The Group A Streptococcus (GAS) is a Gram-positive pathogen that is the causative agent of a greater variety of human disease than any other bacterial species. Such versatility requires the ability to coordinately regulate the expression and production of numerous factors in rapid response to host and environmental signals. The specific hypothesis to be addressed by the proposed research is that the Streptococcal Regulator of Virulence (Srv) is a transcriptional activator of metabolic and virulence factors that promote GAS survival in the host. The hypothesis is based on three observations. First, an isogenic mutant strain lacking srv exhibits significantly attenuated virulence in a mouse model of GAS bacteremia. Second, Microarray analysis indicated that 21% (354/1678) of GAS genes examined are down regulated in vitro in the srv mutant strain while only 7 genes were up regulated. Third, only a single Srv protein variant has been identified in a set of diverse strains isolated from different cases of human GAS infection. Understanding the basic mechanisms of Srv gene regulation will provide insight into how GAS mediates interactions with the host and may lead to novel therapeutic approaches designed to interrupt virulence gene expression or alter GAS colonization. Thus, we propose the following specific aims: (1) To determine how Srv mediates transcription and distinguish between those genes directly and indirectly controlled by Srv. (2) To determine the effect of Srv-mediated gene regulation on GAS survival in vivo. An in vivo genomic binding site analysis will be used to identify the set of target sequences able to be bound by Srv. Real-time reverse transcriptase-PCR studies will be used to study the role of Srv in mediating gene transcription in vitro and in a murine model of GAS disease. Electrophoretic mobility shift assays (EMSA) and DNase I footprinting studies will confirm the interaction of Srv with the identified targets and characterize the specific amino acid residues required for DNA binding. Site-directed mutant(s) incapable of binding DMAwill be used to test the hypothesis that DNA binding is required for Srv function. Relevance: The decade of the 1980's witnessed a marked increase in the incidence of invasive GAS disease which has not declined in the years following with 10,000 invasive infections and 15 million noninvasive infections occurring annually in the US. Understanding the mechanism of Srv-dependent gene regulation will provide new insights into the control of GAS disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI063453-03
Application #
7384432
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Rubin, Fran A
Project Start
2006-04-01
Project End
2010-03-31
Budget Start
2008-04-01
Budget End
2009-03-31
Support Year
3
Fiscal Year
2008
Total Cost
$307,555
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
Roberts, Amity L; Connolly, Kristie L; Kirse, Daniel J et al. (2012) Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage. BMC Pediatr 12:3
Connolly, Kristie L; Roberts, Amity L; Holder, Robert C et al. (2011) Dispersal of Group A streptococcal biofilms by the cysteine protease SpeB leads to increased disease severity in a murine model. PLoS One 6:e18984
Connolly, Kristie L; Braden, Amy K; Holder, Robert C et al. (2011) Srv mediated dispersal of streptococcal biofilms through SpeB is observed in CovRS+ strains. PLoS One 6:e28640
Roberts, Amity L; Connolly, Kristie L; Doern, Christopher D et al. (2010) Loss of the group A Streptococcus regulator Srv decreases biofilm formation in vivo in an otitis media model of infection. Infect Immun 78:4800-8
Doern, Christopher D; Roberts, Amity L; Hong, Wenzhou et al. (2009) Biofilm formation by group A Streptococcus: a role for the streptococcal regulator of virulence (Srv) and streptococcal cysteine protease (SpeB). Microbiology 155:46-52
Doern, Christopher D; Holder, Robert C; Reid, Sean D (2008) Point mutations within the streptococcal regulator of virulence (Srv) alter protein-DNA interactions and Srv function. Microbiology 154:1998-2007