The goal of this research is to characterize expression and assembly of Treponema denticola (Td) surface proteins that mediate interactions with host tissue, thereby gaining insight into mechanisms of periodontal disease. We focus on analysis of two Td protein complexes that directly affect host cells: the oligomeric Msp protein and the PrtP lipoprotein protease complex. The overall hypothesis is that Msp and PrtP are major contributors to Td cytopathic behavior in periodontal disease. Expression of Msp and the PrtP complex are interdependent. To understand their roles in microbe-host interactions, we must understand how these molecules are expressed and interact in the spirochete. We propose genetic analysis of structural features and interactions of these complexes and their components. Our approach is to define the mechanisms of interdependence between Msp and the PrtP complex in order to characterize their roles in microbe-host interactions.
Aim 1 : Determine requirements for assembly and activity of the PrtP complex. We will construct a series of specific mutations in the protease locus that will enable us to characterize posttranslational processing of PrcA and PrtP, expression of PrcB, localization of protease complex proteins and assembly of the active protease complex.
Aim 2 : Determine the role of Msp in biosynthesis of protease complex proteins, and the role of the protease in oligomerization of Msp. We will characterize interactions between Msp and protease operon proteins that influence expression and activity of each outer membrane complex.
Aim 3 : Characterize interactions of Td outer membrane components with epithelial cells. To further studies of host cell responses to Td challenge, we will characterize interactions between Msp and a putative receptor identified on epithelial cells and characterize early events in epithelial cell innate responses to the PrtP protease complex and Msp, with the goal of differentiating between roles of Msp and the protease complex in cellular responses to Td. This project, involving genetic and biochemical analyses of Td outer membrane complex expression and analysis of Td effects on host cells, will contribute to molecular analysis of microbe-host interactions in the oral environment. Our laboratory is uniquely positioned to conduct these studies. Completion of these Aims will contribute to both basic knowledge of spirochete molecular biology and to understanding of microbe-host interactions in chronic infections such as periodontal diseases. Public Health Significance: This project will provide information on how a key bacterium involved in initiation periodontal disease interacts with human cells. The studies will first characterize the process of protein complex assembly on the bacterial surface. Then we will determine which specific proteins on human cells these complexes bind to, and then assay cellular responses to bacterial protein complexes binding to cell surface receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE018221-02
Application #
7826782
Study Section
Special Emphasis Panel (ZRG1-MOSS-E (02))
Program Officer
Lunsford, Dwayne
Project Start
2009-05-05
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2012-04-30
Support Year
2
Fiscal Year
2010
Total Cost
$386,250
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biology
Type
Schools of Dentistry
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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Goetting-Minesky, M P; Godovikova, V; Li, J J et al. (2013) Conservation and revised annotation of the Treponema denticola prcB-prcA-prtP locus encoding the dentilisin (CTLP) protease complex. Mol Oral Microbiol 28:181-91
Cogoni, Valentina; Morgan-Smith, Alex; Fenno, J Christopher et al. (2012) Treponema denticola chymotrypsin-like proteinase (CTLP) integrates spirochaetes within oral microbial communities. Microbiology 158:759-70
Fenno, J Christopher (2012) Treponema denticola interactions with host proteins. J Oral Microbiol 4:
Bian, Jiang; Fenno, J Christopher; Li, Chunhao (2012) Development of a modified gentamicin resistance cassette for genetic manipulation of the oral spirochete Treponema denticola. Appl Environ Microbiol 78:2059-62
McDowell, J V; Frederick, J; Miller, D P et al. (2011) Identification of the primary mechanism of complement evasion by the periodontal pathogen, Treponema denticola. Mol Oral Microbiol 26:140-9
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Godovikova, Valentina; Goetting-Minesky, M Paula; Fenno, J Christopher (2011) Composition and localization of Treponema denticola outer membrane complexes. Infect Immun 79:4868-75
Goetting-Minesky, M Paula; Fenno, J Christopher (2010) A simplified erythromycin resistance cassette for Treponema denticola mutagenesis. J Microbiol Methods 83:66-8
Godovikova, Valentina; Wang, Hong-Tao; Goetting-Minesky, M Paula et al. (2010) Treponema denticola PrcB is required for expression and activity of the PrcA-PrtP (dentilisin) complex. J Bacteriol 192:3337-44