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Role of Vibrio cholerae neuraminidase in the function of cholera toxin.

ABSTRACT: Vibrio cholerae neuraminidase (NANase) is hypothesized to act synergistically with cholera toxin (CT) and increase the severity of a secretory response by increasing the binding and penetration of CT to enterocytes. To test this hypothesis, the NANase gene (nanH) from V. cholerae Ogawa 395 was first cloned and sequenced. Isogenic wild-type and NANase- V. cholerae 395 strains were then constructed by using suicide vector-mediated mutagenesis. The influence of NANase on CT binding and penetration was examined in vitro by using culture filtrates from these isogenic strains. Fluorescence due to binding of fluorescein-conjugated CT to C57BL/6 and C3H mouse fibroblasts exposed to NANase+ filtrates increased five- and eightfold, respectively, relative to that with NANase- filtrates. In addition, NANase+ filtrates increased the short-circuit current measured in Ussing chambers 65% relative to that with NANase- filtrates, although this difference decreased as production of CT increased. The role of NANase in V. cholerae pathogenesis was examined in vivo by intragastric inoculation of the isogenic strains into CD1 suckling mice. No difference in fluid accumulation ratios was seen at doses of 10(4) to 10(8) CFU, but NANase+ strains produced 18% higher fluid accumulation ratios at 10(9) CFU than NANase- strains when inoculated into nonfasted suckling mice. It is concluded that NANase plays a subtle but significant role in the binding and uptake of CT by susceptible cells under defined conditions.

SUBMITTER: Galen JE

PROVIDER: S-EPMC257643 | biostudies-other | 1992 Feb

REPOSITORIES: biostudies-other

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Role of Vibrio cholerae neuraminidase in the function of cholera toxin.

Galen J E JE Ketley J M JM Fasano A A Richardson S H SH Wasserman S S SS Kaper J B JB

Infection and immunity 19920201 2

Vibrio cholerae neuraminidase (NANase) is hypothesized to act synergistically with cholera toxin (CT) and increase the severity of a secretory response by increasing the binding and penetration of CT to enterocytes. To test this hypothesis, the NANase gene (nanH) from V. cholerae Ogawa 395 was first cloned and sequenced. Isogenic wild-type and NANase- V. cholerae 395 strains were then constructed by using suicide vector-mediated mutagenesis. The influence of NANase on CT binding and penetration ...[more]

PMID: 1730470

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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

Role of Vibrio cholerae neuraminidase in the function of cholera toxin.

Publications

Role of Vibrio cholerae neuraminidase in the function of cholera toxin.

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