Biomedical subjects

W W Kay

Publications and source records attributed to W W Kay.

At least 55 records · Page 3Linked to original sources

PubMed1992

S-layer-mediated association of Aeromonas salmonicida with murine macrophages.

The interaction of Aeromonas salmonicida with the murine macrophage (M phi) cell line P388D1 was used as a convenient model to study the involvement of the bacterial crystalline surface array (or A-layer) in the association with M phi s. A-layer-positive (A+) cells readily associated with M phi s in phosphate-buffered saline, whereas A- mutants were unable to do so, even when the bacterium-M phi interaction was forced by centrifugation. M phi s selectively interacted with A+ cells when challenged with mixtures of A+ and excess A- cells. Electron microscopy indicated that in phosphate-buffered saline only A+ bacteria were readily internalized, although by a nonconventional mechanism, suggesting that efficient phagocytosis in the absence of opsonins was A-layer mediated. Latex beads coated with a partially assembled A-layer were more efficiently taken up than uncoated or A-protein-coated beads, indicating that an organized A-layer was essential for M phi uptake. The reduced ability of M phi s plated on a substratum coated with the A-layer to bind A+ bacteria also suggested that association was both A-layer and receptor mediated. In the presence of tissue culture medium, competent M phi s interacted efficiently with A- bacteria and internalized them through conventional phagocytosis. A+ cells were markedly cytotoxic to M phi s, whereas the A-protein or A-layer was not. A- cells were cytotoxic to a lesser extent, suggesting that cytotoxicity was targeted.

Aeromonas↗

PubMed1992

Interaction of the fish pathogen Aeromonas salmonicida with rainbow trout macrophages.

A procedure was developed to culture rainbow trout macrophages (M phi) on supported glass coverslips. Using this method and a variety of well-characterized Aeromonas salmonicida strains with normal or altered cell surfaces, we investigated the role of this unusual bacterial surface in the bacterium-M phi interaction. An intact crystalline protein array, the A-layer, mediated adherence of A. salmonicida cells to M phi even in the absence of opsonins. In contrast, unopsonized cells of an A-layer-negative (A-) mutant with a smooth lipopolysaccharide (LPS) layer were unable to interact with M phi. However, this ability was recovered when the A-layer was reconstituted onto the smooth LPS surface of these A- LPS+ cells. Two A. salmonicida mutants possessing the A-layer in different disorganized states had a reduced ability to interact with M phi. A+ cells grown under calcium limitation produced A-layers locked into an alternative conformation which mediated the highest levels of M phi association in the absence of opsonins or any other surface coating. Coating A+ cells with hemin greatly increased their levels of M phi association, and bacterial cells grown on trout blood agar plates also had a dramatic increase in their ability to interact with M phi. Only A+ A. salmonicida cells were highly cytotoxic to trout M phi, especially after being coated with hemin, presumably due to a more focused targeting of the bacterial cell onto the M phi surface and/or into the intracellular regions of the M phi.

Aeromonas↗

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↗

PubMed1991

Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida.

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.

Aeromonas↗

PubMed1991

Form and functions of the regular surface array (S-layer) of Aeromonas salmonicida.

The principal virulence factor of Aeromonas salmonicida is its S-layer (A-layer) which is comprised of tetragonally arrayed approximately 50,000 Mr protein subunits tethered to the cell surface via LPS. The detailed composition of its LPS is known, as is the primary sequence, and three-dimensional disposition of the A protein subunits. The A-layer physically protects the cell against bacteriophage, proteases, as well as immune and non-immune complement. The A-layer appears to be uniquely adapted towards binding biologically important molecules such as heme, and to various basement membrane proteins. In addition, the A-layer is required for macrophage infiltration and resistance. Specific mutants containing a disorganized A-layer are avirulent and provide significant protection to salmonids when applied by immersion.

Aeromonas↗

PubMed1991

Surface-disorganized, attenuated mutants of Aeromonas salmonicida as furunculosis live vaccines.

A slow-growing, aminoglycoside-resistant mutant and a rapidly-growing pseudo-revertant were isolated from Aeromonas salmonicida, the causative agent of salmonid furunculosis. These mutants continued to elicit a variety of classical virulence factors associated with A. salmonicida pathogenesis. They differed morphologically from the wild-type and from one another with respect to A-layer organization, membrane antagonist sensitivity and particularly to aerobic metabolism. Both mutants were drastically altered in the architecture of the 2D crystalline surface array (A-layer), although both were similar to wild-type with respect to cell surface composition. The slow-growing, antibiotic-resistant mutant differed significantly from the wild-type by the apparent loss of virtually all aerobic metabolism; the pseudo-revertant had partially recovered the ability to aerobically metabolize certain carbon sources. Both mutants were avirulent and incapable of tissue persistence. The rapidly-growing, antibiotic-sensitive pseudo-revertant, when administered either intraperitoneally or by immersion, effectively protected salmonid fish from challenge by a heterologous virulent stain suggesting its candidature as a live, attenuated furunculosis vaccine.

Aeromonas↗

PubMed1991

Type 1 fimbriae of Salmonella enteritidis.

Salmonella enteritidis was previously shown to produce fimbriae composed of 14,000-molecular-weight (Mr) fimbrin monomers (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Another distinct fimbrial structure, comprising 21,000-Mr fimbrin monomers, has now been identified. These fimbriae are simply designated as SEF 14 and SEF 21, respectively (for S. enteritidis fimbriae and the Mr [in thousands] of the fimbrin monomer). A simple method for the purification of both structures was developed by using the different biochemical properties of these fimbriae. SEF 21 remained intact after being boiled in sodium dodecyl sulfate but readily dissociated into subunits of 21,000 Mr at pH 2.2. The overall amino acid composition and the N-terminal amino acid sequence of the SEF 21 fimbrin were distinct from those of SEF 14 but were virtually identical to the predicted sequence for type 1 fimbrin of Salmonella typhimurium. Immunoelectron microscopy of S. enteritidis clearly revealed fimbrial structures that reacted with immune serum specific to the 21,000-Mr fimbrin. Immune sera raised against this subunit were cross-reactive with type 1 fimbrins found in whole-cell lysates of S. typhimurium, Salmonella illinois, and Salmonella cubana. However, there was no cross-reaction with Escherichia coli type 1 fimbriae or with other fimbrins produced by S. enteritidis. Under certain growth conditions, S. enteritidis produced both SEF 14 and SEF 21. However, when S. enteritidis was grown at 30 degrees C or lower, only the 21,000-Mr SEF 21 fimbrin could be detected. There was a direct correlation between mannose-sensitive hemagglutination and the presence of SEF 21.

Amino Acid Sequence↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1989

Three-dimensional structure of an open form of the surface layer from the fish pathogen Aeromonas salmonicida.

Cell-free culture supernatants of a lipopolysaccharide (LPS) O-polysaccharide-deficient, single-insertion transposon mutant of the tetragonal surface protein array (S layer)-containing fish pathogen Aeromonas salmonicida were examined by electron microscopy. Negative staining showed that the S layer was released as sheets of tetragonal material, indicating that although surface retention of assembled S layer requires the presence of wild-type LPS oligosaccharides, initial assembly of S-layer subunits into sheets does not require the presence of O-polysaccharide chains. The three-dimensional structure of the S layer was reconstructed from tilted micrographs of the released sheets. Horizontal sections through this reconstruction showed that the released sheets were composed of two identical S layers that were perfectly in register. The reconstructed layer had a lattice constant of 12.5 nm. At a resolution of 1.6 nm, the layer consisted of a major tetragon at one fourfold axis of symmetry and a minor tetragon at the second fourfold axis of symmetry. The core, composed of four of the major domains, contained a large depression and was located toward the inside of the layer. The minor tetragon provided connectivity within the layer and was located toward the outer surface of the layer. Projections through the double layer gave a type I (closed) pattern (M. Stewart, T. J. Beveridge, and T. J. Trust, J. Bacteriol. 166:120-127, 1986), yet projections through the single layer indicated that the type II (open) pattern was present. This open pattern was indistinguishable from that seen in S layer released from the surfaces of wild-type cells.

Aeromonas↗

PubMed1989

Genetic regulation of the tricarboxylate transport operon (tctI) of Salmonella typhimurium.

Tricarboxylates are transported into Salmonella typhimurium by a binding protein-dependent transport system known as TctI. Genetically, it comprises three structural genes, tctCBA, as well as a fourth gene of unknown function (tctD), which is transcribed divergently from tctC (K. A. Widenhorn, J. M. Somers, and W. W. Kay, J. Bacteriol. 170:3223-3227, 1988). Deletions in tctD strongly reduced expression of tctC or of tctC-lacZ transcriptional fusions; however, expression was restored when tctD was present in trans. Expression of tctD-lacZ transcriptional fusions was strongly repressed in the presence of D-glucose but could be alleviated by the addition of cyclic AMP. Furthermore, transcription of tctD was found not to be autogenously regulated. Thus, tctD is considered to be regulated by catabolite repression and encodes a transcriptional activator of tctCBA expression. From the DNA sequence of tctD, the predicted gene product was hydrophilic and shared distinct homologies with other globally regulated transcriptional activators such as OmpR and NtrC.

Amino Acid Sequence↗

PubMed1989

Fimbriation genes of Salmonella enteritidis.

From a cosmid clone, a 5.3-kilobase (kb) HindIII fragment of Salmonella enteritidis DNA containing the fimA gene was subcloned into bacteriophage T7 promoter vectors in both orientations. Predominantly mature fimbrin (14,000 Mr) was produced by clones containing the 5.3-kb insert, whereas the original cosmid clone predominantly accumulated a prefimbrin precursor (16,500 Mr). T7 RNA polymerase-dependent expression of the 5.3-kb insert and of a series of nested deletions revealed several membrane-localized polypeptides (80,000, 40,000, 29,000, 25,000, and 16,500 Mrs) transcribed in the same direction as fimA as well as a single polypeptide (9,000 Mr) transcribed in the opposite direction. Mini-Mu and TnphoA (Tn5 IS50L::phoA) transposon mutagenesis was used to identify a 2- to 3.5-kb region downstream of fimA that affected fimbrin production and processing. A more distant region (greater than 7 kb), revealed by Tn10 and Mu dI mutagenesis, was also required for fimbriation but did not hybridize with the 5.3-kb fragment. Yet another distant region did hybridize to the 5.3-kb fragment, suggesting the existence of other fimbriation-related genes.

Bacterial Proteins↗

PubMed1988

Immunoglobulin binding by the regular surface array of Aeromonas salmonicida.

The cell surface of Aeromonas salmonicida is covered by a regular surface array composed of a single species of protein, the A-protein (Phipps, B. M., Trust, T. J., Ishiguro, E. E., and Kay, W. W. (1983) Biochemistry 22, 2934-2939). The array, known as the A-layer, is the key virulence factor for this organism. Cells containing the A-layer specifically bound rabbit IgG and human IgM with high affinity (KD = 1.0 X 10(-6) M and 3.3 X 10(-6) M, respectively), but neither isogenic A-protein-deficient strains nor an Aeromonas hydrophila strain also possessing a regular surface array had binding activity. Selective removal of A-protein at pH 2.2 inactivated IgG binding. Structurally intact IgG was requisite for binding since both Fab and Fc fragments were inactive. Aeromonas A-protein did not share the same IgG binding sites as Staphylococcus aureus protein A. Purified A-protein bound IgG only weakly, but reassembled A-layer regained binding activity. Protein modification and perturbation of the A-layer indicated that no single amino acid residue was critical for binding, and that the binding site consisted of a native arrangement of at least four A-protein monomers in the layer.

Aeromonas↗

PubMed1988

Cloning and properties of the Salmonella typhimurium tricarboxylate transport operon in Escherichia coli.

The tricarboxylate transport operon (tctI) was cloned in Escherichia coli as a 12-kilobase (kb) fragment from an EcoRI library of the Salmonella typhimurium chromosome in lambda gtWES. It was further subcloned as a 12-kb fragment into pACYC184 and as an 8-kb fragment into pBR322. By insertional mutagenesis mediated by lambda Tn5, restriction mapping, and phenotypic testing, the tctI operon was localized to a 4.5-kb region. The tctC gene which encodes a periplasmic binding protein (C protein) was located near the center of the insert. E. coli/tctI clones on either multicopy or single-copy vectors grew on the same tricarboxylates as S. typhimurium, although unusually long growth lags were observed. E. coli/tctI clones exhibited similar [14C]fluorocitrate transport kinetics to those of S. typhimurium, whereas E. coli alone was virtually impermeable to [14C]fluorocitrate. The periplasmic C proteins (C1 and C2 isoelectric forms) were produced in prodigious quantities from the cloned strains. Motile E. coli/tctI clones were not chemotactic toward citrate, whereas tctI deletion mutants of S. typhimurium were. Taken together, these observations indicate that tctI is not an operon involved in chemotaxis.

Biological Transport↗

PubMed1988

Unmasking of bacteriophage Mu lipopolysaccharide receptors in Salmonella enteritidis confers sensitivity to Mu and permits Mu mutagenesis.

The human pathogen Salmonella enteritidis 3b was found to be highly resistant to phage P22 and Mu derivatives. The Mu sensitivity (musA1) allele from Salmonella typhimurium could be transferred to S. enteritidis 3b at low frequency by cotransduction with hisG::Tn10. Sensitivity to Mu resulted in a large reduction in the number of lipopolysaccharide core-region oligosaccharides that were substituted with O-antigen polysaccharide. The residual high-molecular-weight lipopolysaccharide appeared to be a hybrid displaying O antigens which were immunologically related to those of S. typhimurium and not to those of S. enteritidis. Consequently, Mu d1(Ap lac) could then be transduced into Mus strains forming stable lysogens. On temperature induction, Mu transposition could easily be used to generate mutations in genes coding for cell surface antigens including fimbriae, lipopolysaccharide, and flagella.

Antigens, Surface↗

PubMed1988

Expression of the divergent tricarboxylate transport operon (tctI) of Salmonella typhimurium.

Membrane-associated gene products of shock-sensitive bacterial transport operons are often difficult to detect. A 4.5-kilobase DNA fragment, known to completely encode the Salmonella typhimurium tctI operon, was cloned in both orientations behind the T7 phage promoter phi 10 and expressed by using the T7 polymerase-promoter system of Tabor and Richardson (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078, 1985). Under these conditions, five proteins were clearly demonstrated. One DNA strand was shown to encode the periplasmic (29,000-Mr) C protein (as a 31,000-Mr precursor), a 19,000-Mr protein, and a 40,000- to 45,000-Mr protein which ran as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The opposite strand carried the information for two additional proteins of 29,000 and 14,000 Mr. By Tn5 mutagenesis, subcloning of Tn5 insertions, and subcloning of various deletion mutants it was shown that the tctI system is divergently transcribed. The periplasmic binding protein (C protein) is the first product of one operon, followed by the 19,000-Mr and 45,000-Mr integral inner membrane proteins. On the opposite strand only the 29,000-Mr protein was essential for tctI function, and it was found to be weakly attached to the inner membrane. Thus tctI encodes four proteins, one periplasmic, two integral, and one peripheral to the cytoplasmic membrane, with the genes arranged as tctA tctB tctC tctD.

Biological Transport↗

PubMed1988

Cloning and expression of a Salmonella enteritidis fimbrin gene in Escherichia coli.

A gene bank of DNA from a human isolate of Salmonella enteritidis was constructed in the cosmid pHC79 in Escherichia coli HB101. Five clones containing 35- to 45-kilobase inserts of S. enteritidis DNA reacted in colony immunoblot assays with a polyclonal antiserum prepared against purified S. enteritidis fimbriae. Electron microscopy showed that none of the five fimbrin-producing clones produced fimbriae, yet radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis located the 14,400-molecular-weight S. enteritidis in the outer membrane fraction of three of the clones and in the periplasmic fraction of all five clones. By using an oligonucleotide probe homologous to the 5' region of the fimbrin structural gene, the fimbrin gene was located on a 5.3-kilobase HindIII fragment. In vitro transcription-translation analysis verified that this HindIII fragment subcloned into plasmid pTZ18R produced unprocessed S. enteritidis fimbrin of molecular weight 16,400. Dot blot hybridization against a selection of strains of the family Enterobacteriaceae indicated a limited distribution of the S. enteritidis fimbrin gene.

Autoradiography↗

PubMed1986

Tellurite susceptibility and non-plasmid-mediated resistance in Escherichia coli.

Tellurite (TeO3(2-)) is highly toxic toward Escherichia coli (MIC, approximately 1 microgram ml-1). Mutants (Tel) that were resistant to low levels of TeO3(2-) (MIC, approximately 10 micrograms ml-1) and collaterally resistant to arsenate were isolated. These Tel mutants were unable to grow on media containing low levels of Pi, which supported growth of the parent strain. When grown at much higher Pi levels they exhibited depressed levels of the outer membrane phoE protein and the periplasmic phoS protein, as well as several other proteins indicative of Pi starvation. Tel mutants were markedly defective in 32Pi transport, and TeO3(2-) was shown to be a potent competitive inhibitor of 32Pi transport in the parent strain. The Tel phenotype could be complemented by an F' plasmid harboring the phoR, phoB, and phoA loci, and curing of the F' plasmid completely restored TeO3(2-) resistance. Of a variety of well-characterized Pi transport mutants, only phoB mutants were equally resistant to TeO3(2-), and susceptibility could also be restored in strains carrying an F' plasmid for the phoB region and lost once more after F' curing. The tel and phoB loci were equally cotransducible with lac. Tel mutants still synthesized alkaline phosphatase, the phoA gene product, after Pi starvation, suggesting that the phoB locus per se was not involved because phoB is a positive regulatory gene for phoA expression. The results indicate that TeO3(2-) is transported into E. coli by a phosphate transport system and that resistance to TeO3(2-) specifically selects for as yet uncharacterized mutants in the phoB-phoA region of the chromosome.

Alkaline Phosphatase↗

PubMed1986

Heme requirement for growth of fastidious atypical strains of Aeromonas salmonicida.

The growth of fastidious atypical strains of the fish pathogen Aeromonas salmonicida on both solid and liquid media was dependent specifically on a source of heme which was apparently required for initiation of growth at low inoculum densities. Thus, hemin enhanced the plating efficiencies of such strains on solid medium and significantly reduced their inoculum-size-dependent lag times in broth. The heme requirement could also be satisfied by hematoporphyrin and, less effectively, by hemoglobin. Since the requirement was a stable property of all 17 strains tested, it may prove to be another taxonomic criterion by which the atypical strains can be differentiated from the typical strains of A. salmonicida.

Aeromonas↗