Akt Inhibition by RNAi reverses P. gingivalis-induced-survival of epithelial cells

Porphyromonas gingivalis, a Gram-negative anaerobe, successfully colonizes oral tissues and survives for extended periods in primary gingival epithelial cells (GECs). P. gingivalis infection of GECs also inhibits apoptosis induced by pro-apoptotic agents such as staurosporine. Activation of the anti-apoptotic phosphatidylinositol-3-kinase and its downstream signaling partner Akt appear to play a critical role for survival of infected GECs. Objective: This study investigated whether Akt protein has a significant functional importance in P. gingivalis-induced protection of GECs against cell death. Methods: Expression of endogenous Akt in GECs was knocked down using siRNA technology and examined by RT-PCR, Western-blot and immunofluorescence microscopy following 48h transfection. The Akt-deficient cells and siRNA-untreated cells were then infected with P. gingivalis at MOI 100 for 24h to determine the amount of cell death. The analyses were performed using Annexin-V and propidium Iodide double staining by fluorescence microscopy. In addition, siRNA treated and untreated cells were infected with P. gingivalis for 30 min or 1h. The samples were used as controls evaluating bacterial invasion efficiencies by immunofluorescence microscopy using NIH image analysis. Results: The siRNA treatment of GECs caused significant amount of reduction in the level of Akt. However, Akt-deficient cells had infection levels comparable to siRNA untreated cells suggesting Akt deficiency did not impact the internalization efficiency of P. gingivalis. On the other hand, there was approximately a 3-fold increase in the level of cell death in the infected cells that were deficient in Akt protein compared to siRNA untreated and P. gingivalis infected cells. Conclusion: Akt acts as an important survival mechanism for P. gingivalis induced anti-apoptotic activity in GECs. Identifying the specific signal transduction pathway(s) in P. gingivalis' colonization and survival in the oral tissues will be important for pharmacological intervention in treatment of periodontal diseases

This work supported by NIDCR R01DE016593 grant and UFCD-Student-Summer-Research-Fellowship.