Ronald K. Taylor

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.

Organization of tcp, acf, and toxT genes within a ToxT-dependent operon

The toxin coregulated pilus (TCP) is required for Vibrio cholerae to colonize the human intestine. The expression of the pilin gene, tcpA, is dependent upon ToxR and upon ToxT. The toxT gene was recently mapped within the TCP biogenesis gene cluster and shown to be capable of activating a tcpA::TnphoA fusion when cloned in Escherichia coli. In this study, we determined that ToxR/ToxT activation occurs at the level of tcpA transcription. ToxT expressed in E. coli could activate a tcp operon fusion, while ToxR, ToxR with ToxS, or a ToxR‐PhoA fusion failed to activate the tcp operon fusion and we could not demonstrate binding of a ToxR extract to the tcpA promoter region in DNA mobility‐shift assays. The start site for the regulated promoter was shown by primer extension to lie 75 bp upstream of the first codon of tcpA. An 800‐base tcpA message was identified, by Northern analysis, that correlates by size to the distance between the transcriptional start and a hairpin‐loop sequence between tcpA and tcpB. The more‐sensitive assay of RNase protection analysis demonstrated that a regulated transcript probably extends through the rest of the downstream tcp genes, including ToxT and the adjacent accessory colonization factor (acf) genes. An in‐frame tcpA deletion, but not a polar tcpA::TnphoA fusion, could be complemented for pilus surface expression by providing tcpA in trans. This evidence suggests that the tcp genes, including toxT, are organized in an operon directly activated by ToxT in a ToxR‐dependent manner. Most of the ToxT expression under induced conditions requires transcription of the tcpA promoter. Further investigation of how tcp::TnphoA insertions that are polar on ToxT expression retain regulation showed that a low basal level of toxT expression is present in toxR and tcp::TnphoA strains. Overall, these observations support the ToxR/ToxT cascade of regulation for tcp. Once induced, toxT expression becomes autoregulatory via the tcp promoter, linking tcp expression to that of additional colonization factors, exotoxin production, and genes of unknown function in cholera pathogenesis.

Safe, live Vibrio cholerae vaccines?

Mutants of Vibrio cholerae defective in intestinal colonization have been constructed. Characterization of these mutants has led to the identification of a gene cluster involved in the assembly of a pilus colonization factor called TCP. The tcp operon has been cloned and strains of V. cholerae have been constructed that overproduce this pilus and the B subunit of cholera toxin. Together these studies may contribute to the eventual construction of efficient live and killed, oral cholera vaccines.

Biogenesis and regulation of the Vibrio cholerae toxin-coregulated pilus: analogies to other virulence factor secretory systems

Biogenesis of the toxin-coregulated pilus (TCP) of Vibrio cholerae 01 is essential for successful bacterial colonization of the small intestine. Pilus assembly requires the products of at least seven genes located on the chromosome adjacent to the pilin-encoding gene, tcpA. Previously reported TnphoA insertions in the TCP-assembly-deficient V. cholerae strains, KP2.21 and KP4.2, were isolated from the chromosome for further analysis. Nucleotide sequencing of the tcpE::phoA and tcpF::phoA fusions and corresponding clones of the region containing the intact genes revealed the presence of two open reading frames (ORFs) of 340 and 338 amino acids, designated TcpE and TcpF, respectively. The partial sequence of an ORF downstream from the TcpF coding sequence was determined to correspond to the global virulence regulator, ToxT. Proteins corresponding to the observed ORFs were visualized with the T7 promoter/RNA polymerase expression system. Computer-generated alignment algorithms predict that a homology exists between TcpE and the Klebsiella pneumoniae pullulanase secretion proteins PulD and PulF, the Xanthomonas campestris extracellular enzyme secretion factor XpsF, the Bacillus subtilis DNA competence protein ComG-ORF2, and the Yersinia enterocolitica Yop secretion determinant YscC. These observations provide a model to investigate further the relationship between the secretion mechanisms utilized by these seemingly diverse virulence determinants. Additionally, an extreme C-terminal segment of TcpE shows striking homology to the transmembrane segment of the eukaryotic integrin beta-1 chain, which could imply a role for TcpE in not only TCP secretion, but also host cell interaction.

Production and characterization of monoclonal antibodies to the toxin coregulated pilus (TCP) of Vibrio cholerae that protect against experimental cholera in infant mice

Abstract Six monoclonal antibodies (MAbs) directed against purified TCP (toxin coregulated pilus) of classical V. cholerae strain 0395 were developed and characterized. All of these MAbs recognized the major pilin subunit (TcpA) by western immunoblot. In the infant mouse experimental cholera model, only two of the MAbs (169·1 and 16·1) were shown to be protective although all six recognized TCP quaternary structure as determined by ELISA or immunoelectron microscopy. The results suggest that potentially protective epitopes can be identified in the TCP structure, specifically within TcpA, and that neutralization of the corresponding functional domains is sufficient to provide protection from cholera.

987P fimbrial gene identification and protein characterization by T7 RNA polymerase‐induced transcription and TnphoA mutagenesis

The 987P fimbrial gene cluster has been previously cloned as a 12 kb fragment from prototype strain 987. Gene products encoded by the whole clone were analysed by utilizing an in vivo system based on the induction of transcription by T7 RNA polymerase. The sensitivity of this technique permitted us to identify new proteins involved in 987P fimbriation. In total, eight proteins were detected, their genes (fasA to fasH) were mapped and their orientation of transcription determined. Several of the gene products demonstrated typical properties of exported proteins. Precursor and processed forms could be correlated after inhibiting protein transport with ethanol. The detection of enzymatically active fusion proteins after TnphoA (Tn5IS50L::phoA) mutagenesis supported and complemented these results. One protein encoded by the 12kb fragment was found not to be related to fimbriation but rather the product of the STla gene, identified as a component of a Tn1681‐like transposon.

TCP Pilus Expression and Biogenesis by Classical and El Tor Biotypes of Vibrio Cholerae 01

The mechanisms utilized by Vibrio cholerae for adherence and colonization of the human intestine have remained elusive to investigators for many years. This is probably due to the overall complexity of colonization and the lack of genetic systems that allowed the introduction of defined mutations to eliminate the expression of individual components possibly involved in the process, thereby allowing for the assessment of each one’s role. The most likely molecules to mediate attachment include a number of bacterial cell surface and soluble hemagglutinins that vary with regards to the source of erythrocytes they hemagglutinate, the carbohydrate sensitivity of the binding, and the stage of growth during which they are expressed (1). The other potential mediators of colonization are several different pili (fimbriae), some of which may be the same as certain hemagglutinins, although this correlation has yet to be determined (3). The expression of these pili varies with growth conditions. One of these pili types, that we have recently reported on is shown in Fig. 1. We have named this pilus TCP for toxin coregulated pilus, because its expression parallels that of toxin production (12).