Sharon C. Clouthier

Antigenic Characterization of the Fish Pathogen Flavobacterium psychrophilum

ABSTRACT Flavobacteria are a poorly understood and speciated group of commensal bacteria and opportunistic pathogens. The psychrotrophFlavobacterium 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 ∼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 ∼20-kDa protein. This study represents the first description at the molecular level of the surface characteristics and potential vaccine targets of confirmed F. psychrophilumstrains.

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.

CHARACTERIZATION OF THE AGFBA FIMBRIAL OPERON ENCODING THIN AGGREGATIVE FIMBRIAE OF SALMONELLA ENTERITIDIS

Of the several fimbrial types produced by the human enteropathogen Salmonella enteritidis, the thin aggregative fimbriae, SEF17, are particularly interesting being highly immunogenic, aggregative, extremely stable, cell surface structures which are widely distributed among Salmonella serovars (Collinson et al., 1991; Doran et al., 1993). These fimbriae are composed of a 17,000 Da fimbrin, AgfA, and require treatment with 90% formic acid before depolymerization occurs (Collinson et al., 1991). Diarrheagenic Escherichia coli strains produce biochemically and serologically related, thin, aggregative fimbriae (Collinson et al., 1992) called curli which are composed of polymerized CsgA fimbrins (Arnqvist et al., 1994). An agfA-specific, oligonucleotide probe capable of distinguishing agfA from csgA forms the basis for a valuable, new, Salmonella-specific, diagnostic test (Doran et al., 1993). The role(s) of SEF17 in Salmonella pathogenesis remain(s) to be elucidated but purified SEF17 binds fibronectin in vitro suggesting a possible role in facilitating bacterial-tissue interactions (Collinson et al., 1993).

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.