Streptococcus pneumoniae (pneumococcus) is an important gram positive human respiratory pathogen that is developing antibiotic resistance. The essential VicRK two component system (TCS) and its associated third component VicX are required for pneumococcal virulence. The long-term goal of this proposal is to determine the signal transduction pathways used by the VicRKX system to regulate genes encoding an essential murein biosynthetic enzyme and established virulence factors on the pneumococcal cell surface. Our new results show that this regulation is direct and mediated by phosphorylation of the VicR response regulator (RR). Other new results suggest that defective cell wall biosynthesis may generate metabolic signals sensed by the VicRKX system, possibly by the VicK histidine kinase (HK), which lacks an extracellular sensing domain but contains a PAS domain, or by other phosphoryl group donors.
Five Specific Aims will be achieved in this five-year grant:
Aim I, We will characterize the binding of phosphorylated VicR and the resulting transcription activation at promoter regions of key regulon gene members using in vitro biochemical methods.
Aim II. We will determine the expression levels of VicRKX proteins and the virulence properties of vicRKX mutants to understand why the VicK HK is not essential in S. pneumoniae growing in culture, but is required for virulence. We will use genetic approaches to determine which other donors phosphorylate the VicR RR in the absence of the VicK HK.
Aim III. We will use biochemical approaches to determine the signal(s) sensed by the VicK HK and whether the VicK HK possesses a VicR-P phosphatase activity. We will construct lacZ reporter fusions to key regulon genes to determine culture and stress conditions that may be sensed by the VicRKX system. Genetic screens and selections will be used to identify possible signals sensed by the VicRKX system and other modes of regulation of these virulence factor genes.
Aim I V. We will use biochemical and genetic approaches to determine the roles of the VicX third component and putative VicK phosphatase activity in VicRKX signal transduction. We will determine whether the (3-lactamase fold in VicX plays a role in sensing additional signals.
Aim V. We will use biochemical and microarray methods to determine new members of the VicRKX regulon that were missed in previous studies. This grant will provide fundamental new knowledge about the regulation of important cell wall biosynthesis and virulence factor genes in a serious human pathogen. It will provide insights into the multiple mechanisms of signal transduction used by the VicRKX system in pneumococcus and likely other species of streptococcus to communicate between the cytoplasm and cell surface. Understanding the unusual features of VicRKX signal transduction will extend the paradigm of TCS regulation. Finally, gene products in the VicRKX regulon are promising surface targets for future antibiotic and vaccine development.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Khambaty, Farukh M
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Indiana University Bloomington
Schools of Arts and Sciences
United States
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Wayne, Kyle J; Li, Shuo; Kazmierczak, Krystyna M et al. (2012) Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and limiting cross-talk in Streptococcus pneumoniae D39 cells. Mol Microbiol 86:645-60
Sham, Lok-To; Tsui, Ho-Ching T; Land, Adrian D et al. (2012) Recent advances in pneumococcal peptidoglycan biosynthesis suggest new vaccine and antimicrobial targets. Curr Opin Microbiol 15:194-203
Biller, Steven J; Wayne, Kyle J; Winkler, Malcolm E et al. (2011) The putative hydrolase YycJ (WalJ) affects the coordination of cell division with DNA replication in Bacillus subtilis and may play a conserved role in cell wall metabolism. J Bacteriol 193:896-908
Nichols, Robert J; Sen, Saunak; Choo, Yoe Jin et al. (2011) Phenotypic landscape of a bacterial cell. Cell 144:143-56
Land, Adrian D; Winkler, Malcolm E (2011) The requirement for pneumococcal MreC and MreD is relieved by inactivation of the gene encoding PBP1a. J Bacteriol 193:4166-79
Sham, Lok-To; Barendt, Skye M; Kopecky, Kimberly E et al. (2011) Essential PcsB putative peptidoglycan hydrolase interacts with the essential FtsXSpn cell division protein in Streptococcus pneumoniae D39. Proc Natl Acad Sci U S A 108:E1061-9
Tsui, Ho-Ching Tiffany; Keen, Susan K; Sham, Lok-To et al. (2011) Dynamic distribution of the SecA and SecY translocase subunits and septal localization of the HtrA surface chaperone/protease during Streptococcus pneumoniae D39 cell division. MBio 2:
Jacobsen, Faith E; Kazmierczak, Krystyna M; Lisher, John P et al. (2011) Interplay between manganese and zinc homeostasis in the human pathogen Streptococcus pneumoniae. Metallomics 3:38-41
Barendt, Skye M; Sham, Lok-To; Winkler, Malcolm E (2011) Characterization of mutants deficient in the L,D-carboxypeptidase (DacB) and WalRK (VicRK) regulon, involved in peptidoglycan maturation of Streptococcus pneumoniae serotype 2 strain D39. J Bacteriol 193:2290-300
Hsueh, Yi-Huang; Cozy, Loralyn M; Sham, Lok-To et al. (2011) DegU-phosphate activates expression of the anti-sigma factor FlgM in Bacillus subtilis. Mol Microbiol 81:1092-108

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