The role of enolase as a virulence factor of Tannerella forsythia

Abstract

Objectives Periodontal disease is a chronic inflammatory disease in the periodontium caused by multi-species oral bacteria. The progression of the disease leads to destruction of periodontal tissues and alveolar bone loss. Tannerella forsythia is considered to be a periodontopathogen because it has been detected more frequently in periodontitis patients than in healthy subjects. In spite of the association of T. forsythia with periodontal disease, its virulence factors have not been fully studied. Although enolase presents mainly in the cytosol of many organisms, it is a multiple-functional moonlighting protein that exists in distinct locations. Some bacterial enolase has been reported to be transported to extracellular space. A secreted protein of bacteria is tightly correlated with host cell infection and might be an excellent candidate of virulence factors. In addition, recent research has demonstrated that some enolases function as a human plasminogen receptor. The purpose of this study was to elucidate the pathogenic potential of T. forsythia enolase. Methods The viability of T. forsythia was examined by measuring the growth curve and live/dead staining. To identify the secreted proteins, the bacteria were cultured in new oral spirochete (NOS) medium and the culture supernatants were obtained by centrifugation. The culture supernatants were filtered using a membrane filter with pore size of 0.22㎛. The collected culture supernatants were then concentrated by using a 3 kDa-Centricon and subjected to SDS-PAGE to determine the secreted proteins. The secreted proteins of T. forsythia were analyzed by MALDI-TOF. Recombinant T. forsythia enolase was expressed in Escherichia coli, recombinant proteins were purified and endotoxin decontamination was verified. To examine whether T. forsythia enolase is exposed on the bacterial surface, immunoblotting and flow cytometry analysis were performed using bacterial enolase antibody. The binding ability of T. forsythia enolase to plasminogen was analyzed, as well as the activating ability of the enolase-bound plasminogen. Fibronectin degradation by T. forsythia enolase-activated plasmin was analyzed using immunoblotting. To find out the effect of T. forsythia enolase on the proinflammatory responses in THP-1, the cells were treated with T. forsythia enolase for 24 h. The expression of IL-1β, IL-6, IL-8 and TNF-α was determined by real-time RT-PCR and ELISA. Results The secreted proteins were identified in the culture supernatants of a T. forsythia 24 h culture which was in the exponential phase. Enolase was identified as one of the secreted proteins. T. forsythia enolase was not only expressed on the bacterial surface but also secreted out of the bacteria. T. forsythia enolase bound to human plasminogen, and a plasminogen activator activated the enolase-bound plasminogen to plasmin. T. forsythia enolase-activated plasmin degraded fibronectin secreted from human gingival fibroblasts. T. forsythia enolase significantly induced IL-1β, IL-6, IL-8 and TNF-α in THP-1 cells at the gene and protein level. Conclusion T. forsythia enolase has a pathogenic potential to host by plasminogen binding and activation as well as induction of proinflammatory cytokines. These results suggest that T. forsythia enolase might induce tissue destruction and inflammatory response which could exaggerate inflammation, a characteristic of periodontitis.