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R K Taylor

Publications and source records attributed to R K Taylor.

At least 55 records · Page 3Linked to original sources
PubMed1991

Localization of protective epitopes within the pilin subunit of the Vibrio cholerae toxin-coregulated pilus.

From a collection of monoclonal antibodies (MAbs) that recognize the native structure of the toxin-coregulated pilus of Vibrio cholerae, two protective MAbs (16.1 and 169.1) were used to localize the corresponding epitopes on the pilus. These MAbs were shown to specifically recognize the carboxyl half of the TcpA pilin subunit, as determined by their recognition of proteolytic fragments and hybrid pilin proteins. The positions of the epitopes were precisely determined through the use of overlapping synthetic peptides corresponding to this region of the pilin. The MAbs were found to recognize adjacent peptides, delineating a region between residues 157 and 199. Since the protective nature is specific for these two antibodies, the findings suggest that this region defines a domain that participates in toxin-coregulated pilus-mediated colonization and therefore represents a target for studies of its potential as an immunogen for incorporation into a component cholera vaccine.

Amino Acid Sequence↗
PubMed1991

Shared antigenicity and immunogenicity of type 4 pilins expressed by Pseudomonas aeruginosa, Moraxella bovis, Neisseria gonorrhoaea, Dichelobacter nodosus, and Vibrio cholerae.

Immunoblotting with polyclonal rabbit antibodies raised against pilins expressed by Pseudomonas aeruginosa, Moraxella bovis, Neisseria gonorrhoeae, Dichelobacter nodosus, and Vibrio cholerae was used to demonstrate that these polypeptides display conserved antigenic and, in most cases, immunogenic determinants. These determinants appear to be localized to the highly homologous amino-terminal domains (residues 1 to 25).

Animals↗
PubMed1991

Immunogenicity of Vibrio cholerae O1 toxin-coregulated pili in experimental and clinical cholera.

A functional tcpA gene, encoding the major subunit of toxin-coregulated pili (TCP), is necessary for Vibrio cholerae O1 Ogawa strain 395 to colonize the human intestine and confer protective immunity to virulent challenge. The immunogenicity of TCP and other antigens in experimental and naturally acquired cholera was determined. Seroconversion to cholera toxin (CT), whole cell preparations, and to Ogawa lipopolysaccharide but not to purified native TCP or to a TcpA mimiotope was found in volunteers. Local intestinal secretory immunoglobulin A from volunteers showed conversions to cholera toxin and lipopolysaccharide but not to TCP. Cholera patients in Indonesia showed a seroconversion rate to TCP of 3 of 6 and a seroconversion to a TcpA mimiotope of 1 of 6. Volunteer and patient sera showed similar vibriocidal seroconversions when assayed against either TCP-positive and TCP-negative V. cholerae O1 strains, suggesting that TCP do not contribute demonstrably to the vibriocidal antigen. We conclude that although seroconversion to TCP can occur in naturally acquired cholera, solid long-term protection can be engendered in the absence of a detectable anti-TCP immune response.

Amino Acid Sequence↗
PubMed1991

Genetic analysis of 987P adhesion and fimbriation of Escherichia coli: the fas genes link both phenotypes.

The 987P fimbrial gene cluster has recently been shown to contain eight genes (fasA to fasH) clustered on large plasmids of enterotoxigenic Escherichia coli and adjacent to a Tn1681-like transposon encoding the heat-stable enterotoxin STIa. Different genetic approaches were used to study the relationship between 987P fimbriation and adhesion. TnphoA mutagenesis, complementation assays, and T7 RNA polymerase-promoted gene expression indicated that all of the fas genes were involved in fimbrial expression and adhesion. In contrast to other fimbrial systems, the lack of expression of any single fas gene never resulted in the dissociation of fimbriation and adhesion, indicating that the adhesin is required for fimbrial expression and suggesting that FasA, the fimbrial structural subunit itself, is the adhesin. In addition, fimbrial length was shown to be modulated by the levels of expression of different fas genes.

Animals↗
PubMed1991

Bacterial adhesion to mucosal surfaces.

Bacteria utilize one or more of several mechanisms to adhere to mucosal surfaces. Members of the enterobacteriaceae and related bacteria typically elaborate adhesive filamentous appendages from their surfaces termed fimbriae (or pili). Vibrio cholerae expresses at least three types of these structures that are likely involved in various stages of colonizing the intestine. So far one of these, termed TCP for toxin coregulated pilus, has been shown to be essential for colonization and virulence in humans and in an infant mouse model. The major pilin subunit, TcpA, is a member of the type 4 pilin class, common to a number of diverse pathogenic bacteria. To understand how these pili function at the molecular level, a transposon mutagenesis was undertaken which identified at least a dozen genes involved in TCP biogenesis, function, and regulation. Despite this complexity, passive immunization of infant mice using polyclonal and monoclonal antibodies directed against TCP suggests that the adhesive moiety lies within the major pilin subunit itself, rather than as a minor pilus adhesin protein as in the case of the PAP and type 1 fimbriae of Escherichia coli. Pilin or peptides corresponding to the epitopes recognized by the monoclonal antibodies represent possible immunogens for improved cholera vaccines.

Animals↗
PubMed1990

Antibodies directed against the toxin-coregulated pilus isolated from Vibrio cholerae provide protection in the infant mouse experimental cholera model.

Pathogenic strains of Vibrio cholerae O1 elaborate a toxin-coregulated pilus, designated TCP, that is required for the bacteria to colonize the human intestine and cause disease. The possibility that antibodies directed against TCP might block colonization and thereby potentially prevent infection was investigated. The pilus was purified and polyclonal antiserum raised against it was shown to react preferentially with the 20.5-kDa major pilin subunit, TcpA. This antiserum inhibited attachment of the bacteria to epithelial cells in vitro. In a cholera animal model system, these pilus-specific antibodies efficiently protected infant mice from challenge with virulent V. cholerae strains of different serotypes and biotypes. Western immunoblot analysis of available killed, whole-cell vaccine preparations using TcpA-specific antibodies failed to detect pilin in either preparation. The results suggest that inclusion of TCP in cholera vaccines would provide a common antigen to induce immunity to the strains associated with human infection and potentially increase vaccine efficacy.

Animals↗
PubMed1990

The 987P fimbrial gene cluster of enterotoxigenic Escherichia coli is plasmid encoded.

A clone containing the 987P fimbrial gene cluster was selected from a cosmid library of total DNA of the prototype Escherichia coli strain 987 by using 987P-specific antiserum. A subclone of 12 kilobases containing all of the genes required for fimbrial expression on a nonfimbriated K-12 strain of E. coli and a DNA fragment internal to the fimbrial subunit gene were used to probe the prototype strain and various isolates of 987P-fimbriated enterotoxigenic E. coli. All strains had several plasmids, as shown by agarose gel electrophoresis, and each of five strains which expressed 987P fimbriae showed a plasmid of 35 to 40 megadaltons (MDa) hybridizing to both 987P-specific probes. Hybridization to restricted DNA of strain 987 supported a plasmid origin for the cloned 987P gene cluster. Moreover, an isogenic strain which had lost its 35-MDa plasmid was no longer capable of synthesizing fimbrial subunits, but regained fimbrial expression after reintroduction of the TnphoA (Tn5 IS50L::phoA)-tagged 35-MDa plasmid. Absence of fimbrial subunit synthesis in K-12 strains transformed with the 35-MDa plasmid alone suggested the requirement of regulatory elements existing in strain 987 but missing in K-12 strains. A probe for the heat-stable enterotoxin STIa hybridized in each of the 987P-fimbriated strains to the plasmid containing the 987P genes and in most of these strains to an additional plasmid which contained the gene for the heat-stable enterotoxin STII. Occurrence of the 987P and STIa genes on the same replicon correlates with epidemiological observations, STIa being the most prevalent toxin produced by 987P-fimbriated E. coli.

Bacterial Toxins↗
PubMed1990

Vibrio cholerae O395 tcpA pilin gene sequence and comparison of predicted protein structural features to those of type 4 pilins.

Vibrio cholerae O1 expresses a pilus that is coordinately regulated with cholera toxin production and hence termed TCP, for toxin-coregulated pilus. Insertion of Tn5 IS50L::phoA (TnphoA) into the major pilin subunit gene, tcpA, has previously been shown to render the strain avirulent as a result of its inability to colonize. One such insertion was isolated and used as a probe to screen for clones containing the intact tcpA gene. The DNA sequence of tcpA was determined by using the intact gene and several tcpA-phoA gene fusions. The deduced protein sequence agreed completely with that previously determined for the TcpA N terminus and with the size of the mature pilin protein. The reported homology with N-methylphenylalanine (type 4) pilins near the N terminus was extended and shown to include components of the atypical leader peptide as well as overall predicted structural similarities in other regions of the pilins. In contrast to the modified N-terminal phenylalanine residue found in all characterized type 4 pilins, the corresponding position in tcpA contains a Met codon, thus implying that the previously uncharacterized amino acid corresponding to the N-terminal position of the mature TcpA pilin is a modified form of methionine. Except for this difference, mature TcpA has the overall predicted structural motifs shared among type 4 pilins.

Amino Acid Sequence↗
PubMed1989

Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae.

Gene fusions between the cholera toxin structural genes and phoA, which encodes bacterial alkaline phosphatase, were identified after TnphoA mutagenesis of the cloned genes in Escherichia coli and were then mobilized into Vibrio cholerae. The activities of the hybrid proteins were detectable in V. cholerae and suggested that, like cholera toxin, they were secreted beyond the cytoplasm. To extend the utility of TnphoA to identify additional genetic export signals in V. cholerae and other gram-negative bacteria, TnphoA delivery vectors utilizing broad-host-range plasmids were developed. By using V. cholerae as a model system, insertion mutants carrying active phoA gene fusions were identified as colonies expressing alkaline phosphatase, which appeared blue on agar containing the indicator 5-bromo-4-chloro-3-indolyl phosphate. Since alkaline phosphatase is active only upon export from the cytoplasm, PhoA+ colonies resulting from the mutagenesis procedure were enriched for insertions in genes that encode secreted proteins. Insertion mutations were identified in the gene encoding a major outer membrane protein, OmpV, and in tcpA, which encodes a pilus (fimbrial) subunit. Mutant strains harboring chromosomal insertions isolated in this manner can be used to assess the role of the corresponding inactivated gene products on survival of V. cholerae in vivo. The expression of the hybrid proteins as determined by measuring alkaline phosphatase activity also allowed the convenient study of virulence gene expression.

Alkaline Phosphatase↗
PubMed1988

Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans.

Isogenic mutant strains of V. cholerae O1 lacking elements of a genetic regulon controlled by toxR and implicated in virulence were tested in volunteers. A deletion mutation in ctxA, the gene encoding the A subunit of cholera toxin, markedly attenuated disease symptoms without affecting intestinal colonization. Deletion of toxR, the gene encoding the cholera toxin-positive regulatory protein resulted in a diminution in colonizing capacity. A deletion mutation in tcpA, encoding the major subunit of the toxin coregulated pilus (regulated by toxR), abolished the colonizing capacity of this strain. These results show for the first time the role of a specific pilus structure in colonization of the human intestine by V. cholerae O1 and exemplify the significance of a genetic regulon in pathogenesis.

Adult↗
PubMed1987

Cholera toxin transcriptional activator toxR is a transmembrane DNA binding protein.

The toxR gene encodes a transcriptional activator controlling cholera toxin, pilus, and outer-membrane protein expression in V. cholerae. Nucleotide sequence and mutational analysis has identified the toxR gene product as a 32,527 dalton protein. Hydropathicity analysis of the derived amino acid sequence of ToxR predicts a transmembrane structure. The properties of hybrid proteins composed of N-terminal fragments of ToxR fused to the periplasmic enzyme alkaline phosphatase provide additional evidence for the transmembrane topology of the ToxR protein. These fusion proteins also allowed the localization of the transcriptional activation and DNA binding domains of the ToxR protein to its cytoplasmically located N-terminal portion. DNA binding assays and a deletion analysis of the cholera toxin promoter support a model for transcriptional activation that involves ToxR binding to a tandemly repeated 7 bp DNA sequence 56 bp upstream of the transcriptional start point.

Bacterial Proteins↗
PubMed1987

Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin.

The transposon TnphoA was used to generate fusions between phoA, the gene for alkaline phosphatase (PhoA), and genes encoding proteins that are secreted by Vibrio cholerae. One of the PhoA+ mutants isolated showed a dramatic reduction in its ability to colonize the intestines of suckling mice. This mutant no longer produced a 20.5-kDa protein (TcpA) that we show is the major subunit of a V. cholerae pilus. Amino-terminal sequence analysis of the TcpA pilus subunit showed that it shares amino acid homology with the pilins produced by several other pathogenic bacteria. The TcpA pilus was coordinately expressed with cholera toxin under various culture conditions, and this effect appeared to be dependent on the transcriptional activator encoded by the toxR gene. We conclude that the toxR gene plays a central role in the transcriptional regulation of multiple virulence genes of V. cholerae.

Alkaline Phosphatase↗
PubMed1985

Isolation and characterization of delta ompB strains of Escherichia coli by a general method based on gene fusions.

We isolated a series of delta ompR delta envZ mutants by inducing a strain carrying a lambda prophage in the closely linked gene malP and screening the bacterial survivors for loss of the major outer membrane porins OmpF and OmpC. Characterization of these deletion strains showed that both OmpR and EnvZ were necessary for transcription of ompF and ompC and that neither gene was essential for cell viability. Moreover, the deletion strains did not exhibit the pleiotropic membrane protein deficiency observed with certain envZ mutants. The method described should allow the simple isolation of deletions in any region of the chromosome.

Bacterial Outer Membrane Proteins↗
PubMed1985

Selection for mutants altered in the expression or export of outer membrane porin OmpF.

Strains in which the lacZ gene (which specifies beta-galactosidase) is fused to a gene encoding an envelope protein often exhibit a phenotype termed overproduction lethality. In such strains, high-level synthesis of the cognate hybrid protein interferes with the process of protein export, and this leads ultimately to cell death. A variation of this phenomenon has been discovered with lacZ fusions to the gene specifying the major outer membrane porin protein OmpF. In this case, we find that lambda transducing phage carrying an ompF-lacZ fusion will not grow on a host strain that constitutively overexpresses ompF. We have exploited this observation to develop a selection for ompF mutants. Using this protocol, we have isolated mutants altered in ompF expression and have identified mutations that block OmpF export. Our results suggest that it should be possible to adapt this selection for use with other genes specifying exported proteins.

Bacterial Outer Membrane Proteins↗
PubMed1985

Mutations that define the promoter of ompF, a gene specifying a major outer membrane porin protein.

Expression of the major porin structural genes, ompF and ompC, is influenced by medium osmotic strength and requires the products of two regulatory genes, ompR and envZ. To help define the sites required for the expression of both porin genes, we have used a novel selection to identify mutations that decrease ompF transcription. From our assignment of the mRNA start site by the primer extension method, these mutations appear to delineate poorly conserved -10 and -35 consensus promoter regions. In addition, one mutation provides the first genetic evidence that an A residue at position -45 may be important for RNA polymerase recognition.

Bacterial Outer Membrane Proteins↗
PubMed1985

Water intoxication: a possible complication during endurance exercise.

Four athletes developed water intoxication (hyponatremia) during endurance events lasting more than 7 h. The etiology of the condition appears to be voluntary hyperhydration with hypotonic solutions combined with moderate sweat sodium chloride losses. The reason why the fluid excess in these runners was not corrected by increased urinary losses is unknown. When advised to drink less during prolonged exercise, three of the athletes have subsequently completed prolonged endurance events uneventfully.

Adult↗