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R K Taylor

Publications and source records attributed to R K Taylor.

At least 19 recordsLinked to original sources
PubMed2003

Cold storage affects survival and growth of Erwinia amylovora on the calyx of apple.

AIMS: To determine the effect of cold storage on the survival of Erwinia amylovora. METHODS AND RESULTS: The survival of E. amylovora was assessed during storage at 2 degrees C. Populations of E. amylovora inoculated into phosphate-buffered saline remained static, whereas in nutrient media populations increased at low temperatures. In contrast, populations of E. amylovora on tissue in the apple calyx decreased during cold storage. CONCLUSIONS: Erwinia amylovora has the ability, in nutrient media, to multiply at low temperatures. However, populations of E. amylovora on tissue in the apple calyx decrease with the time spent in cold storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Cold storage of apples will provide assurance that mature fruit from orchards, free of fire blight, or even with low levels of fire blight, may be exported with a negligible risk of introducing the disease into countries free of fire blight.

Cold Temperature↗
PubMed2001

The mannose-sensitive hemagglutinin of Vibrio cholerae promotes adherence to zooplankton.

The bacterium Vibrio cholerae, the etiological agent of cholera, is often found attached to plankton, a property that is thought to contribute to its environmental persistence in aquatic habitats. The V. cholerae O1 El Tor biotype and V. cholerae O139 strains produce a surface pilus termed the mannose-sensitive hemagglutinin (MSHA), whereas V. cholerae O1 classical biotype strains do not. Although V. cholerae O1 classical does not elaborate MSHA, the gene is present and expressed at a level comparable to that of the other strains. Since V. cholerae O1 El Tor and V. cholerae O139 have displaced V. cholerae O1 classical as the major epidemic strains over the last fifteen years, we investigated the potential role of MSHA in mediating adherence to plankton. We found that mutation of mshA in V. cholerae O1 El Tor significantly diminished, but did not eliminate, adherence to exoskeletons of the planktonic crustacean Daphnia pulex. The effect of the mutation was more pronounced for V. cholerae O139, essentially eliminating adherence. Adherence of the V. cholerae O1 classical mshA mutant was unaffected. The results suggest that MSHA is a factor contributing to the ability of V. cholerae to adhere to plankton. The results also showed that both biotypes of V. cholerae O1 utilize factors in addition to MSHA for zooplankton adherence. The expression of MSHA and these additional, yet to be defined, adherence factors differ in a serogroup- and biotype-specific manner.

Animals↗
PubMed2001

Anti-class II monoclonal antibody-targeted Vibrio cholerae TcpA pilin: modulation of serologic response, epitope specificity, and isotype.

Toxin-coregulated pilus (TCP) is a colonization factor required for cholera infection. It is not a strong immunogen when delivered in the context of whole cells, yet pilus subunits or TcpA derivative synthetic peptides induce protective responses. We examined the efficacy of immunizing mice with TCP conjugated to anti-class II monoclonal antibodies (MAb) with or without the addition of cholera toxin (CT) or anti-CD40 MAb to determine if the serologic response to TcpA could be manipulated. Anti-class II MAb-targeted TCP influenced the anti-TCP peptide serologic response with respect to titer and isotype. Responses to TcpA peptide 4 were induced with class II MAb-targeted TCP and not with nontargeted TCP. Class II MAb-targeting TcpA reduced the response to peptide 6 compared to the nontargeted TCP response. Class II MAb-targeted TcpA, if delivered with CT, enhanced the serologic response to TcpA peptides. The effectiveness of the combination of targeted TCP and CT was reduced if anti-CD40 MAb were included in the primary immunization. These data establish the need to understand the role of TCP presentation in the generation of B-cell epitopes in order to optimize TcpA-based cholera vaccines.

Adjuvants, Immunologic↗
PubMed2001

Immune response genes modulate serologic responses to Vibrio cholerae TcpA pilin peptides.

Cholera is an enteric disease caused by Vibrio cholerae. Toxin-coregulated pilus (TCP), a type 4 pilus expressed by V. cholerae, is a cholera virulence factor that is required for host colonization. The TCP polymer is composed of subunits of TcpA pilin. Antibodies directed against TcpA are protective in animal models of cholera. While natural or recombinant forms of TcpA are difficult to purify to homogeneity, it is anticipated that synthesized TcpA peptides might serve as immunogens in a subunit vaccine. We wanted to assess the potential for effects of the immune response (Ir) gene that could complicate a peptide-based vaccine. Using a panel of mice congenic at the H-2 locus we tested the immunogenicity of TcpA peptide sequences (peptides 4 to 6) found in the carboxyl termini of both the classical (Cl) and El Tor (ET) biotypes of TCP. Cl peptides have been shown to be immunogenic in CD-1 mice. Our data clearly establish that there are effects of the Ir gene associated with both biotypes of TcpA. These effects are dynamic and dependent on the biotype of TcpA and the haplotypes of the host. In addition to the effects of the classic class II Ir gene, class I (D, L) or nonclassical class I (Qa-2) may also affect immune responses to TcpA peptides. To overcome the effects of the class II Ir gene, multiple TcpA peptides similar to peptides 4, 5, and 6 could be used in a subunit vaccine formulation. Identification of the most protective B-cell epitopes of TcpA within a particular peptide and conjugation to a universal carrier may be the most effective method to eliminate the effects of the class II and class I Ir genes.

Amino Acid Sequence↗
PubMed2001

Evaluation of cholera vaccines formulated with toxin-coregulated pilin peptide plus polymer adjuvant in mice.

Cholera is an acute diarrheal disease that is caused by the gram-negative bacterium Vibrio cholerae. The low efficacy of currently available killed-whole-cell vaccines and the reactinogenicity coupled with potential reversion of live vaccines have thus far precluded widespread vaccination for the control of cholera. Recent studies on the molecular nature of the virulence components that contribute to V. cholerae pathogenesis have provided insights into possible approaches for the development of a defined subunit cholera vaccine. Genetic analysis has demonstrated that the toxin-coregulated pilus (TCP) is the major factor that contributes to colonization of the human intestine by V. cholerae. In addition, polyclonal and several monoclonal antibodies directed against TCP have been shown to provide passive immunity to disease in the infant mouse cholera model. In the present study, synthetic peptides corresponding to portions of the C-terminal disulfide region of TcpA pilin were formulated with polymer adjuvants currently in clinical trials and used to actively immunize adult female CD-1 mice. The experimental vaccine formulations elicited high levels of antigen-specific immunoglobulin G (IgG), including a broad spectrum of subclasses (IgG1, IgG2a, IgG2b, and IgG3), and lower levels of IgA. Infant mice born to the immunized mothers showed 100% protection against a 50% lethal dose (1 LD(50)) challenge and 50% protection against a 10-LD(50) challenge with virulent strain O395. These results indicate that specific regions of TcpA, including those delineated by the peptides used in this study, have the potential to be incorporated into an effective defined subunit vaccine for cholera.

Adjuvants, Immunologic↗
PubMed2001

Molecular and morphological reassessment of relationships within the Vittadinia group of Astereae (Asteraceae).

Morphological and ITS (internal transcribed spacer) sequence data for 40 species of the Austral-Pacific genera Camptacra, Kippistia, Minuria, Peripleura, Tetramolopium, and Vittadinia as well as one semiherbaceous species of Olearia were subjected to cladistic analysis, separately and together. Minuria, Peripleura, and Tetramolopium are paraphyletic as currently defined. Tetramolopium vagans from Australia appears to represent an undescribed genus. Both Kippistia suadefolia and Peripleura diffusa show close affinity to Minuria species, and Minuria macrorhiza appears to contain two distinct but closely related species. Vittadinia and the remaining species of Tetramolopium and Peripleura form a strong affinity group. The distribution of indels and the combined analysis each provide evidence that the Hawaiian and Cook Island species of Tetramolopium are descended from New Guinea species. The combined analysis also suggests that the Cook Island species T. mitiaroense is sister to the Hawaiian clade. Olearia arguta groups strongly with Camptacra and shows no close affinity with either of the arborescent species of Olearia used to root these analyses. Marked homoplasy among morphological characters indicates why generic delimitation in the group has been problematic.

Journal Article↗
PubMed2000

The type 4 prepilin peptidases comprise a novel family of aspartic acid proteases.

Type 4 prepilins or prepilin-like-proteins are secreted by a wide range of bacterial species and are required for a variety of functions including type 4 pilus formation, toxin and other enzyme secretion, gene transfer, and biofilm formation. A distinctive feature of these proteins is the presence of a specialized leader peptide that is cleaved off by a cognate membrane-bound type 4 prepilin peptidase (TFPP) during the process of secretion. In this report we show that the TFPPs represent a novel family of bilobed aspartate proteases that is unlike any other protease. The active site pairs of aspartic acids of the two TFPPs in Vibrio cholerae are found at positions 125 and 189 of TcpJ and 147 and 212 of VcpD. Corresponding aspartate residues are completely conserved throughout this extensive peptidase family.

Amino Acid Sequence↗
PubMed2000

Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae.

The toxin-co-regulated pilus (TCP), a type 4 pilus that is expressed by epidemic strains of Vibrio cholerae O1 and O139, is required for colonization of the human intestine. The TCP structure is assembled as a polymer of repeating subunits of TcpA pilin that form long fibres, which laterally associate into bundles. Previous passive immunization studies have suggested that the C-terminal region of TcpA is exposed on the surface of the pilus fibre and has a critical role in mediating the colonization functions of TCP. In the present study, we have used site-directed mutagenesis to delineate two domains within the C-terminal region that contribute to TCP structure and function. Alterations in the first domain, termed the structural domain, result in altered pilus stability or morphology. Alterations in the second domain, termed the interaction domain, affect colonization and/or infection by CTX-bacteriophage without affecting pilus morphology. In vitro and in vivo analyses of the tcpA mutants revealed that a major function of TCP is to mediate bacterial interaction through direct pilus-pilus contact required for microcolony formation and productive intestinal colonization. The importance of this function is supported by the finding that intragenic suppressor mutations that restore colonization ability to colonization-deficient mutants simultaneously restore pilus-mediated bacterial interactions. The alterations resulting from the suppressor mutations also provide insight into the molecular interactions between pilin subunits within and between pilus fibres.

Alleles↗
PubMed2000

Vibrio cholerae H-NS silences virulence gene expression at multiple steps in the ToxR regulatory cascade.

H-NS is an abundant nucleoid-associated protein involved in the maintenance of chromosomal architecture in bacteria. H-NS also has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. In this study we demonstrate a role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon of Vibrio cholerae. Deletion of hns resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin-coregulated pilus, and the ToxT virulence gene regulatory protein. For the cholera toxin- and ToxT-encoding genes, elevated expression in an hns mutant was found to occur in the absence of the cognate activator proteins, suggesting that H-NS functions directly at these promoters to decrease gene expression. Deletion analysis of the region upstream of toxT suggests that an extensive region located far upstream of the transcriptional start site is required for complete H-NS-mediated repression of gene expression. These data indicate that H-NS negatively influences multiple levels of gene expression within the V. cholerae virulence cascade and raise the possibility that the transcriptional activator proteins in the ToxR regulon function to counteract the repressive effects of H-NS at the various promoters as well as to recruit RNA polymerase.

Bacterial Proteins↗
PubMed1999

A new level in the Vibrio cholerae ToxR virulence cascade: AphA is required for transcriptional activation of the tcpPH operon.

The expression of the ToxR virulence regulon is dependent upon the regulatory proteins ToxR/ToxS, TcpP/TcpH and ToxT. We describe here a previously unidentified gene in Vibrio cholerae, aphA (activator of tcpP and tcpH expression), which is required for the transcription of the tcpPH operon. Under conditions normally optimal for virulence gene expression, an in frame aphA deletion decreased the expression of a cholera toxin promoter fusion (ctx-lacZ) and prevented the production of the toxin co-regulated pilus (TCP). Plasmids producing ToxT or TcpP/H, but not ToxR, restored ctx-lacZ expression and TCP production in the delta aphA strain, suggesting that the mutation interferes with toxT expression by influencing the transcription of tcpPH. Indeed, the expression of a chromosomal tcpP-lacZ fusion was reduced in the delta aphA mutant and increased in both V. cholerae and Escherichia coli by introducing aphA expressed from an inducible promoter. These results support a model in which AphA functions at a previously unknown step in the ToxR virulence cascade to activate the transcription of tcpPH. TcpP/TcpH, together with ToxR/ToxS, then activate the expression of toxT, resulting ultimately in the production of virulence factors such as cholera toxin and TCP.

Alkaline Phosphatase↗
PubMed1999

Differential transcription of the tcpPH operon confers biotype-specific control of the Vibrio cholerae ToxR virulence regulon.

Epidemic strains of Vibrio cholerae O1 are divided into two biotypes, classical and El Tor. In both biotypes, regulation of virulence gene expression depends on a cascade in which ToxR activates expression of ToxT, and ToxT activates expression of cholera toxin and other virulence genes. In the classical biotype, maximal expression of this ToxR regulon in vitro occurs at 30 degrees C at pH 6.5 (ToxR-inducing conditions), whereas in the El Tor biotype, production of these virulence genes only occurs under very limited conditions and not in response to temperature and pH; this difference between biotypes is mediated at the level of toxT transcription. In the classical biotype, two other proteins, TcpP and TcpH, are needed for maximal toxT transcription. Transcription of tcpPH in the classical biotype is regulated by pH and temperature independently of ToxR or ToxT, suggesting that TcpP and TcpH couple environmental signals to transcription of toxT. In this study, we show a near absence of tcpPH message in the El Tor biotype under ToxR-inducing conditions of temperature and pH. However, once expressed, El Tor TcpP and TcpH appear to be as effective as classical TcpP and TcpH in activating toxT transcription. These results suggest that differences in regulation of virulence gene expression between the biotypes of V. cholerae primarily result from differences in expression of tcpPH message in response to environmental signals. We present an updated model for control of the ToxR virulence regulon in V. cholerae.

Bacterial Proteins↗
PubMed1999

Genetic and transcriptional analyses of the Vibrio cholerae mannose-sensitive hemagglutinin type 4 pilus gene locus.

The mannose-sensitive hemagglutinin (MSHA) of the Vibrio cholerae O1 El Tor biotype is a member of the family of type 4 pili. Type 4 pili are found on the surface of a variety of gram-negative bacteria and have demonstrated importance as host colonization factors, bacteriophage receptors, and mediators of DNA transfer. The gene locus required for the assembly and secretion of the MSHA pilus has been localized to a 16.7-kb region of the V. cholerae chromosome. Sixteen genes required for hemagglutination, including five that encode prepilin or prepilin-like proteins, have been identified. Examination of MSHA-specific cDNAs has localized two promoters that drive expression of these genes. This evidence indicates that the MSHA gene locus is transcriptionally organized into two operons, one encoding the secretory components and the other encoding the structural subunits, an arrangement unique among previously characterized type 4 pilus loci. The genes flanking the MSHA locus encode proteins that show homology to YhdA and MreB of Escherichia coli. In E. coli, the yhdA and mreB genes are adjacent to each other on the chromosome. The finding that the MSHA locus lies between these two E. coli homologs and that it is flanked by a 7-bp direct repeat suggests that the MSHA locus may have been acquired as a mobile genetic element.

Bacterial Proteins↗
PubMed1998

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation.

Cholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae. Although the eps gene locus encodes several type four prepilin-like proteins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepilin peptidase from the V. cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa. Plasmid disruption or deletion of this peptidase gene in either EI Tor or classical V. cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion. In the case of the EI Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose-sensitive haemagglutination is abolished. The cloned V. cholerae peptidase processes either EpsI or MshA preproteins when co-expressed in E. coli. Mutation of the V. cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU. The V. cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepilin peptidase required for pilus assembly and cytolysin secretion in V. vulnificus. Accordingly, the V. cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.

Adhesins, Bacterial↗
PubMed1998

Investigation of the roles of toxin-coregulated pili and mannose-sensitive hemagglutinin pili in the pathogenesis of Vibrio cholerae O139 infection.

In this study, adult volunteers were fed tcpA and mshA deletion mutants of V. cholerae O139 strain CVD 112 to determine the role of toxin-coregulated pili (TCP) and mannose-sensitive hemagglutinin (MSHA) in intestinal colonization. Eight of 10 volunteers who received CVD 112 or CVD 112 delta mshA shed the vaccine strains in their stools; the geometric mean peak excretion for both groups was 1.4 x 10(5) CFU/g of stool. In contrast, only one of nine recipients of CVD 112 delta tcpA shed vibrios in his stool (P < 0.01); during the first 24 h after inoculation, 3 x 10(2) CFU/g was recovered from this volunteer. All recipients of CVD 112 and 8 (80%) of the recipients of CVD 112 delta mshA developed at least a fourfold rise in vibriocidal titer after immunization. In contrast, only one (11%) of the nine recipients of CVD 112 delta tcpA developed a fourfold rise in vibriocidal titer (P < 0.01). We conclude that TCP are an important colonization factor of V. cholerae O139 and probably of El Tor V. cholerae O1. In contrast, MSHA does not appear to promote intestinal colonization in humans.

Adult↗
PubMed1998

The Vibrio cholerae mannose-sensitive hemagglutinin is the receptor for a filamentous bacteriophage from V. cholerae O139.

We previously isolated from a 1994 isolate of Vibrio cholerae O139 a filamentous lysogenic bacteriophage, choleraphage 493, which inhibits pre-O139 but not post-O139 El Tor biotype V. cholerae strains in plaque assays. We investigated the role of the mannose-sensitive hemagglutinin (MSHA) type IV pilus as a receptor in phage 493 infection. Spontaneous, Tn5 insertion, and mshA deletion mutants are resistant to 493 infection. Susceptibility is restored by mshA complementation of deletion mutants. Additionally, the 493 phage titer is reduced by adsorption with MSHA-positive strains but not with a DeltamshA1 strain. Monoclonal antibody against MSHA inhibits plaque formation. We conclude that MSHA is the receptor for phage 493. The emergence and decline of O139 in India and Bangladesh are correlated with the susceptibility and resistance of El Tor strains to 493. However, mshA gene sequences of post-O139 strains are identical to those of susceptible pre-O139 isolates, indicating that phage resistance of El Tor is not due to a change in mshA. Classical biotype strains are (with rare exceptions) hemagglutinin negative and resistant to 493 in plaque assays. Nevertheless, they express the mshA pilin gene. They can be infected with 493 and produce low levels of phage DNA, like post-O139 El Tor strains. Resistance to 493 in plaque assays is thus not equivalent to resistance to infection. The ability of filamentous phages, such as 493, to transfer large amounts of DNA provides them, additionally, with the potential for quantum leaps in both identity and pathogenicity, such as the conversion of El Tor to O139.

Bacterial Proteins↗
PubMed1998

Mutations in toxR and toxS that separate transcriptional activation from DNA binding at the cholera toxin gene promoter.

ToxR and ToxS are integral membrane proteins that activate the transcription of virulence genes in Vibrio cholerae. ToxR can be separated into three different domains: an N-terminal cytoplasmic DNA binding domain, a central transmembrane domain, and a C-terminal periplasmic domain. ToxS is thought to enhance ToxR-mediated transcriptional activation through a periplasmic interaction. By P22 challenge phage selection for DNA binding, in combination with a screen for cholera toxin gene transcription, 12 toxR and toxS positive control mutants producing variant ToxR proteins from the toxRS operon that bind to the cholera toxin promoter but that fail to activate transcription were isolated. One mutation in toxR specifies an E82K change in the predicted helix-loop-helix DNA binding domain and destroys ToxR-mediated activation. Seven toxR mutations included frameshifts and stop codons introduced into the periplasmic domain, and six of these mutations appeared to produce proteolytically processed shorter forms of ToxR, suggesting that even short periplasmic deletions alter the folding of ToxR in the periplasm. Deletion of toxS did not alter the steady-state level of ToxR, and ToxR was found to be capable of binding to DNA in the absence of ToxS even though it did not activate transcription. However, the ToxS L33S variant rendered ToxR susceptible to proteolysis, suggesting that the natural function of ToxS is to complex with ToxR. Therefore, certain alterations that map to the ToxR cytoplasmic DNA binding domain, to the periplasmic domain, or to ToxS separate DNA binding activity from activator function. These data support a model where proper assembly or stability of the periplasmic domain of ToxR is enhanced by ToxS. This chaperone-like activity of ToxS may be required for the formation of the transcriptional activation complex but not the ToxR-DNA complex.

Alleles↗
PubMed1997

Sequence and functional analysis of the gene encoding Vibrio cholerae cAMP receptor protein.

We describe here the cloning, nucleotide sequence, and functional expression of the crp gene of Vibrio cholerae (Vc) encoding the cyclic AMP receptor protein (CRP). The Vc crp gene shows 81% identity with the crp gene from Escherichia coli (Ec) and its deduced amino acid sequence shows 95% identity with the Ec protein. When expressed from inducible promoters, the cloned Vc gene produced an approximately 20-kDa protein which complemented the carbohydrate-negative and growth-defective phenotypes of both Ec and Vc crp mutants. In the Vc crp mutant, the cloned crp gene also restored the normal repression of ToxR-regulated virulence genes which occurs under certain environmental conditions.

Amino Acid Sequence↗