The continuing long-term goal of research conducted under R01 DE015931 is to identify the in vivo conditions of the gingival sulcus that promote the growth and virulence of P. gingivalis, so triggering periodontitis. The hypothesis is that the role of P. gingivalis in this host-pathogen interaction is manifested through its responses to environmental conditions that prevail in the subgingival crevice. Like other bacteria, in P. gingivalis these responses are regulated by two-component signal transduction systems. In the simplest system the first component, a histidine kinase (HK), responds to a specific environmental stimulus by auto-phosphorylating a designated histidine residue in its sequence. Information is relayed by transfer of activated phosphate from the HK histidine to a designated aspartate residue in the cognate response regulator (RR). The activated RR then binds to promoters of its target genes (the regulon) and regulates their expression.
In Specific Aim 1 of this application the regulons of P. gingivalis RRs will be identified by ChIP-on-chip and complementary assays.
In Specific Aim 2, direct interaction between RRs and promoters of target genes will be confirmed by gel shift assays, and target promoter sequences will be analyzed. There is relatively limited information on the inducers of two-component systems compared to that available on the molecular mechanisms of gene regulation. A goal of this application is to fill this knowledge gap and identify environmental conditions to which P. gingivalis RRs respond. At this point in the project validated information on each RR and its regulon will be used to make informed decisions on potential conditions that trigger activation. Therefore, in Specific Aim 3 the environmental cues that activate individual HK-RR systems will be identified using reporter gene constructs. It is claimed that histidine kinases and response regulators are good therapeutic targets because of their multiple roles in bacterial physiology. Though the development of specific inhibitors has been slow, small molecule inhibitors for bacterial transcriptional regulators were recently discovered in high through-put screens of chemical libraries, reviving interest in these potential targets. In the future, such inhibitors may have a positive impact on general human health and influence treatments for periodontal disease replacing less effective and riskier antibiotic therapies. PROJECT NARRATIVE: The goal of this research is to identify conditions within the gingival pocket that promote the growth of bacteria that cause periodontitis. Bacterial responses to these conditions are regulated by signal relay systems comprised of two components: histidine kinases that sense changes in the environment and regulators that co- ordinate the response to these changes. Such two-component systems are prime targets for new inhibitor therapeutics that positively impact human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE015931-09
Application #
8299177
Study Section
Oral, Dental and Craniofacial Sciences Study Section (ODCS)
Program Officer
Lunsford, Dwayne
Project Start
2004-04-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2014-07-31
Support Year
9
Fiscal Year
2012
Total Cost
$484,967
Indirect Cost
$241,266
Name
Forsyth Institute
Department
Type
DUNS #
062190616
City
Cambridge
State
MA
Country
United States
Zip Code
02142
Boisvert, Heike; Lorand, Laszlo; Duncan, Margaret J (2014) Transglutaminase 2 is essential for adherence of Porphyromonas gingivalis to host cells. Proc Natl Acad Sci U S A 111:5355-60
Krishnan, Karthik; Duncan, Margaret J (2013) Role of sodium in the RprY-dependent stress response in Porphyromonas gingivalis. PLoS One 8:e63180
Scott, Jodie C; Klein, Brian A; Duran-Pinedo, Ana et al. (2013) A two-component system regulates hemin acquisition in Porphyromonas gingivalis. PLoS One 8:e73351
Stipp, Rafael N; Boisvert, Heike; Smith, Daniel J et al. (2013) CovR and VicRK regulate cell surface biogenesis genes required for biofilm formation in Streptococcus mutans. PLoS One 8:e58271
Klein, Brian A; Tenorio, Elizabeth L; Lazinski, David W et al. (2012) Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis. BMC Genomics 13:578
Duque, Cristiane; Stipp, Rafael N; Wang, Bing et al. (2011) Downregulation of GbpB, a component of the VicRK regulon, affects biofilm formation and cell surface characteristics of Streptococcus mutans. Infect Immun 79:786-96
Boisvert, Heike; Duncan, Margaret J (2010) Translocation of Porphyromonas gingivalis gingipain adhesin peptide A44 to host mitochondria prevents apoptosis. Infect Immun 78:3616-24
Nishikawa, Kiyoshi; Duncan, Margaret J (2010) Histidine kinase-mediated production and autoassembly of Porphyromonas gingivalis fimbriae. J Bacteriol 192:1975-87
Chen, Weilin; Kajiya, Mikihito; Giro, Gabriela et al. (2010) Bacteria-derived hydrogen sulfide promotes IL-8 production from epithelial cells. Biochem Biophys Res Commun 391:645-50
Duran-Pinedo, Ana E; Nishikawa, Kiyoshi; Duncan, Margaret J (2007) The RprY response regulator of Porphyromonas gingivalis. Mol Microbiol 64:1061-74