Taewan Kim
Kathleen Boesze-Battaglia
Bruce, J Shenker
| Taewan Kim | Kathleen Boesze-Battaglia | Bruce, J Shenker |
Taewan Kim1, Kathleen Boesze-Battaglia2, Bruce J Shenker2
1Periodontics, University of Pennsylvania, School of Dental Medicine; 2Basic & Translational Sciences, University of Pennsylvania
Introduction
A cohort of localized aggressive periodontitis (LAP) patients are characterized by persistence of Aa in high numbers in tooth surface associated biofilms, inflammation, and the destruction of periodontal tissue. Aa creates an environment suitable for other pathogens which collectively contribute to LAP pathogenesis. Our study focuses on one of three Aa toxins, the cytolethal distending toxin (Cdt), shown to cause cell cycle arrest, apoptosis in T cells and enhanced secretion of pro-inflammatory cytokines in macrophages. Cdt exerts its toxic effect through the active, CdtB subunit, which acts as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase, converting PIP3 to phosphatidylinsoitol-3,4-diphosphate (PI3,4P2) thereby depleting PIP3 pools. The aim of the study is to determine if shift from PIP3 to PI3,4P2 not only causes toxicity via pathological cell signaling but it also disrupts one of the host’s defense mechanisms, phagocytosis, and phago-lysosomal degradation of pathogens.
Methods
To determine if CdtB phosphatase activity alters PI pool, THP1 macrophages were treated with CdtBWT or CdtBR117A (phosphatase activity deficient mutant), or media (untreated control). Verification of PI pool changes were analyzed using multi-fluor confocal imaging and ELISA. To observe the changes in phagolysosome fusion, opsonized E. Coli with pH sensitive fluorochrome (pHrodo) was used and fluorescence was measured as a phagolysosome fusion. Lastly, to determine if CdtB phosphatase modulates macrophage susceptibility to bacterial infection, THP1 macrophages were treated with/without CdtBWT and inoculated for two hours with wild type Aa (D7S-SA) or mutant Aa lacking Cdt (D7S-SA CHE001) before lysis and bacterial culture. Colony forming units were counted and interpreted as a bactericidal function of THP1 macrophage.
Results
Confocal images and PI pool ELISA of CdtBWT treated THP1 macrophages showed increased PI3,4P2 level and decreased PI3,4,5P3 level, while the PI4,5P2 level was maintained unchanged. Phagolysosome formation study revealed decreased pHrodo fluorescence activity with CdtBWT treated THP1 macrophage as compared to CdtBR117A and untreated control. Aa survivability challenge with/without CdtBWT treatment against THP1 macrophage revealed that there is a significant decrease in bactericidal function of THP1 macrophage when it is treated with CdtBWT.
Conclusion
CdtB activity specifically and significantly increases intracellular PI3,4P2 levels and decreases host phagocytic function. Lastly, increased in Aa survivability with CdtBWT treatment showed that disruption in phagocytosis leading to reduced local host defense.