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Effects of changes in membrane sodium flux on virulence gene expression in Vibrio cholerae
1Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
The expression of several virulence factors of Vibrio cholerae is coordinately regulated by the ToxT molecule and the membrane proteins TcpP/H and ToxR/S, which are required for toxT transcription. To identify proteins that negatively affect toxT transcription, we screened transposon mutants of V. cholerae carrying a chromosomally integrated toxT::lacZ reporter construct for darker blue colonies on media containing 5-bromo-4-chlor-3-indolyl beta-D galactoside (X-gal). Two mutants had transposon insertions in a region homologous to the nqr gene cluster of Vibrio alginolyticus, encoding a sodium-translocating NADH-ubiquinone oxidoreductase (NQR). In V. alginolyticus, NQR is a respiration-linked Na+ extrusion pump generating a sodium motive force that can be used for solute import, ATP synthesis, and flagella rotation. Inhibition of NQR enzyme function in V. cholerae by the specific inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) resulted in elevated toxT::lacZ activity. Increased toxT::lacZ expression in an nqr mutant strain compared with the parental strain was observed when the TcpP/H molecules alone were strongly expressed, suggesting that the negative effect of the NQR complex on toxT transcription is mediated through TcpP/H. However, the ability of the TcpP/H proteins to activate the toxT::lacZ reporter construct was greatly diminished in the presence of high NaCl concentrations in the growth medium. The flagellar motor of V. cholerae appears to be driven by a sodium motive force, and modulation of flagella rotation by inhibitory drugs, high media viscosity, or specific mutations resulted in increases of toxT::lacZ expression. Thus, the regulation of the main virulence factors of V. cholerae appears to be modulated by endogenous and exogenous sodium levels in a complex way.
Insights
The sodium-translocating NADH-ubiquinone oxidoreductase (NQR) complex negatively regulates Vibrio cholerae virulence factor expression. NQR inhibition or mutation increases toxT transcription, mediated by TcpP/H and influenced by sodium levels and flagellar rotation.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Pathogenesis
Background:
- Vibrio cholerae virulence factor expression is tightly regulated by ToxT and membrane proteins TcpP/H and ToxR/S.
- Understanding negative regulators of toxT transcription is crucial for deciphering Vibrio cholerae pathogenesis.
- The sodium-translocating NADH-ubiquinone oxidoreductase (NQR) complex is involved in respiration and ion transport in bacteria.
Purpose of the Study:
- To identify proteins that negatively regulate toxT transcription in Vibrio cholerae.
- To investigate the role of the NQR complex in the regulation of virulence factors.
- To elucidate the interplay between NQR, TcpP/H, sodium levels, and flagellar motor function in virulence gene expression.
Main Methods:
- Screening of Vibrio cholerae transposon mutants using a toxT::lacZ reporter construct.
- Inhibition of NQR enzyme function using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO).
- Assessing toxT::lacZ expression under varying conditions, including different NaCl concentrations and flagellar motor modulation.
Main Results:
- Transposon insertions in the nqr gene cluster led to increased toxT::lacZ expression.
- NQR inhibition with HQNO elevated toxT::lacZ activity, suggesting a negative regulatory role.
- The negative effect of NQR on toxT transcription is mediated through TcpP/H and influenced by sodium levels and flagellar rotation.
Conclusions:
- The NQR complex acts as a negative regulator of toxT transcription in Vibrio cholerae.
- Virulence factor regulation in Vibrio cholerae is complex, involving modulation by sodium levels and flagellar motor activity.
- These findings provide new insights into the intricate regulatory networks governing Vibrio cholerae pathogenesis.