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Effects of treponema denticola on an oral epithelial cell model

University of British Columbia

Periodontal diseases are destructive inflammatory conditions of the tissues supporting the tooth. The inflammatory responses are induced by mixed bacterial infection leading to eventual destruction of the periodontal tissue. Oral spirochetes of the genus Treponema are one of the bacterial groups that are closely related to these diseases. This study investigated the interaction of a periodontal pathogen, T. denticola, its whole cells and outer membrane (OM) proteins with cultured eukaryotic cells. Cultured porcine periodontal ligament epithelial cells (PLE, a junctional epithelium model), and various cell lines were challenged with T. denticola ATCC 35405 whole cells or with the major OM protein (Msp) complex, gel purified Msp, and chymotrypsin-like proteinase (CTLP). Msp complex was isolated from whole T. denticola by detergent extraction, autoproteolysis, ultrafiltration and detergent removal. The location of the Msp and CTLP on T. denticola OM was studied. Hexagonal array was observable on negative-stained T. denticola OM under electron microscope where Msp probably constituted the main building block of the regular pattern together with the previously reported OM located CTLP. Native T. denticola Msp is an oligomer containing 53 kDa protein subunits which had porin activity in an artificial lipid bilayer. CTLP was previously characterized. Microscopic and modified ELISA assays showed that spirochetes adhered to PLE cells whether prefixed with glutaraldhyde (fixed PLE-FPLE) or not. Pre-treatment of T. denticola with the protease inhibitor TV-tosyl-L-phenylalanine chloromethyl ketone or phenylmethanesulfonyl fluoride blocked this binding and chymotrypsin-like activity. T. denticola intact or sonicated whole cells were cytotoxic to PLE cells and to several cell lines. T. denticola cytotoxicity was measured by microculture tetrazolium assay or by quantifying lactate dehydrogenase release upon cell lysis (LDH assay). T. denticola induced PLE cellular changes were observed by light microscopy, image analysis and electron microscopy. These included: transient cell size increase of non-detached PLE cells leading to confluency maintenance, membrane disruption, vacuolation, loss of cell contacts, loss of cell size control, cytoskeletal rearrangement, and apoptosis of PLE cells. Msp complex, Msp and CTLP could attach to PLE/FPLE and were found to be cytotoxic to PLE cells. Adherence of Msp was partially blocked by specific antibodies. Adherence of CTLP was partially blocked by serine protease inhibitors and was further inhibited by specific antibodies. Cytotoxicity of Msp and CTLP was inhibited by the same treatments that inhibited adherence. Standard patch clamp recording methods were used to study the effects of Msp complex on HeLa cells. Msp bound to several HeLa cell proteins, including a 65 kDa surface protein and a 96 kDa cytoplasmic protein. The Msp complex depolarized and increased the conductance of the HeLa cell membrane in a manner that was not strongly selective for Na+, K+ , Ca2+, and CI" ions. Cell-attached patches of HeLa cell membrane exposed to Msp complex exhibited short-lived channels with a slope conductance of 0.4 nS in physiologically normal saline. These studies show that T. denticola ATCC 35405 and its major OM protein elements, namely Msp and CTLP, could attach to a cell model that resembles junctional epithelium and could produce cytopathic and cytotoxic effects. It was hypothesized that the strong proteolytic activities of CTLP and pore-forming activity of the Msp complex were responsible for the cytopathic and cytotoxic effects of the putative periodontopathogen T. denticola on PLE cells.

Thesis/Dissertation

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2009-06-22

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http://hdl.handle.net/2429/9525

Dental Science

University of British Columbia

UBCV

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