Vibrio cholerae causes the fatal epidemic diarrhea! disease cholera. The expression of its primary virulence factors, toxin-coregulated pilus and cholera toxin, occurs via a transcriptional cascade involving several activator proteins and serves as a paradigm for the regulation of bacterial virulence. AphA and AphB initiate the expression of the cascade by a novel interaction at the tcpPH promoter. AphA is a member of a new regulator family and AphB is a LysR-type activator, one of the largest transcriptional regulatory families. Once expressed, cooperation between TcpP/TcpH and the homologous transmembrane activators ToxR/ToxS activates the toxT promoter. ToxT, an AraC-type regulator, then directly activates the promoters of the primary virulence factors. Transcriptional activation at these various promoters occurs only in response to certain environmental stimuli. One such stimulus, cell density, influences the virulence cascade through the quorum sensing system regulator HapR which represses the expression of the aphA promoter. The long term goals of this proposal are to understand the molecular basis of virulence gene regulation so as to facilitate the development of better strategies to prevent and cure bacterial diseases. Achieving these goals requires an understanding of how the specific regulatory proteins function at their cognate promoters to control gene expression and, ultimately, how they are influenced by environmental stimuli. Through a collaborative effort of laboratories with expertise in structural biology, virulence gene regulation and pathogenesis, we have obtained the crystal structure of AphA at 2.2 A resolution. Its structure reveals the presence of a winged-helix DMA binding domain and a topologically unique dimerization domain.
Aim 1 focuses on obtaining high resolution structures of (1) AphA with its cognate binding site, (2) AphB alone and in the presence of its cognate binding site, and (3) AphA and AphB together in a ternary complex with DNA. In addition, specific structural predictions will be tested using site-directed mutagenesis.
Aims 2 and 3 focus on obtaining high resolution structures of ToxT and HapR in the absence and presence of their binding sites and mutagenesis will also be carried out to test structural predictions. This proposed work will significantly increase our understanding of how these proteins regulate virulence gene expression in order to facilitate efforts to identify new molecules that interfere with their functions and which may serve as novel antivirulence drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI072661-03
Application #
7534978
Study Section
Special Emphasis Panel (ZRG1-IDM-H (02))
Program Officer
Hall, Robert H
Project Start
2006-12-15
Project End
2011-11-30
Budget Start
2008-12-01
Budget End
2009-11-30
Support Year
3
Fiscal Year
2009
Total Cost
$392,155
Indirect Cost
Name
Dartmouth College
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Privett, Britney R; Pellegrini, Maria; Kovacikova, Gabriela et al. (2017) Identification of a Small Molecule Activator for AphB, a LysR-Type Virulence Transcriptional Regulator in Vibrio cholerae. Biochemistry 56:3840-3849
Woodbrey, Anne K; Onyango, Evans O; Pellegrini, Maria et al. (2017) A new class of inhibitors of the AraC family virulence regulator Vibrio cholerae ToxT. Sci Rep 7:45011
Midgett, Charles R; Almagro-Moreno, Salvador; Pellegrini, Maria et al. (2017) Bile salts and alkaline pH reciprocally modulate the interaction between the periplasmic domains of Vibrio cholerae ToxR and ToxS. Mol Microbiol 105:258-272
Shi, Wei; Kovacikova, Gabriela; Lin, Wei et al. (2015) The 40-residue insertion in Vibrio cholerae FadR facilitates binding of an additional fatty acyl-CoA ligand. Nat Commun 6:6032
Taylor, Jennifer L; De Silva, Rukman S; Kovacikova, Gabriela et al. (2012) The crystal structure of AphB, a virulence gene activator from Vibrio cholerae, reveals residues that influence its response to oxygen and pH. Mol Microbiol 83:457-70
Lowden, Michael J; Skorupski, Karen; Pellegrini, Maria et al. (2010) Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes. Proc Natl Acad Sci U S A 107:2860-5
De Silva, Rukman S; Kovacikova, Gabriela; Lin, Wei et al. (2007) Crystal structure of the Vibrio cholerae quorum-sensing regulatory protein HapR. J Bacteriol 189:5683-91
De Silva, Rukman S; Kovacikova, Gabriela; Lin, Wei et al. (2005) Crystal structure of the virulence gene activator AphA from Vibrio cholerae reveals it is a novel member of the winged helix transcription factor superfamily. J Biol Chem 280:13779-83
Myskowski, P L (1993) Kaposi's sarcoma. Where do we go from here? Arch Dermatol 129:1320-3