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S K Collinson

Publications and source records attributed to S K Collinson.

At least 19 recordsLinked to original sources
PubMed2003

Extracellular polysaccharides associated with thin aggregative fimbriae of Salmonella enterica serovar enteritidis.

Lipopolysaccharide (LPS) O polysaccharide was identified as the principle factor impeding intercellular formation of intact thin aggregative fimbriae (Tafi) in Salmonella enterica serovar Enteritidis. The extracellular nucleation-precipitation assembly pathway for these organelles was investigated by quantifying fimbrial formation between deltaagfA (AgfA recipient) and deltaagfB (AgfA donor) cells harboring mutations in LPS (galE::Tn10) and/or cellulose (deltabcsA) synthesis. Intercellular complementation could be detected between deltaagfA and deltaagfB strains only when both possessed the galE mutation. LPS O polysaccharide appears to be an impenetrable barrier to AgfA assembly between cells but not within individual cells. The presence of cellulose did not restrict Tafi formation between cells. Transmission electron microscopy of w+ S. enterica serovar Enteritidis 3b cells revealed diffuse Tafi networks without discernible fine structure. In the absence of cellulose, however, individual Tafi fibers were clearly visible, appeared to be occasionally branched, and showed the generally distinctive appearance described for Escherichia coli K-12 curli. A third extracellular matrix component closely associated with cellulose and Tafi was detected on Western blots by using immune serum raised to whole, purified Tafi aggregates. Cellulose was required to tightly link this material to cells. Antigenically similar material was also detected in S. enterica serovar Typhimurium and one diarrheagenic E. coli isolate. Preliminary analysis indicated that this material represented an anionic, extracellular polysaccharide that was distinct from colanic acid. Therefore, Tafi in their native state appear to exist as a complex with cellulose and at least one other component.

Arabidopsis Proteins↗
PubMed2001

Structure and characterization of AgfB from Salmonella enteritidis thin aggregative fimbriae.

The agfBAC operon of Salmonella enteritidis encodes thin aggregative fimbriae, fibrous, polymeric structures primarily composed of AgfA fimbrins. Although uncharacterized, AgfB shows a 51 % overall amino acid sequence similarity to AgfA. Using AgfB epitope-specific antiserum, AgfB was detected as a minor component of whole, purified fimbriae. Like AgfA, AgfB was released from purified fimbriae by >70 % formic acid, whereupon both AgfA-AgfA and AgfA-AgfB dimers as well as monomers were detected. This suggested that AgfB may form specific, highly stable, structural associations with AgfA in native fimbrial filaments, associations that were weakened in structurally unstable fibers derived from AgfA chimeric fimbrial mutants. Detailed sequence comparisons between AgfA and AgfB showed that AgfB harbored a similar fivefold repeated sequence pattern (x(6)QxGx(2)NxAx(3)Q), and contained structural motifs similar to the parallel beta helix model proposed for AgfA. Molecular modeling of AgfB revealed a 3D structure remarkably similar to that of AgfA, the structures differing principally in the surface disposition of non-conserved, basic, acidic and non-polar residues. Thus AgfB is a fimbrin-like structural homologue of AgfA and an integral, minor component of native thin aggregative fimbrial fibers. AgfB from an agfA deletion strain was detected as a non-fimbrial, SDS-insoluble form in the supernatant and was purified. AgfA from an agfB deletion strain was found in both SDS-soluble and insoluble, non-fimbrial forms. No AgfA-AgfA dimers were detected in the absence of AgfB. Fimbriae formation by intercellular complementation between agfB and agfA deletion strains could not be shown under a variety of conditions, indicating that AgfA and AgfB are not freely diffusible in S. enteritidis. This has important implications on the current assembly hypothesis for thin aggregative fimbriae.

Amino Acid Motifs↗
PubMed2000

Salmonella enteritidis fimbriae displaying a heterologous epitope reveal a uniquely flexible structure and assembly mechanism.

Two distinct Salmonella fimbrins, AgfA and SefA, comprising thin aggregative fimbriae SEF17 and SEF14, respectively, were each genetically engineered to carry PT3, an alpha-helical 16-amino acid Leishmania T-cell epitope derived from the metalloprotease gp63. To identify regions within AgfA and SefA fimbrins amenable to replacement with this epitope, PCR-generated chimeric fimbrin genes were constructed and used to replace the native chromosomal agfA and sefA genes in Salmonella enteritidis. Immunoblot analysis using anti-SEF17 and anti-PT3 sera demonstrated that all ten AgfA chimeric fimbrin proteins were expressed by S. enteritidis under normal growth conditions. Immunoelectron microscopy confirmed that eight of the AgfA::PT3 proteins were effectively assembled into cell surface-exposed fimbriae. The PT3 replacements in AgfA altered Congo red (CR) binding, cell-cell adhesion and cell surface properties of S. enteritidis to varying degrees. However, these chimeric fimbriae were still highly stable, being resistant to proteinase K digestion and requiring harsh formic acid treatment for depolymerization. In marked contrast to AgfA, none of the chimeric SefA proteins were expressed or assembled into fimbriae. Since each PT3 replacement constituted over 10% of the AgfA amino acid sequence and all ten replacements collectively represented greater than 75% of the entire AgfA primary sequence, the ability of AgfA to accept large sequence substitutions and still assemble into fibers is unique among fimbriae and other structural proteins. This structural flexibility may be related to the novel fivefold repeating sequence of AgfA and its recently proposed structure Proper formation of chimeric fimbrial fibers suggests an unusual assembly mechanism for thin aggregative fimbriae which tolerates aberrant structures. This study opens a range of possibilities for Salmonella thin aggregative fimbriae as a carrier of heterologous epitopes and as an experimental model for studies of protein structure.

Amino Acid Sequence↗
PubMed1999

Structural predictions of AgfA, the insoluble fimbrial subunit of Salmonella thin aggregative fimbriae.

The unusually stable and multifunctional, thin aggregative fimbriae common to all Salmonella spp. are principally polymers of the fimbrin subunit, AgfA. AgfA of Salmonella enteritidis consists of two domains: a protease-sensitive, 22 amino acid residue N-terminal region and a protease-resistant, 109 residue C-terminal core. The unusual amino acid sequence of the AgfA core region comprises two-, five- and tenfold internal sequence homology patterns reflected in five conserved, 18-residue tandem repeats. These repeats have the consensus sequence, Sx5QxGx2NxAx3Q and are linked together by four or five residues, (x)xAx2. The predicted secondary structure for this unusual arrangement of tandem repeats in AgfA indicates mainly extended conformation with the beta strands linked by four to six residues. Candidate proteins of known structure with motifs of alternating beta strands and short loops were selected from folds described in SCOP as a source of coordinates for AgfA model construction. Three all-beta class motifs selected from the Serratia marcescens metalloprotease, myelin P2 protein or vitelline membrane outer protein I were used for initial AgfA homology build-up procedures ultimately resulting in three structural models; beta barrel, beta prism and parallel beta helix. The beta barrel model is a compact, albeit irregular structure, with the beta strands arranged in two antiparallel beta sheet faces. The beta prism model does not reflect the 5 or 10-fold symmetry of the AgfA primary sequence. However, the favored, parallel beta helix model is a compact coil of ten helically arranged beta strands forming two parallel beta sheet faces. This arrangement predicts a regular, potentially stable, C-terminal core region consistent with the observed tandem repeat sequences, protease-resistance and strong tendency of this fimbrin to oligomerize and aggregate. Positional conservation of amino acid residues in AgfA and the Escherichia coli AgfA homologue, CsgA, provides strong support for this model. The parallel beta helix model of AgfA offers an interesting solution to a multifunctional fimbrin molecular surface having solvent exposed areas, regions for major and minor subunit interactions as well as fiber-fiber interactions common to many bacterial fimbriae.

Amino Acid Sequence↗
PubMed1999

High efficiency gene replacement in Salmonella enteritidis: chimeric fimbrins containing a T-cell epitope from Leishmania major.

A simple, high frequency chromosomal gene replacement method of general utility was developed for Salmonella enteritidis. This system uses an unstable, imperfectly segregating, temperature-sensitive replicon, pHSG415, as a carrier of the recombinant gene of interest. It also allows for site-specific replacement of chromosomal genes without the need for antibiotic resistance markers in the recombinant genes or the use of specific bacterial strains. This strategy was used to replace the chromosomal sefA and agfA fimbrin genes of S. enteritidis 3b with recombinant genes containing a 48 bp DNA fragment encoding PT3, an immunoprotective T-cell epitope from GP63 of Leishmania major. The fidelity of chimeric fimbrial replacements were confirmed by DNA sequence analysis. Nearly 30% of the S. enteritidis clones selected in the final stage of sefA mutagenesis contained the sefA::PT3 recombinant gene, whereas for agfA the efficiency was as high as 10%. To our knowledge, this is the first report of fimbrial epitope replacement in the Salmonellae and the first chimeric fimbrin genes that have been reconstituted into a wild-type genetic background for any organism. As such, this model represents a promising 'organelle' expression system for epitope display in vaccinology.

Amino Acid Sequence↗
PubMed1998

Periplasmic and fimbrial SefA from Salmonella enteritidis.

Salmonella enteritidis produces thin, filamentous fimbriae composed of the fimbrin subunit SefA. Although insoluble in most detergents and chaotropic agents, these fimbriae were soluble at pH 10.5. Furthermore, in sodium dodecyl sulfate, these fibers depolymerized into monomers, dimers and other multimers of SefA, which precipitated on removal of the detergent. In contrast, unassembled periplasmic SefA fimbrins purified from Escherichia coli expressing cloned sefA and sefB were readily soluble in aqueous solution. Fimbrial and periplasmic SefA also differed in their reaction with an anti-SEF14 monoclonal antibody and in their surface hydrophobicity, indicating that the two forms had different properties. Precise mass measurements of periplasmic and fimbrial SefA by mass spectroscopy showed that these variations were not due to post-translational modifications. Periplasmic SefA consisted primarily of intact as well as some N-terminally truncated forms. The main 24 amino acid, N-terminally truncated form of periplasmic SefA was present as a 12.2 kDa monomer which had a low tendency to dimerize whereas intact periplasmic SefA was present as a 34.1 kDa homodimer. Intact periplasmic SefA also formed stable multimers at low concentrations of chemical cross-linker but multimerization of the truncated form required high concentrations of protein or cross-linker. Thus, SefA fimbrins appear to multimerize through their N-termini and undergo a conformational change prior to assembly into fibers. Within these fibers, subunit-subunit contact is maintained through strong hydrophobic interactions.

Bacterial Proteins↗
PubMed1998

Thin aggregative fimbriae enhance Salmonella enteritidis biofilm formation.

Salmonella enteritidis enteropathogens produce a variety of potentially adherent fimbrial types including SEF14, SEF17, SEF18 and SEF21 (type I). In a simplified, pure culture, biofilm generating system the virulent isolate, S. enteritidis 3b, readily adhered to Teflon (polytetrafluoroethylene) and stainless steel forming thick cell aggregates. The inability of an isogenic SEF17-deficient mutant to form thick biofilms suggested a role for SEF17 in stabilizing cell-cell interactions during biofilm formation. Epifluorescent detection of SEF17 in biofilms confirmed the association of these fimbriae with aggregated cells but not with adherent mutants unable to produce SEF17. The reduced adherence observed with an isogenic SEF14/SEF21-deficient strain implicated the involvement of additional cell surface adherence factors, possibly including SEF21 (type I) fimbriae in the adherence of S. enteritidis to stainless steel or Teflon. The role of SEF17 fimbriae in biofilm formation and the contributions of SEF17 to the persistence of Salmonellae on surfaces and in food are discussed.

Bacterial Adhesion↗
PubMed1998

tRNA(Arg) (fimU) and expression of SEF14 and SEF21 in Salmonella enteritidis.

A Tn10 insertion affecting SEF14 fimbrial synthesis in Salmonella enteritidis was located 13 bp upstream of a gene designated fimU. The 77-bp DNA sequence of fimU from S. enteritidis was identical to that of fimU encoding tRNA(Arg) (UCU) from Salmonella typhimurium and 96% identical to that of the Escherichia coli argU homolog. Furthermore, the open reading frame adjacent to and overlapping the 3' end of fimU was similar to the prophage DLP12 integrase gene. The fimU-encoded transcript comigrated with total cellular tRNA and was predicted to form a tRNA-like cloverleaf structure containing the arginine anticodon UCU. Thus, fimU encoded a tRNA(Arg) specific for the rare codon AGA. fimU mapped to the SEF21 fim operon located 15 C's from the sef14 gene cluster. Although fimU was located within the SEF21 fim gene cluster, the fimU Tn10 insertion mutant of S. enteritidis was found to be defective in SEF14 as well as SEF21 (type 1) fimbria production. SEF17 and SEF18 fimbria production was not affected. Complementation of this mutant with plasmid-borne fimU restored normal production of the fimbrins SefA and FimA as well as their respective fimbriae SEF14 and SEF21. This is the first description of tRNA simultaneously controlling the production of two distinct fimbriae.

Antigens, Bacterial↗
PubMed1996

The location of four fimbrin-encoding genes, agfA, fimA, sefA and sefD, on the Salmonella enteritidis and/or S. typhimurium XbaI-BlnI genomic restriction maps.

Four fimbrin-encoding genes, fimA (type-1 or SEF21 fimbriae), agfA (thin aggregative or SEF17 fimbriae), sefA (SEF14 fimbriae and sefD (SEF18 fimbriae) from Salmonella enteritidis (Se) 27655-3b were located onto the XbaI-BlnI genomic restriction maps of Salmonella typhimurium (St) LT2 and Se strains SSU7998 and 27655-3b. The XbaI or BlnI genomic fragments carrying these genes were identified by hybridization with labeled oligodeoxyribonucleotides or fimbrin-encoding genes. The fimbrin-encoding genes were not encoded by the virulence plasmids, but were located on chromosomal DNA fragments. The position of each gene on a given XbaI fragment was determined by hybridization of a series of XbaI-digested genomic DNA samples from previously characterized Tn10 mutants of Se and St with its respective probe. The fimA gene mapped near 13 centisomes (Cs) between purE884::Tn10 at 12.6 Cs (11.8 min) and apeE2::Tn10 at 12.8 Cs (12.3 min) beside the first XbaI site at 13.0 Cs in St or between purE884::Tn10 at 12.6 Cs and the XbaI site at 13.6 Cs in Se. The agfA gene mapped near 26 Cs between putA::Tn10 and pyrC691::Tn10 in St, but near 40 Cs between pncX::Tn10 and the XbaI site at 43.3 Cs in Se. This difference in map position was due to the location of agfA near one end of the 815-kb chromosomal fragment inverted between Se and St. The sefA and sefD genes mapped precisely at 97.6 Cs in Se, but were absent from the genome of St LT2. To verify the mapping procedures used herein, tctC was also mapped in both Salmonella serovars. As expected, tctC mapped near 60 Cs in both St and Se, thereby confirming previous studies.

Antigens, Bacterial↗
PubMed1996

Diagnostic potential of sefA DNA probes to Salmonella enteritidis and certain other O-serogroup D1 Salmonella serovars.

Salmonella enteritidis thin fimbriae, SEF14, were found to be restricted to S. dublin and the predominantly poultry-associated members of the Salmonella O-serogroup D1, S. enteritidis, S. berta, S. gallinarum and S. pullorum, when tested by Western and ELISA analysis from among 90 Salmonella isolates of 42 serovars, as well as from members of several related genera of the Enterobacteriaceae. These five serovars and a single isolate of S. typhi (D1) were also detected by hybridization of genomic DNA from 732 Salmonella isolates of 117 serogroups to gene probes derived from the S. enteritidis sefA (fimbrin gene), sefB (chaperone) or sefC (outer membrane protein) genes encoding proteins involved in SEF14 biosynthesis. None of 250 Enterobacteriaceae or 27 other eubacterial isolates tested hybridized to the sef probes. The sefA, sefB and sefC genes were amplified from these six Salmonella serovars by PCR using primer pairs designed from sefA, sefB or sefC of S. enteritidis. DNA sequencing of sefA genes from these five serovars indicated limited sequence variability among sefA genes and recognition of individual base pairs which could potentially differentiate certain strains of S. enteritidis, S. dublin and S. gallinarum.

Animals↗
PubMed1996

Salmonella enteritidis agfBAC operon encoding thin, aggregative fimbriae.

Salmonella enteritidis produces thin, aggregative fimbriae, named SEF17, which are composed of polymerized AgfA fimbrin proteins. DNA sequence analysis of a 2-kb region of S. enteritidis DNA revealed three contiguous genes, agfBAC. The 453-bp agfA gene encodes the AgfA fimbrin, which was predicted to be 74% identical and 86% similar in primary sequence to the Escherichia coli curli structural protein, CsgA. pHAG, a pUC18 derivative containing a 3.0-kb HindIII fragment encoding agfBAC, directed the in vitro expression of the major AgfA fimbrin, with an M(r) of 17,000, and a minor AgfB protein, with an M(r) of 16,000, encoded by the 453-bp agfB gene. AgfA was not expressed from pDAG, a pUC18 derivative containing a 3.1-kb DraI DNA fragment encoding agfA but not agfB. Primer extension analysis identified two adjacent transcription start sites located immediately upstream of agfB in positions analogous to those of the E. coli curlin csgBA operon. No transcription start sites were located immediately upstream of agfA or agfC. Northern (RNA) blot analysis confirmed that transcription of agfA was initiated from the agfB promoter region. Secondary-structure analysis of the putative mRNA transcript for agfBAC predicted the formation of a stem-loop structure (delta Gzero, -22 kcal/mol [-91 kJ/mol]) in the intercistronic region between agfA and agfC, which may be involved in stabilization of the agfBA portion of the agfBAC transcript. agfBAC and flanking regions had a high degree of sequence similarity with those counterparts of the E. coli curlin csgBA region for which sequence data are available. These data are demonstrative of the high degree of similarity between S. enteritidis SEF17 fimbriae and E. coli curli with respect to fimbrin amino acid sequence and genetic organization and, therefore, are indicative of a common and relatively recent ancestry.

Amino Acid Sequence↗
PubMed1994

fimA and tctC based DNA diagnostics for Salmonella.

Immunochemical analyses of 85 isolates of 17 Salmonella serovars using polyclonal antiserum to SEF21, the type 1 fimbriae of Salmonella enteritidis, demonstrated antigenic relatedness among both type 1 and type 2 fimbriae of Salmonella. However, anti-SEF21 antiserum was not entirely suitable as a Salmonella diagnostic probe due either to a variability of, or a rare deficiency of, detectable fimbriae. Partial amino acid sequence analyses of the SEF21 structural fimbrin protein revealed 99% homology to Salmonella typhimurium FimA. Therefore, oligonucleotide probes for Salmonella detection were designed following sequencing of S. enteritidis fimA and comparison to the corresponding genes of S. typhimurium, Escherichia coli, Klebsiella pneumoniae and Serratia marcescens. One oligonucleotide probe hybridized to all 612 Salmonella isolates of 89 serovars tested while two other probes detected 97.5% and 99.7% of the isolates. Three consistently weak positive reactions were obtained, therefore, inclusivity was optimized by identification of a Salmonella-specific tctC DNA probe that detected 609 of 612 Salmonella isolates. No hybridization of these Salmonella probes was detected to 250 other Enterobacteriaceae isolates or to 14 other eubacterial species. Therefore, in combination, DNA probes to fimA and tctC proved to be highly reliable diagnostics for Salmonella bacteria. Accordingly, PCR assays targeting fimA and tctC were developed.

Amino Acid Sequence↗
PubMed1994

Unique fimbriae-like structures encoded by sefD of the SEF14 fimbrial gene cluster of Salmonella enteritidis.

The SEF14 gene cluster of Salmonella enteritidis was recently shown to contain three genes, sefABC, encoding a unique fimbrin, and proteins homologous to fimbrial chaperones and outer membrane proteins (ushers), respectively. A fourth open reading frame, designated sefD, was found immediately downstream of sefABC and overlapping sefC. The translated protein sequence of sefD was unique, but the composition was similar to that of other bacterial fimbriae. SefD was produced in abundance by wild-type S. enteritidis as shown by Western blot analysis using antibodies raised to affinity-purified, recombinant SefD. Furthermore, unusually long, thin, fimbriae-like structures were evident on S. enteritidis and Escherichia coli by immunoelectron microscopy, but in other bacterial species SefD was expressed as amorphous material. Therefore, in S. enteritidis and E. coli, SefD is the predominant structural subunit of SEF18. The SEF18 fimbriae-like structures were shown to be serologically distinct from the three known S. enteritidis fimbriae SEF14, SEF17 and SEF21. Furthermore, SEF18 was still produced in sefA insertion mutants, indicating that SEF14 and SEF18 were structurally distinct. Thus, the SEF14 gene cluster is the first example in the Enterobacteriaceae of a gene cluster that encodes two fimbrin-like proteins, which are assembled into two distinct cell-surface structures, SEF14 and SEF18. DNA hybridization and Western blot analyses showed that SefD was widely distributed among the Enterobacteriaceae and was present in E. coli, Shigella, Enterobacter, Citrobacter, Erwinia, Hafnia, Klebsiella, Providencia, and Proteus but not in the non-Enterobacteriaceae Gram-negative bacteria Pseudomonas and Aeromonas, or in Gram-positive bacteria Bacillus or Staphylococcus.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1993

Thin, aggregative fimbriae mediate binding of Salmonella enteritidis to fibronectin.

The binding of human fibronectin and Congo red by an autoaggregative Salmonella enteritidis strain was found to be dependent on its ability to produce thin, aggregative fimbriae, named SEF 17 (for Salmonella enteritidis fimbriae with an apparent fimbrin molecular mass of 17 kDa). Two other fimbrial types produced by S. enteritidis, SEF 14 and SEF 21, were not responsible for the aggregative phenotype or for fibronectin binding. SEF 17-negative TnphoA mutants which retained the ability to produce SEF 14 and SEF 21 were unable to bind human fibronectin or Congo red and lost the ability to autoaggregate. Only purified SEF 17 but not purified SEF 14 or SEF 21 bound fibronectin in a solid-phase binding assay. Furthermore, only SEF 17 was able to inhibit fibronectin binding to S. enteritidis whole cells in a direct competition enzyme-linked immunosorbent assay. These results indicate that SEF 17 are the fimbriae responsible for binding fibronectin by this enteropathogen.

Alkaline Phosphatase↗
PubMed1993

Characterization of three fimbrial genes, sefABC, of Salmonella enteritidis.

Salmonella enteritidis produces thin, filamentous fimbriae designated SEF14. A 3.9-kb region of a 5.3-kb fragment encoding genes responsible for SEF14 biosynthesis was sequenced and found to contain three genes, sefABC. sefA encoded a novel fimbrin, the structural subunit of SEF14 fimbriae. sefB and sefC encoded proteins homologous to Escherichia coli and Klebsiella pneumoniae fimbrial periplasmic chaperone proteins and fimbrial outer membrane proteins, respectively, and are the first such genes to be characterized from Salmonella spp. in vitro expression directed by the 5.3-kb DNA fragment identified SefA, SefB, and SefC as approximately 14,000-, 28,000-, and 90,000-M(r) proteins, respectively, which correlated with their predicted amino acid sequences. sefB and sefC were not expressed in the absence of sefA. Primer extension analysis of sefABC revealed two major transcription start sites located upstream of sefA. Transcription of sefBC also initiated from the sefA promoter region. Secondary-structure analysis of the mRNA transcript for sefABC predicted the formation of two stable stem-loop structures in the intercistronic region between sefA and sefB indicative of differential regulation of SefA, SefB, and SefC translation. E. coli cells carrying the 5.3-kb DNA fragment of S. enteritidis DNA were unable to assemble distinguishable SEF14 fimbriae; however, immunogold-labelled SEF14 fimbriae were displayed on E. coli clones containing a 44-kb DNA fragment which encompassed the 5.3-kb region. Therefore, sefABC genes make up part of a complex sef operon responsible for the expression and assembly of SEF14 fimbriae.

Amino Acid Sequence↗
PubMed1993

DNA-based diagnostic tests for Salmonella species targeting agfA, the structural gene for thin, aggregative fimbriae.

Salmonella enteritidis 27655-3b and a few diarrheagenic Escherichia coli strains produce morphologically and antigenically related, thin, aggregative fimbriae, collectively named GVVPQ fimbriae (S. K. Collinson, L. Emödy, T. J. Trust, and W. W. Kay, J. Bacteriol. 174:4490-4495, 1992). To determine whether GVVPQ fimbriae are common to Salmonella spp. and other enteropathogenic members of the family Enterobacteriaceae, 113 isolates were phenotypically screened for Congo red binding and aggregative colony morphology. Presumptive positive and representative negative strains were examined by Western blotting (immunoblotting) by using antiserum to SEF 17, the native GVVPQ fimbria of S. enteritidis. Only four S. enteritidis strains and six E. coli isolates possessed substantial amounts of GVVPQ fimbriae after 24 h of incubation on T medium. Following 5 days of incubation, 56 of 93 Salmonella isolates (60%) and 1 of 7 additional E. coli clinical isolates possessed detectable levels of GVVPQ fimbriae. Since variable expression of GVVPQ fimbriae was observed among Salmonella isolates and some E. coli strains produced scant amounts, as revealed by immunoelectron microscopy, the ability to produce these fimbriae was evaluated by genotypic screening. The structural gene for the SEF 17 fimbrin, agfA, was amplified by the polymerase chain reaction, cloned, and sequenced to provide a characterized DNA probe. An agfA DNA fragment hybridized strongly to 603 of 604 (99.8%) Salmonella isolates but very weakly to 31 of 266 other members of the family Enterobacteriaceae including 26 of 137 E. coli strains, 3 of 14 Citrobacter spp., and single isolates of Shigella sonnei and Enterobacter cloacae. The agfA DNA probe proved to be a valuable diagnostic tool for Salmonella isolates arrayed on hydrophobic grid membrane filters. Unique agfA sequences were targeted in the development of a polymerase chain reaction assay specific for Salmonella spp.

Amino Acid Sequence↗
PubMed1992

Thin aggregative fimbriae from diarrheagenic Escherichia coli.

Four strains of diarrheagenic Escherichia coli originally isolated from distinct geographic regions were found to produce unusual thin aggregative fimbriae requiring depolymerization in formic acid prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy of native fimbriae and Western blot (immunoblot) analysis of the corresponding 18-kDa fimbrins showed that these E. coli fimbriae were serologically cross-reactive with SEF 17 (Salmonella enteritidis fimbriae with a fimbrin molecular mass of 17 kDa). The E. coli and S. enteritidis fimbrins had similar total amino acid compositions and highly conserved N-terminal amino acid sequences. These results indicate that E. coli and S. enteritidis produce biochemically related, aggregative fimbriae which constitute a new type of intergenerically distributed fimbriae for which we propose the descriptive name GVVPQ fimbriae on the basis of the conserved N-terminal amino acid sequence.

Amino Acid Sequence↗