Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Immunology and Microbiology

First Advisor

Jeffrey H. Withey

Abstract

Vibrio cholerae is the etiologic agent of the severe diarrheal disease cholera. The aquatic bacterium is ingested by humans through contaminated water or food. Disease initiation depends on the production of the major virulence factors: cholera toxin (CT) and the toxin co-regulated pilus (TCP). The bacterium responds to signals in the human host that activate a virulence regulatory cascade termed the "ToxR Regulon". The ToxR regulon consists of various transcription regulators whose activity culminates in the production of the major virulence regulator, ToxT. ToxT directly activates transcription of CT and TCP, as well as many other gene products involved in disease. ToxT is a 276 amino acid AraC/XylS family member that binds to 13 base pair degenerate sequences, called toxboxes, to initiate transcription. Post-transcriptional modulation of ToxT activity occurs via negative and positive effector molecules present in the human upper small intestine where V. cholerae colonizes. Negative effectors of ToxT activity include bile and the unsaturated fatty acid (UFA) components of bile. Conversely, the positive effector, bicarbonate, enhances ToxT activity. These positive and negative effectors provide in vivo signals to the bacterium to initiate transcription of virulence genes in the appropriate location. This dissertation is focused on identifying the molecular mechanism of positive modulation of ToxT activity by bicarbonate. Additionally, the role of bicarbonate in controlling transcription of ToxT-independent genes associated with colonization and host survival is explored.

Chapter one elucidates the mechanism for bicarbonate-mediated enhancement of ToxT activity. The ToxT-dependent promoters contain toxboxes that exist in various orientations, including direct and inverted repeat configurations. Additionally, the promoter of the ToxT-dependent genealdA, contains only a single toxbox. We have shown that bicarbonate enters the V. cholerae cell where it can interact with ToxT in the cytoplasm. Furthermore, we have shown that bicarbonate can enhance the activity of ToxT regardless of toxbox orientation. This is achieved through an increase in ToxT binding affinity for the promoters it activates.

Chapter two characterizes an unstructured region in the N-terminal domain of ToxT that controls the response of ToxT to effectors, as well as mediating proteolysis. Through mutagenesis of this region we have discovered a ToxT mutant that has a decreased response to both the negative and positive effectors of ToxT activity. The highly active ToxT N106F mutant can no longer be activated by bicarbonate. Similarly, activity of the mutant is unresponsive to negative modulation of activity by bile and UFAs. In addition to its importance in responding to effector molecules, this region of ToxT is essential for normal proteolysis. Many mutations in this region alter proteolysis, with the ToxT M103A mutant completely abolishing proteolysis.

Chapter three discusses alterations in the transcriptome of V. cholerae with the addition of external bicarbonate. RNA-Seq was performed to assess these changes. Bicarbonate mediated the up-regulation of all the known ToxT-dependent genes, as well as other genes important for host survival and colonization. Conversely, bicarbonate caused down-regulation of genes that are unnecessary or detrimental for host colonization and survival. The findings in this chapter signify the importance of the host molecule bicarbonate to initiate essential changes in the V. cholerae transcriptome.

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