Antigenic characterization of Henneguya salminicola.
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Amino Acid Sequence↗
Biomedical subjects
W W Kay
Publications and source records attributed to W W Kay.
Small cryptic plasmids of multiplasmid, clinical Escherichia coli.
Clinical isolates of Escherichia coli were found to host a multiplicity of plasmids. These were resolved from plasmid gel profiles, from the properties of various transconjugants and transformants of E. coli DH1, by the topoisomerase I relaxation of covalently closed circle plasmid DNA, by electron microscopy, and by the determination of their compatibilities. The majority of these were unusually small, cryptic plasmids (SCPs). From one strain, KL4, 13 electrophoretic bands were resolved to five plasmids, three of which were SCPs. SCPs were phenotypically barren, and the smallest of these, pKL1, contained barely enough information for self-replication. A derivative of pKL1, pKL1Km, in which the transposon was restricted to a small 350-bp region, was stably maintained in Shigella, Salmonella, Serratia, and Citrobacter species and its replication was polA independent. pKL1 encoded only a single protein, RepA (Mr 17960), which specifically bound to pKL1 DNA. No apparent homologies with other RepA protein sequences could be detected. Thus the SCP, pKL1, is a novel minimal plasmid replicon encoding only enough information to ensure perpetuation. A hypothesis is presented describing SCPs as a class of selfish DNA that persists simply due to its ability to replicate and to its stability based on high copy number.
Conjugation, Genetic↗
Characterization of the agfBA fimbrial operon encoding thin aggregative fimbriae of Salmonella enteritidis.
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Bacterial Adhesion↗
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↗
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↗
Antigenic characterization of the salmonid pathogen Piscirickettsia salmonis.
Piscirickettsia salmonis, the etiological agent of salmonid rickettsial septicemia, was purified from infected immortal chinook salmon (Oncorhynchus tshawytscha) embryo cells by a combination of differential and Percoll density gradient centrifugation. Immune sera from rabbits immunized with purified whole cells of P. salmonis reacted with four protein antigens and two carbohydrate antigens with relative molecular sizes of 65, 60, 54, 51, 16, and approximately 11 kDa, respectively. The carbohydrate antigens appear to be mainly core region lipo-oligosaccharide with lesser amounts of lipopolysaccharide. Serum from convalescent rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) reacted with several minor immunoreactive protein antigens between 10 and 70 kDa in size and a carbohydrate antigen with a relative molecular size of approximately 11 kDa. The salmonid immune system did not appear to elicit a strong humoral response against this intracellular pathogen. Indirect immunofluorescence microscopy, immunogold transmission electron microscopy, and biotin labeling of intact P. salmonis cells suggest that the immunoreactive antigens identified with rabbit antisera are surface exposed and differ significantly from those identified with salmonid antisera.
Alphaproteobacteria↗
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↗
Physical and functional S-layer reconstitution in Aeromonas salmonicida.
The various functions attributed to the S-layer of Aeromonas salmonicida have been previously identified by their conspicuous absence in S-layer-defective mutants. As a different approach to establish the multifunctional nature of this S-layer, we established methods for reconstitution of the S-layer of A. salmonicida. Then we investigated the functional competence of the reconstituted S-layer. S-layers were reconstituted in different systems: on inert membranes or immobilized lipopolysaccharide (LPS) from purified S-layer protein (A-protein) or on viable cells from either A-protein or preassembled S-layer sheets. In the absence of divalent cations and LPS, purified A-protein in solution spontaneously assembled into tetrameric oligomers and, upon concentration by ultrafiltration, into macroscopic, semicrystalline sheets formed by oligomers loosely organized in a tetragonal arrangement. In the presence of Ca2+, purified A-protein assembled into normal tetragonal arrays of interlocked subunits. A-protein bound with high affinity (Kd, 1.55 x 10(-7) M) and specificity to high-molecular-weight LPS from A. salmonicida but not to the LPSs of several other bacterial species. In vivo, A-protein could be reconstituted only on A. salmonicida cells which contained LPS, and Ca2+ affected both a regular tetragonal organization of the reattached A-protein and an enhanced reattachment of the A-protein to the cell surface. The reconstitution of preformed S-layer sheets (produced by an S-layer-secreting mutant) to an S-layer-negative mutant occurred consistently and efficiently when the two mutant strains were cocultured on calcium-replete solid media. Reattached A-protein (exposed on the surface of S-layer-negative mutants) was able to bind porphyrins and an S-layer-specific phage but largely lacked regular organization, as judged by its inability to bind immunoglobulins. Reattached S-layer sheets were regularly organized and imparted the properties of porphyrin binding, hydrophobicity, autoaggregation, adherence to and invasion of fish macrophages and epithelial cells, and resistance to macrophage cytotoxicity. However, cells with reconstituted S-layers were still sensitive to complement and insensitive to the antibiotics streptonigrin and chloramphenicol, indicating incomplete functional reconstitution.
Aeromonas↗
Capsulated cells of Aeromonas salmonicida grown in vitro have different functional properties than capsulated cells grown in vivo.
When grown in vivo in the peritoneal cavity of rainbow trout, Aeromonas salmonicida produces a clearly defined capsule with virulence-related functions. Aeromonas salmonicida grown in vitro in a glucose-rich medium (GRM) has also been reported to reproduce capsular material. Because in vitro mimicry of in vivo induced traits is highly desirable in vaccine design, the extent to which growth in GRM mimicked in vivo growth was examined. Antibodies specific to in vivo grown cells partially labeled the surface of GRM-grown cells, as well as two distinct proteins (81,700 and 41,000 Mr) in immunoblots of mutants with S-layer or lipopolysaccharide defects. GRM-grown strains showed an increased sensitivity to trout serum in contradistinction to the complete serum resistance of in vivo grown cells; as well, GRM-grown cells were more adherent to trout macrophages. Thus in spite of possessing some surface antigens normally expressed in vivo, cells grown on solid GRM did not possess all functional properties of in vivo grown cells.
Aeromonas↗
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↗
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↗
Physiological consequences of the S-layer of Aeromonas salmonicida in relation to growth, temperature, and outer membrane permeation.
S-layers are paracrystalline protein multimers that cover the entire cell surface of many bacterial species. The presence of an S-layer in Aeromonas salmonicida (also known as A-layer) predisposed this bacterium to apparently unrelated physiological consequences: inhibition of growth at 30 degrees C, enhanced cell filamentation at 37 degrees C, and enhanced uptake of the hydrophobic antibiotics streptonigrin and chloramphenicol. Growth inhibition or enhanced filamentation was not observed when the native A-layer was missing or its arrangement altered, as in Ca(2+)-limited or Ca(2+)- and Mg(2+)-limited cells, in A-layer-negative (A-) cells with an artificially reconstituted A-layer, or in mutants unable to correctly assemble this layer. A-layer-positive cells (A+) were far more sensitive to the intracellularly acting antibiotics streptonigrin and chloramphenicol than were A- cells, and streptonigrin-resistant mutants were predominantly A-. Hemin, a compound known to specifically bind to the A-layer, alleviated streptonigrin toxicity to A+, but not A-, cells. As well, Ca(2+)- and Mg(2+)-limited cells, or mutants harboring A-layer defects had a reduced sensitivity to streptonigrin, and A- cells with reconstituted A-layers remained resistant to streptonigrin and chloramphenicol. Thus, the presence of a native A-layer arrangement on the cell surface, and not the mere presence of A-layer protein subunits, predisposed A. salmonicida toward the aforementioned physiological consequences. The A-layer is suggested to specifically effect these consequences, in particular the permeation of streptonigrin or chloramphenicol, by a specific interaction of A-layer subunits with the outer membrane.
Aeromonas↗
Novel antigens expressed by Aeromonas salmonicida grown in vivo.
Virulent and avirulent Aeromonas salmonicida strains grown inside intraperitoneal implants in Rainbow trout (Oncorhynchus mykiss) were examined for unique antigen expression. Western blots (immunoblots), performed with immune rabbit serum raised against in vivo-grown cells, revealed several unique antigens. With the exception of lipopolysaccharide (LPS), these novel antigens were destroyed after proteinase K treatment. The majority of these antigens were not induced in vitro in response to either iron limitation or anaerobiosis. In addition, electron microscopy demonstrated the presence of a putative capsule on in vivo-grown cells. Purification and fractionation of this carbohydrate material from cells grown in carbon-rich synthetic media resulted in the isolation and separation of an antigenically distinct LPS not seen with cells grown in standard media. Antiserum raised against in vivo-grown cells recognized both this LPS and the typical LPS of A. salmonicida apparent in in vitro-grown cells. Antiserum raised against in vitro-grown cells recognized only the LPS expressed in vitro. Antiserum directed against in vivo-grown cells was approximately 10 times more sensitive than serum directed against in vitro-grown cells in detecting A. salmonicida in infected fish kidney tissue.
Aeromonas↗
Aeromonas salmonicida grown in vivo.
The virulent fish pathogen Aeromonas salmonicida was rapidly killed in vivo when restricted inside a diffusion chamber implanted intraperitoneally in rainbow trout. After a period of regrowth, the survivors had acquired resistance to host-mediated bacteriolysis, phagocytosis, and oxidative killing, properties which were subsequently lost by growth in vitro. Resistance to bacteriolysis and phagocytosis was associated with a newly acquired capsular layer revealed by acidic polysaccharide staining and electron microscopy. This capsular layer shielded the underlying, regular surface array (S-layer) from immunogold labeling with a primary antibody to the S-layer protein. Resistance to oxidative killing was mediated by a mechanism not associated with the presence of the capsular layer. An attenuated vaccine strain of A. salmonicida grown in vivo failed to express the capsular layer. Consequently, the in vivo-grown cells of this attenuated strain remained as sensitive to bacteriolysis, and as avidly adherent to macrophages, as the in vitro-grown cells. The importance of these new virulence determinants and their relation to the known virulence factors of A. salmonicida are discussed.
Aeromonas↗
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↗
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↗
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↗
Fate of the fish pathogen Aeromonas salmonicida in the peritoneal cavity of rainbow trout.
A model was developed to study the fate of the fish pathogen Aeromonas salmonicida in vivo, inside a specialized intraperitoneal chamber implanted in rainbow trout, Oncorhynchus mykiss. Although normally recalcitrant to lytic agents in vitro, owing to the presence of its regular surface array (S layer), A. salmonicida was rapidly killed in the peritoneal cavity by a host-derived, soluble lytic activity present in peritoneal fluid. Peritoneal fluid was also found to kill other bacteria and lyse various types of erythrocytes, but was particularly lytic to A. salmonicida. Intraperitoneal survival of injected (free) A. salmonicida cells was several orders of magnitude higher than survival of implanted (restrained) cells. Injected free cells could evade the lytic activity of peritoneal fluid because they readily spread, initiating lethal infections. One evasion strategy was envisioned to be the penetration of peritoneal and (or) tissue macrophages. In spite of the killing mechanisms of these phagocytic cells, A. salmonicida was still able to survive and even replicate inside head kidney macrophages, thereby supporting the notion of A. salmonicida as a facultatively intracellular pathogen. Intraperitoneal chambers in rainbow trout may constitute a valuable experimental tool for studying the in vivo fate of A. salmonicida, and perhaps of other fish pathogens as well.
Aeromonas↗