Dataset Information

Production of putative enhanced oral cholera vaccine strains that express toxin-coregulated pilus.

ABSTRACT: The use of whole cell killed (WCK) oral cholera vaccines is an important strategy for cholera prevention in endemic areas. To overcome current vaccine limitations, we engineered strains of V. cholerae to be non-toxigenic and to express the protective protein colonization factor, toxin-coregulated pilus (TCP), under scale-up conditions potentially amenable to vaccine production. Two V. cholerae clinical strains were selected and their cholera toxin genes deleted. The tcp operon was placed under control of a rhamnose-inducible promoter. Production and stability of TCP were assessed under various conditions. The strains lack detectable cholera toxin production. The addition of 0.1% rhamnose to the growth medium induced robust production of TCP and TcpA antigen. The strains produced intact TCP in larger growth volumes (1 L), and pili appeared stable during heat-killing or acid treatment of the bacterial cultures. To date, no WCK cholera vaccines have included TCP. We have constructed putative strains of V. cholerae for use in a vaccine that produce high levels of stable TCP antigen, which has not previously been achieved.

SUBMITTER: Hauke CA

PROVIDER: S-EPMC5383245 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Production of putative enhanced oral cholera vaccine strains that express toxin-coregulated pilus.

Hauke Caitlyn A CA Taylor Ronald K RK

PloS one 20170406 4

The use of whole cell killed (WCK) oral cholera vaccines is an important strategy for cholera prevention in endemic areas. To overcome current vaccine limitations, we engineered strains of V. cholerae to be non-toxigenic and to express the protective protein colonization factor, toxin-coregulated pilus (TCP), under scale-up conditions potentially amenable to vaccine production. Two V. cholerae clinical strains were selected and their cholera toxin genes deleted. The tcp operon was placed under c ...[more]

PMID: 28384206

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Project description:Coordinate expression of many virulence genes in the human pathogen Vibrio cholerae is controlled by the ToxR, TcpP, and ToxT proteins. These proteins function in a regulatory cascade in which ToxR and TcpP, two inner membrane proteins, are required to activate toxT and ToxT is the direct activator of virulence gene expression. ToxT-activated genes include those whose products are required for the biogenesis of cholera toxin (CTX) and the toxin-coregulated pilus, the major subunit of which is TcpA. This work examined control of toxT transcription. We tested a model whereby activation of toxT by ToxR and TcpP is required to prime an autoregulatory loop in which ToxT-dependent transcription of the tcpA promoter reads through a proposed terminator between the tcpF and toxT genes to result in continued ToxT production. Primer extension analysis of RNA from wild-type classical strain O395 showed that there are two products encoding toxT, one of which is longer than the other by 105 bp. Deletion of the toxT promoter (toxTDeltapro) resulted in the abolishment of toxT transcription, as predicted. Deletion of the tcpA promoter (tcpADeltapro) had no effect on subsequent detection of the smaller toxT primer extension product, but the larger toxT product was not detected, indicating that this product may be the result of transcription from the tcpA promoter and not of initiation directly upstream of toxT. Neither mutant strain produced detectable TcpA, but the CTX levels of the strains were different. The toxTDeltapro strain produced little detectable CTX, while the tcpADeltapro strain produced CTX levels intermediate between those of the wild-type and toxTDeltapro strains. Dependence of toxT transcription on TcpP and TcpH was confirmed by analyzing RNAs from strains carrying deletions in the genes encoding these regulators. The tcpP defect resulted in undetectable toxT transcription, whereas the tcpH mutation led to a diminishing of toxT RNA but not complete abolishment. Taken together, these results suggest that toxT transcription is dependent on two different promoters; one is directly upstream and is activated in part by TcpP and TcpH, and the other is much further upstream and is activated by ToxT.

Dataset Information

Production of putative enhanced oral cholera vaccine strains that express toxin-coregulated pilus.

Publications

Production of putative enhanced oral cholera vaccine strains that express toxin-coregulated pilus.

Similar Datasets

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