Antigenic characterization of the fish pathogen Flavobacterium psychrophilum.

Flavobacteria are a poorly understood and speciated group of commensal bacteria and opportunistic pathogens. The psychrotroph Flavobacterium psychrophilum is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic diseases that heavily impact salmonids. Consequently, two verified but geographically diverse isolates were characterized phenotypically and biochemically. A facile typing system was devised which readily discriminated between closely related species and was verified against a pool of recent prospective isolates. F. psychrophilum was found to be enveloped in a loosely attached, strongly antigenic outer layer comprised of a predominant, highly immunogenic, low-molecular-mass carbohydrate antigen as well as several protein antigens. Surface-exposed antigens were visualized by a combination of immunoflourescence microscopy, immunogold transmission, and thin-section electron microscopy and were discriminated by Western blotting using rabbit antisera, by selective extraction with EDTA-polymyxin B agarose beads, and by extrinsic labeling of amines with sulfo-N-hydoxysuccinimide-biotin and glycosyl groups with biotin hydrazide. The predominant approximately 16 kDa antigen was identified as low-molecular-mass lipopolysaccharide (LPS), whereas high-molecular-mass LPS containing O antigen was not as prevalent on whole cells but was abundant in culture supernatants. Rainbow trout convalescent antisera recognized both molecular mass classes of LPS as well as a predominant approximately 20-kDa protein. This study represents the first description at the molecular level of the surface characteristics and potential vaccine targets of confirmed F. psychrophilum strains.

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.

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.

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.

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.

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.

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.

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)

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.