Mitochondrial Calcium Fluxes in Gingival Fibroblasts Exposed to the Major Outer Sheath Protein (Msp) of Treponema denticola

T. denticola Msp induces acute Ca2+ influx, then perturbs store-operated calcium kinetics in human gingival fibroblasts (HGFs). Objective: to determine the effect of Msp exposure of HGFs on the kinetics of Ca2+ uptake in mitochondria, which is known to ‘buffer’ cytosolic Ca2+ concentration ([Ca2+]c) and to regulate store-operated Ca2+ fluxes. Methods: Native Msp complex was enriched from T. denticola 35405. Cultured HGFs were loaded with Rhod-2/AM to report mitochondrial fluorescence intensity (FI) by confocal laser scanning photometry. Localization of Rhod-2 emissions to mitochondria was confirmed by double labeling with MitoTrackerGreen. Fluo-3 was used for reporting [Ca2+]c. For acute experiments, cells were treated with 30 µg/ml Msp or control vehicle; fluorescent emission images were collected immediately. To test the effect of Msp on store-operated Ca2+ fluxes, cells were pretreated with Msp or vehicle for 45 min, then exposed either to ATP or to thapsigargin (Tg) in Ca2+-free buffer. Results: Msp induced an acute transient of mitochondrial FI that reached peak amplitude in 2 min. In response to ATP, Msp-pretreated cells had a rapid, 4x mean rise in mitochondrial FI followed by slow Ca2+ release, similar to controls. In response to Tg, Msp-pretreated cells had a 4.5x mean rise in mitochondrial FI; control cells had only a 1.5x rise. In Msp-pretreated cells, the mean amplitude of mitochondrial FI was 4x higher and rate of increase much faster than that of cytosolic FI; in controls, the cytosolic FI amplitude was 2x higher than the mitochondrial FI amplitude. Conclusions: Mitochondria buffer cytosolic calcium following the acute rise in [Ca2+]c that is due to exposure to Msp. Rapid mitochondrial uptake of Ca2+ released from stores by agonists may partly account for the retarded [Ca2+]c kinetics typical of gingival fibroblasts exposed to T. denticola Msp. Supported by CIHR grant MOP-5619, Group and Strategic Training Grants