Role of Tannerella forsythia NanH sialidase in epithelial cell attachment.

Tannerella forsythia is a Gram-negative oral anaerobe which contributes to the development of periodontitis, an inflammatory disease of the tooth-supporting tissues leading to tooth loss. The mechanisms by which this bacterium colonizes the oral cavity are poorly understood. The bacterium has been shown to express two distinct sialidases, namely, SiaHI and NanH, with the latter being the major sialidase. Bacterial sialidases can play roles in pathogenesis by cleaving sialic acids on host glycoproteins, destroying their integrity, and/or unmasking hidden epitopes on host surfaces for colonization. In the present study, we investigated the roles of the SiaHI and NanH sialidases by generating and characterizing specific deletion mutants. Our results showed that the NanH deficiency resulted in a total loss of sialidase activity associated with the outer-membrane and secreted fractions. On the other hand, the SiaHI deficiency resulted in only a slight reduction in the total sialidase activity, with no significant differences in the levels of sialidase activity in the outer membrane or secreted fractions compared to that in the wild-type strain. The results demonstrated that NanH is both surface localized and secreted. The NanH-deficient mutant but not the SiaHI-deficient mutant was significantly attenuated in epithelial cell binding and invasion abilities compared to the wild-type strain. Moreover, the NanH-deficient mutant alone was impaired in cleaving surface sialic acids on epithelial cells. Thus, our study suggests that NanH sialidase might play roles in bacterial colonization by exposing sialic acid-hidden epitopes on epithelial cells.

The OxyR homologue in Tannerella forsythia regulates expression of oxidative stress responses and biofilm formation.

Tannerella forsythia is an anaerobic periodontal pathogen that encounters constant oxidative stress in the human oral cavity due to exposure to air and reactive oxidative species from coexisting dental plaque bacteria as well as leukocytes. In this study, we sought to characterize a T. forsythia ORF with close similarity to bacterial oxidative stress response sensor protein OxyR. To analyse the role of this OxyR homologue, a gene deletion mutant was constructed and characterized. Aerotolerance, survival after hydrogen peroxide challenge and transcription levels of known bacterial antioxidant genes were then determined. Since an association between oxidative stress and biofilm formation has been observed in bacterial systems, we also investigated the role of the OxyR protein in biofilm development by T. forsythia. Our results showed that aerotolerance, sensitivity to peroxide challenge and the expression of oxidative stress response genes were significantly reduced in the mutant as compared with the wild-type strain. Moreover, the results of biofilm analyses showed that, as compared with the wild-type strain, the oxyR mutant showed significantly less autoaggregation and a reduced ability to form mixed biofilms with Fusobacterium nucleatum. In conclusion, a gene annotated in the T. forsythia genome as an oxyR homologue was characterized. Our studies showed that the oxyR homologue in T. forsythia constitutively activates antioxidant genes involved in resistance to peroxides as well as oxygen stress (aerotolerance). In addition, the oxyR deletion attenuates biofilm formation in T. forsythia.