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Vibrio cholerae toxin-coregulated pilus structure analyzed by hydrogen/deuterium exchange mass spectrometry.

ABSTRACT: The bacterial pathogen Vibrio cholerae uses toxin-coregulated pili (TCP) to colonize the human intestine, causing the severe diarrheal disease cholera. TCP are long, thin, flexible homopolymers of the TcpA subunit that self-associate to hold cells together in microcolonies and serve as the receptor for the cholera toxin phage. To better understand TCP's roles in pathogenesis, we characterized its structure using hydrogen/deuterium exchange mass spectrometry and computational modeling. We show that the pilin subunits are held together by tight packing of the N-terminal alpha helices, but loose packing of the C-terminal globular domains leaves substantial gaps on the filament surface. These gaps expose a glycine-rich, amphipathic segment of the N-terminal alpha-helix, contradicting the consensus view that this region is buried in the filament core. Our results explain extreme filament flexibility, suggest a molecular basis for pilus-pilus interactions, and reveal a previously unrecognized therapeutic target for V. cholerae and other enteric pathogens.

SUBMITTER: Li J

PROVIDER: S-EPMC2238685 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Vibrio cholerae toxin-coregulated pilus structure analyzed by hydrogen/deuterium exchange mass spectrometry.

Li Juliana J Lim Mindy S MS Li Sheng S Brock Melissa M Pique Michael E ME Woods Virgil L VL Craig Lisa L

Structure (London, England : 1993) 20080101 1

The bacterial pathogen Vibrio cholerae uses toxin-coregulated pili (TCP) to colonize the human intestine, causing the severe diarrheal disease cholera. TCP are long, thin, flexible homopolymers of the TcpA subunit that self-associate to hold cells together in microcolonies and serve as the receptor for the cholera toxin phage. To better understand TCP's roles in pathogenesis, we characterized its structure using hydrogen/deuterium exchange mass spectrometry and computational modeling. We show th ...[more]

PMID: 18184591

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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

Vibrio cholerae toxin-coregulated pilus structure analyzed by hydrogen/deuterium exchange mass spectrometry.

Publications

Vibrio cholerae toxin-coregulated pilus structure analyzed by hydrogen/deuterium exchange mass spectrometry.

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