1016 Inflammasome Activation is a Critical Signaling Pathway in Clostridium difficile Toxin-Induced Intestinal Injury Which Requires Conservation of Specific Toxin Processing Events, Toxin Domains and Downstream Signaling

BACKGROUND: Intestinal electroneutral Na absorption involves the brush border (BB) Na/ H exchanger 3 (NHE3). In the intestine, impaired Na absorption results in decreased water absorption and diarrhea. Elevated levels of intracellular Ca2+ ([Ca]i) inhibit NHE3 activity in the intact intestine by ~40%. A role for apical PLC-γ in [Ca]i inhibition of NHE3 activity has been identified: 1) PLC-γ directly binds NHE3 and this interaction is necessary for [Ca]i inhibition of NHE3 activity and 2) the SH2 domains of PLC-γ may scaffold Ca2+ signaling proteins necessary for regulation of NHE3 activity. [Ca]i regulation of NHE3 activity also involves Src; however, the mechanism responsible for this effect remains undetermined. Since Src has been shown to directly bind PLC-γ, we tested the hypothesis that PLCγ links Src to NHE3 containing complexes and mediates Ca2+ regulation of NHE3 activity. METHODS: For the current study, 12 day post-confluent Caco-2/BBe cells grown on filters were transiently infected with a 3HA-NHE3 adenovirus construct and studied 48 hours later. NHE3 activity was measured by fluorimetry using the pH-sensitive dye, BCECF, in Caco-2/BBe cells treated with vehicle or the Src inhibitor, PP2 (10μM), in the presence or absence of 10μM carbachol (CCH) to elevate [Ca]i. Total cell lysates were obtained (with 0.1% Triton X-100) from Caco-2/BBe cells for sucrose density centrifugation (SDC) and coimmunoprecipitation (Co-IP). RESULTS: 1) CCH decreased NHE3 activity by ~40%, an effect that was abolished with PP2 treatment. We also determined whether Src exists in multi-protein complexes. 2) As determined by SDC with Western blot analysis, Src was observed in similar sized multi-protein complexes as both NHE3 and PLC-γ. 3) Co-IP studies in Caco-2/BBe cells demonstrated that Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased after elevated [Ca]i (less than 5 min post CCH), which was prior to dissociation of PLC-γ and NHE3 (after 5 min post CCH). 4) Finally, the association of Src and PLC-γ was not altered by blocking the SH2 domain of PLC-γ, a domain necessary for [Ca]i inhibition of NHE3. SUMMARY: This study demonstrated that Src 1) activity is necessary for [Ca]i inhibition of NHE3 activity, 2) exists in similar sized multi-protein complexes as NHE3 and PLC-γ, 3) associates dynamically with PLC-γ, but not NHE3. CONCLUSIONS: PLC-γ exerts lipase independent effects in its involvement in elevated [Ca]i inhibition of NHE3 activity by scaffolding Src into NHE3 containingmulti-protein complexes prior to dissociation of PLC-γ fromNHE3 and subsequent endocytosis of NHE3.