Vibrio cholerae is the causative agent of the severe diarrheal disease cholera, which affects millions of people worldwide each year. The goal of our research is to understand the mechanisms for controlling expression of the genes involved in V. cholerae virulence. The V. cholerae virulence genes include those encoding cholera toxin (CT), which produces the characteristic symptoms of cholera, the toxin co-regulated pilus (TCP), which is involved in the colonization of the small intestine of the host, together with a collection of other genes whose roles in virulence are poorly understood. Expression of the virulence genes is controlled by a network of transcriptional regulators; however, one protein, ToxT, ultimately activates transcription of nearly all the virulence genes, and understanding how this protein functions is the focus of our work. Experiments described in this application will examine the following: (1) the amino acid residues in ToxT that are involved in transcriptional activation and DNAbinding will be determined using a genetic selection/screening followed by further characterization of mutants in vivo and in vitro. (2) There is mounting evidence that ToxT activity is regulated post-transcriptionally. We will determine how the post-transcriptional control of ToxT is manifested, examine environmental ToxT variants for functionality in epidemic V. cholerae, and assess the effects of constitutive ToxT expression in vitro and in an animal model. (3) We will determine the identities of all proteins whose expression is ToxT dependent by comparing proteomic analysis of V. cholerae having wildtype toxT and V. cholerae having a toxT deletion. These experiments should significantly increase our knowledge of ToxT protein function and of the functions of genes whose expression is activated by ToxT, and could lead to new therapeutic targets for cholera disease. Cholera remains a significant worldwide problem, particularly in areas suffering from the effect of war, natural disaster, or economic hardship, and improved treatments should greatly increase the quality of life for people in these regions. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Career Transition Award (K22)
Project #
5K22AI071011-02
Application #
7352760
Study Section
Microbiology and Infectious Diseases B Subcommittee (MID)
Program Officer
Hall, Robert H
Project Start
2007-02-15
Project End
2010-01-31
Budget Start
2008-02-01
Budget End
2010-01-31
Support Year
2
Fiscal Year
2008
Total Cost
$108,000
Indirect Cost
Name
Wayne State University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
Plecha, Sarah C; Withey, Jeffrey H (2015) [14C] Linoleic Acid Uptake and Fractionation Assay in Vibrio cholerae. Bio Protoc 5:
Plecha, Sarah C; Withey, Jeffrey H (2015) Mechanism for inhibition of Vibrio cholerae ToxT activity by the unsaturated fatty acid components of bile. J Bacteriol 197:1716-25
Thomson, Joshua J; Plecha, Sarah C; Withey, Jeffrey H (2015) A small unstructured region in Vibrio cholerae ToxT mediates the response to positive and negative effectors and ToxT proteolysis. J Bacteriol 197:654-68
Thomson, Joshua J; Withey, Jeffrey H (2014) Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters. J Bacteriol 196:3872-80
Dittmer, Jennifer B; Withey, Jeffrey H (2012) Identification and characterization of the functional toxboxes in the Vibrio cholerae cholera toxin promoter. J Bacteriol 194:5255-63
Abuaita, Basel H; Withey, Jeffrey H (2011) Genetic screening for bacterial mutants in liquid growth media by fluorescence-activated cell sorting. J Microbiol Methods 84:109-13
Abuaita, Basel H; Withey, Jeffrey H (2011) Termination of Vibrio cholerae virulence gene expression is mediated by proteolysis of the major virulence activator, ToxT. Mol Microbiol 81:1640-53
Abuaita, Basel H; Withey, Jeffrey H (2009) Bicarbonate Induces Vibrio cholerae virulence gene expression by enhancing ToxT activity. Infect Immun 77:4111-20
Bellair, Michelle; Withey, Jeffrey H (2008) Flexibility of Vibrio cholerae ToxT in transcription activation of genes having altered promoter spacing. J Bacteriol 190:7925-31