Characterization of the novel auxiliary inhibitor, VieB, in the VieSAB three-component signal transduction system and insights into the regulation of vieSAB in Vibrio cholerae.
cholerae is a facultative, water-borne pathogen that causes a severe diarrheal disease
known as cholera. Its ability to monitor environmental changes as it transitions between
aquatic reservoirs and the human intestine is vital to its pathogenic lifestyle. One way
V. cholerae alters its gene expression is by sensing changing external stimuli through
the action of two-componen... read moret signal transduction systems (TCS). One such system in V.
cholerae, VieSAB, is comprised of the sensor kinase (VieS), response regulator (VieA),
and an auxiliary protein (VieB). VieSAB has been shown to be important in the induction
of virulence genes by controlling the concentration of the secondary messenger,
cyclic-di-GMP (Tischler, Lee et al. 2002; Tischler and Camilli 2005; Tamayo, Schild et
al. 2008). Even though VieSA behave similar to typical two-component systems
(Martinez-Wilson, Tamayo et al. 2008), many questions relating to its function remain
unanswered. Firstly, the role of VieB remains unclear, however preliminary data suggest
that VieB may interact with VieSA to modulate its activity. Secondly, transcriptional
regulation of vieSAB is poorly understood. Previous studies suggest that activated VieA
is autoregulatory, however regulation of this operon has not been characterized. In
order to gain a better understanding of the VieSAB signal transduction system, the first
goal of my thesis project was to identify and characterize the mechanism of VieB. Using
biochemical techniques, I reveal the function and mechanism of action of VieB in the
VieSA TCS. I provide evidence that VieB partially disrupts VieS autophosphorylation and
completely interrupts phosphotransfer. Taken together, we propose a working model
whereby the inhibitory role of VieB in the VieSA phosphorelay provides negative feedback
control over the signal output. Additionally, I describe attempts to characterize vieB
genetically, however no phenotype has been observed thus far, suggesting that vieB may
play only a minor role in controlling virulence gene expression at the end of infection.
In the second half of my thesis, I describe my work in characterizing the regulation of
the vieSAB operon. While I provide evidence that vieA is not autoregulatory and the
master virulence gene transcriptional regulator, toxT, does not control vieSAB
expression under the conditions tested, I reveal the negative regulation of vieSAB by
the cyclic-AMP receptor protein (CRP) during stationary phase. Interestingly, while CRP
appears to up regulate vieB transcript, this does not correlate to VieB protein levels
suggesting that there may be post-transcriptional regulation or differential expression
within the vieSAB operon. Taken together, we hypothesize that incorporation of CRP into
the regulation of vieSAB ties together carbon source availability with the modulation of
virulence gene expression through
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Andrew Camilli.
Keyword: Microbiology.read less