FILAMENTOUS ACTIN DISRUPTION AND DIMINISHED INOSITOL PHOSPHATE RESPONSE IN GINGIVAL FIBROBLASTS CAUSED BY TREPONEMA-DENTICOLA

Previous reports have shown that Treponema denticola causes rearrangem ent of filamentous actin (F-actin) in human gingival fibroblasts (HGF) . The purpose of this investigation was to determine the effect of T, denticola on the generation of inositol phosphates (IPs) in relation t o a time course for F-actin disruption in HGF, Cultured HGF were expos ed to washed cells of T, denticola ATCC 35405 for 140 min. Changes in the fluorescence intensity of rhodamine-phalloidin-labeled F-actin in serial optical sections of single HGF were quantified by confocal micr oscopy image analysis. The percentage of cells with stress fiber disru ption was also determined by fluorescence microscopy, Challenge with T , denticola caused a significant reduction in F-actin within the first hour, especially at the expense of F-actin in the ventral third of th e cells, and a significant increase in the percentage of HGF with alte red stress fiber patterns, Significant concentration-dependent disrupt ion of stress fibers was also caused by HGF exposure to a Triton X-100 extract of T. denticola outer membrane (OM), IPs were measured by a r adiotracer assay based on the incorporation of myo-[H-3]inositol into IPs in HGF incubated with LiCl to inhibit endogenous phosphatases, HGF challenge with several strains of T. denticola and the OM extract of T. denticola ATCC 35405 resulted in a diminished accumulation of radio labeled IPs relative to both 15 and 1% fetal bovine serum, which serve d as strongly positive and background control agonists, respectively, The significantly diminished IP response to T, denticola ATCC 35405 oc curred within 60 min, concomitant with significant reduction of total F-actin and disruption of stress fibers, Pretreatment with the protein ase inhibitor phenylmethylsulfonyl fluoride, which had previously been found to block T, denticola's degradation of endogenous fibronectin a nd detachment of HGF from the extracellular matrix, had little effect on F-actin stress fiber disruption and the IP response, Therefore, in addition to its major surface chymotrypsin-like properties, T, dentico la expresses cytopathogenic activities that diminish the generation of IPs during the time course associated with significant cytoskeletal d isruption in fibroblasts.