antibody against the 53-kDa fimbrial protein reacted with the short fimbriae of the FPG41 and the wild-type strain. However, the 41-kDa long fimbriae of the wild-type strain and the 67-kDa fimbriae of ATCC 33277 did not react with the same antibody. Moreover, the N-terminal amino acid sequence of the 53-kDa fimbrial protein showed only 2 of 15 residues that were identical to those of the 41-kDa fimbrial protein. These results show that the properties of the 53-kDa fimbriae are different from those of the 67-kDa fimbriae of ATCC 33277 as well as those of the 41-kDa fimbriae.     

Genetic manipulation of major P-fimbrial subunits and consequences for formation of fimbriae.

The influence of genetic manipulation of the structural genes coding for major P-fimbrial subunits on the formation of fimbriae in Escherichia coli was studied. Deletion of two regions that code for hypervariable parts of the P fimbrillin resulted in strong reduction or total absence of fimbria production. Replacement of deleted amino acids by other amino acid residues restored the formation of fimbriae. The hypervariable regions may be important for biogenesis of fimbriae by imposing correct spacing between conserved regions of the protein. The potential for substituting amino acids in the P-fimbrial subunit opens interesting possibilities for use of fimbriae as carriers of foreign antigenic determinants. An antigenic determinant of foot-and-mouth disease virus (FMDV) was incorporated in the F11 fimbrial subunit. Hybrid fimbriae, recognized by an FMDV-specific neutralizing monoclonal antibody directed against FMDV, were formed.     

Studies on the ribosomal RNA operons of Listeria monocytogenes

Mannose-resistant hemagglutinating fimbrial antigen F165 is produced by Escherichia coli strains associated with septicemia in piglets and calves. A fimbrial component with an M(r) of 17,200 as determined by SDS-PAGE was purified to homogeneity from F165-positive E. coli strain 4787 of serogroup O115. This fimbrial component of F165 antigen was named F165(2). Separation procedures included fast protein liquid chromatography with a Superose 12 column followed by ultracentrifugation and 0.15 M ethanolamine buffer (pH 10.5) dissociation. Upon removal of ethanolamine, the fimbrial component reassociated into fimbriae. Amino acid composition analysis indicated that the fimbrial component molecule comprised 158 amino acid residues of which 37.3% were hydrophobic. The amino acid composition and the isoelectric point (9.5) were readily distinguishable from those of F1 fimbriae. The amino acid sequence was determined for approximately 40% of the molecule. For the first 33 residues, the F165(2) sequence was identical to that of F1B fimbriae and very similar to that of F1C. Fimbriae F165(2) could nevertheless be differentiated antigenically from F1C fimbriae as demonstrated by the immunodot technique using cross-absorbed antisera.     

Dual function of a tip fimbrillin of Actinomyces in fimbrial assembly and receptor binding

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.     

The fimA gene encoding the type-1 fimbrial subunit of Escherichia coli. Nucleotide sequence and primary structure of the protein

The nucleotide sequence was determined of a region of 1450 base pairs encompassing the fimA gene for the subunit of type 1 fimbriae of Escherichia coli as well as flanking regions containing potential regulator sequences. The 'translated' protein contains a 23-residue signal peptide; the processed fimbrial subunit consists of 158 amino acid residues yielding a relative molecular mass of 15706. The elucidated sequence shows significant homology with those of other E. coli fimbrial proteins.     

Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.

Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.     

Type 3 fimbriae of Klebsiella sp.: molecular characterization and role in bacterial adhesion to plant roots.

Type 3 fimbriae of Klebsiella were purified and characterized. The fimbriae were 4 to 5 nm in diameter and 0.5 to 2 microns long. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the fimbrillin had an apparent molecular weight of 23,500, and it differed from enterobacterial type 1 fimbrillins in its amino acid composition. Hydrophobic amino acids comprised 33.6% of all amino acids in the fimbrillin, which lacked cystine, phenylalanine, and arginine. Serologically, the type 3 fimbriae were also distinct from the type 1 fimbriae. Purified type 3 fimbriae agglutinated tannin-treated human blood group O erythrocytes; this confirms the role of type 3 fimbriae as hemagglutinins. Purified 125I-labeled type 3 fimbriae bound to the roots of Poa pratensis, and this binding could be inhibited by Fab fragments to the purified fimbriae. Anti-type 3 fimbriae Fab fragments also inhibited bacterial adhesion to plant roots. These results demonstrate that type 3 fimbriae mediate adhesion of klebsiellas to plant roots. Eight nitrogen-fixing strains of Klebsiella also produced type 3 fimbriae when grown under anaerobic nitrogen fixation conditions. It is proposed that type 3 fimbriae are involved in the establishment of the plant-bacterium association concerning nitrogen-fixing Klebsiella strains.     

Characterization of two genes encoding antigenically distinct type-1 fimbriae of Klebsiella pneumoniae

A uropathogenic isolate of Klebsiella pneumoniae was shown to exhibit a mannose-sensitive hemagglutinating phenotype and to produce type-1 fimbriae consisting of subunits with a different electrophoretic mobility than those previously investigated. The gene cluster encoding expression of fimbriae was cloned and the genetic organization of the encoded polypeptides was determined. The gene encoding the major fimbrial subunit was localized and further examined by nucleotide sequence analysis. Comparison of two K. pneumoniae fimbrial genes revealed a nucleotide sequence agreement of 73%, and amino acid sequence agreement of 84% for the mature fimbrial subunits. Predictions of putative antigenic sites were correlated with regions demonstrating amino acid variability. In agreement with these predictions, no serological cross-reactivity between both fimbrial proteins could be demonstrated using an enzyme-linked immunosorbent assay (ELISA).     

Sequence homology between the subunits of two immunologically and functionally distinct types of fimbriae of Actinomyces spp.

Nucleotide sequencing of the type 1 fimbrial subunit gene of Actinomyces viscosus T14V revealed a consensus ribosome-binding site followed by an open reading frame of 1,599 nucleotides. The encoded protein of 533 amino acids (Mr = 56,899) was predominantly hydrophilic except for an amino-terminal signal peptide and a carboxy-terminal region identified as a potential membrane-spanning segment. Edman degradation of the cloned protein expressed in Escherichia coli and the type 1 fimbriae of A. viscosus T14V showed that both began with alanine at position 31 of the deduced amino acid sequence. The amino acid compositions of the cloned protein and fimbriae also were comparable and in close agreement with the composition of the deduced protein. The amino acid sequence of the A. viscosus T14V type 1 fimbrial subunit showed no significant global homology with various other proteins, including the pilins of gram-negative bacteria. However, 34% amino acid sequence identity was noted between the type 1 fimbrial subunit of strain T14V and the type 2 fimbrial subunit of Actinomyces naeslundii WVU45 (M. K. Yeung and J. O. Cisar, J. Bacteriol. 170:3803-3809, 1988). This homology included several different conserved sequences of up to eight identical amino acids that were distributed in both the amino- and carboxy-terminal thirds of each Actinomyces fimbrial subunit. These findings indicate that the different types of fimbriae on these gram-positive bacteria share a common ancestry.     

Proteus mirabilis MR/P fimbrial operon: genetic organization, nucleotide sequence, and conditions for expression.

Proteus mirabilis, an agent of urinary tract infection, expresses at least four fimbrial types. Among these are the MR/P (mannose-resistant/Proteus-like) fimbriae. MrpA, the structural subunit, is optimally expressed at 37 degrees C in Luria broth cultured statically for 48 h by each of seven strains examined. Genes encoding this fimbria were isolated, and the complete nucleotide sequence was determined. The mrp gene cluster encoded by 7,293 bp predicts eight polypeptides: MrpI (22,133 Da), MrpA (17,909 Da), MrpB (19,632 Da), MrpC (96,823 Da), MrpD (27,886 Da), MrpE (19,470 Da), MrpF (17,363 Da), and MrpG (13,169 Da). mrpI is upstream of the gene encoding the major structural subunit gene mrpA and is transcribed in the direction opposite to that of the rest of the operon. All predicted polypeptides share > or = 25% amino acid identity with at least one other enteric fimbrial gene product encoded by the pap, fim, smf, fan, or mrk gene clusters.     

Cloning and nucleotide sequence of a gene for Actinomyces naeslundii WVU45 type 2 fimbriae.

A genomic library of Actinomyces naeslundii WVU45 DNA in Escherichia coli was screened for antigen expression with rabbit antibody against A. naeslundii fimbriae. Western blotting (immunoblotting) of one recombinant clone carrying a 13.8-kilobase-pair insert revealed a 59-kilodalton (kDa) immunoreactive protein. A protein of similar electrophoretic mobility was detected from the isolated fimbrial antigen. Expression of the 59-kDa cloned protein in E. coli was directed by a promoter from the insert. The DNA sequence of the subunit gene was determined, and an open reading frame of 1,605 nucleotides was identified which was preceded by a putative ribosome-binding site and followed by two inverted repeats of 14 and 17 nucleotides, respectively. The reading frame encoded a protein of 534 amino acids (calculated molecular weight, 57,074), and the N-terminal sequence resembled that of a signal peptide. The presence of a 32-amino-acid signal peptide was indicated by amino-terminal sequencing of the fimbriae from A. naeslundii. The sequence, as determined by Edman degradation, was identical to that deduced from the DNA sequence beginning at predicted residue 33 of the latter sequence. Moreover, the amino acid composition of the predicted mature protein was similar to that of the isolated fimbriae from A. naeslundii. Thus, the cloned gene encodes a subunit of A. naeslundii fimbriae.