In Vitro Evaluation of Virulence Characteristics of Three Clinical Isolates of Tannerella forsythia Isolated from Patients Enrolled at the UB Dental Clinic
MetadataShow full item record
T. forsythia is a member of “Red Complex” bacteria strongly implicated in the development of periodontitis. The knowledge of its biology and pathogenicity has mainly arisen from studies in the ATCC type type-strain 43037, and thus it is not known if its virulence varies with the type of strain infecting patients. Therefore, this study was the first attempt to identify clinically relevant strains of T. forsythia and to determine their virulence potential. To achieve the goals, plaque samples were collected from the periodontal pockets of chronic periodontitis patients enrolled at the UB School of Dental Medicine. Genomic DNA was isolated from bacterial cultures derived from single colonies of each isolate with sialidase positive activity and 16S rDNA sequencing was performed to confirm the identity of the isolates. The virulence of strains was then evaluated by comparing the ability of strains to induce proinflammatory cytokine gene expression in THP-1 derived macrophages. This study focused on three isolates, namely UB4, UB20 and UB22, out of 20 others with 100% sequence match of their 16S rDNA region with that of T. forsythia strain ATCC 43037. On preliminary examination, the three clinical strains UB4, UB20 and UB22 showed similarity with the ATCC43037 type strain in terms of electrophoretic profiles of their surface (S)-layer glycoproteins, and differences were observed with regard to the expression of the BspA protein, a well characterized bacterial virulence factor. The western immunoblotting data with an anti-BspA antibody raised against the ATCCC43037 BspA protein showed that while the clinical strains UB4 and UB22 expressed strongly reactive bands of expected size, the UB20 strain expressed a weakly cross-reactive truncated protein. These data prompted us to further assess the virulence potential of the strains via in vitro model of inflammation relying on proinflammatoty gene expression profiling in THP-1 derived macrophages. These data demonstrated that the two distinct strains (UB4 and UB20) differentially regulated proinflammatory cytokine genes in macrophage. Strikingly, the gene that was prominently regulated was the cxcl10 gene, encoding the chemokine CXCL10 (also known as IP-10). The expression of this chemokine encoding gene was approximately 20 fold higher in macrophages stimulated by UB20 compared to UB4 or ATCC43037 type strain. The enhancement of CXCL10 expression could not be attributed to the lack of a functional BspA protein alone in the UB20 strain since a BspA-deletion mutant in the ATCC43037 background did not induce CXCL10 expression. Studies currently ongoing in the lab are focusing on LPS moiety as a potential candidate in driving CXCL10 expression.