Master of Science (MSc)
Faculty of Science
Dr. Michael Suits
The progression of human chronic periodontitis within periodontal disease has been often linked to the presence of key pathogens, such as the presence of Treponema denticola, a late colonizer found in the deepening pockets of the gingival sulcus. This pathogen, as well as its associates Porphyromonas gingivalis and Tannerella forsythia, are classified as the ‘red complex’ and exist in a mixed biofilm during infection. It is within this biofilm state that previous transcriptomic analysis revealed a total of 126 genes that had an increase in their expression by 1.5-fold or greater in T. denticola. Three of these genes, Tde0626, Tde1701, and Tde2714, were further investigated for their importance in pathogenicity. Initial bioinformatics analyses suggested putative functions of the gene products to be a polysaccharide lyase for TDE0626, a bacteriocin-like protein for TDE1701, and a large formylglycine-generating enzyme (FGE) for TDE2714. Each of the targets were recombinantly expressed and purified for structural and functional analysis. Both TDE1701 and TDE2714 were able to be successfully crystallized and X-ray diffraction data were and analyzed. The processed data collected for TDE1701 resulted an I/σI value of 2.2 at 2.4Å and CC1/2 value of 0.823. Attempts thus far to obtain a structure of TDE1701 via molecular replacement have been unsuccessful. Similarly, selenomethionine data were collected for TDE2714, processed to 2.7Å resolution with an I/σI value of 2.2. However, the data quality was insufficient to determine appropriate phase information to characterize the structure. In both cases, further crystallization will be required for definitive structural characterization. In order to test the hypothesis that TDE0626 was a protein responsible for microbial dispersal, attempts at visualization of polysaccharide lyase activity for TDE0626 when incubated with isolates of mixed ‘red complex’ biofilm was attempted, but unsuccessful, and further investigation will be required. At present, functional analysis of TDE1701 has yet to be conducted due to the lack of functional hypotheses. Incubation of TDE2714 with a sulfatase consensus sequence (-LCTPSRA-) revealed an unanticipated modification to the serine rather than the cysteine residue. The data showed a mass loss of 2.02 g/mol by ESI-MS/MS on the serine residue suggesting that TDE2714 may be a serine modifying FGE. Further investigation will be conducted to confirm these results.
Nechacov, Jonah, "Structural and Functional Analysis of Three Upregulated Gene Products, TDE0626, TDE1701, and TDE2714 from Treponema denticola During Biofilm Formation" (2019). Theses and Dissertations (Comprehensive). 2139.
Available for download on Sunday, January 30, 2022