The Interaction Of SEC5 With InsP₃R Modulate Innate Immune Response Against Candida Albican

In healthy individuals,Candida species are commensal in nature and colonize mucous membranes and the skin.However,they can cause severe invasive disease when tissue homeostasis is disrupted.Invasive candidiasis,which comprises both candidemia and deep-seated tissue candidiasis,is the most common fungal disease among hospitalized patients.Candidemia is generally viewed as the more common type of the disease,and it accounts for the majority of cases included in clinical trials.Mortality among patients with invasive candidiasis is as high as 40%,even when patients receive antifungal therapy,and it is believed that only adjunctive immune-therapy could further improve the outcome of these infections.The first step in the development of an immune response to Candida species is the recognition of conserved pathogen-associated molecular patterns?PAMPs?by several families of PRRs,such as Toll-like receptors?TLRs?and C-type lectin receptors?CLRs?.C.albicans infection can activate phospholipase C-??PLC-??to generate 1,4,5-triphosphate?InsP₃?.InsP₃ binds to its receptors,Inositol 1,4,5-trisphosphate receptors?InsP₃R?,which release Ca²⁺from intracellular Ca²⁺stores to elevate cytosolic Ca²⁺concentrations([Ca²⁺]_c).Ca²⁺is a ubiquitous second messenger that controls multiple processes in immune cells,including ROS production,the secretion of cytokines and phagocytosis.Our yeast two-hybrid screen showed that the carboxyl terminus of InsP₃R targets three SEC 5 fragments.In this study,we invested the contribution of SEC5 and InsP₃R to the innate immune response against C.albican infection.Part I,Molecular interaction of InsP₃R with SEC5 in native cells.To confirm the yeast two-hybrid results,we performed a GST pull down assay and a coimmuno-precipitation assay,and found that endogenous SEC5 interacted with InsP₃R.Immuno-fluorescence confocal microscopy images indicate that endogenous SEC5 and InsP₃R3colocalizedat perinuclearin the cytoplasm.The fluorescence resonance energy transfer?FRET?between SEC5 and IP3Rs was measured by acceptor photobleaching in the fixed BMDM cells,and the results indicated that SEC5 and InsP₃R are co-localized within 10nm in those specific areas.To verify the SEC5-binding region in InsP₃R,the GST tagged type1 InsP₃R C-terminal fragments?GST-H1/H2/H3/H4?were used to pull down SEC5 from crude cellular extracts,and showed that only GST-H1 successfully bound SEC5.To delineate the interacting sequences in SEC5 that bind to InsP₃R-H1,the full length SEC5protein was divided into four his-tagged fragments,GST-H1 pull down assay showed that SEC5-2 and SEC5-4 fragments bound to H1.These results demonstrated that SEC5directly binds to InsP₃R in native cells.Part II,The interaction of SEC5 with InsP₃R modulates InsP₃R channel gating.To investigate whether SEC5-InsP₃R interaction could regulate InsP₃Rion channel activity,we performed single-cell Ca²⁺imaging in Human embryonic kidney?HEK?293 cells.We found that SEC5 overexpresstion increased carbachol-induced[Ca²⁺]_c elevation.In contrast,when SEC5 expression was knocked down by SEC5 specific siRNA,carbachol-induced[Ca²⁺]_c elevation was significantly decreasedcompared to cells treated with scramble RNA.Pre-incubating HEK293 cells with Araguspongin B?ARB,an InsP₃R specific inhibitor?,inhibited the stimulative effect of SEC5 carbachol-induced[Ca²⁺]_c elevation.To verify that SEC5-IP3R interaction is essential for the intracellular calcium release,we designed a cell-permeable peptide,H1-TAT and H1-scrambled-TAT.Co-immunoprecipitation assay showed that H1-TAT peptidedecreased the amount of binding InsP₃R to SEC5.Single-cell Ca²⁺imaging showed H1-TAT reduced carbacho-induced Ca²⁺elevation in both SEC5-overexpressing HEK293 cells and EGFP-overexpressing HEK293 cells.A patch clamp technique of nuclear membrane electrophysiology was performed,and we found that adding purified SEC5-2 recombinant peptide to the pipette solutionsignificantly enhancedchannel activity.These results demonstrate that SEC5 directly modulates InsP₃R Ca²⁺channel gating.Part III,SEC5 and InsP₃R interaction modulates macrophage phagocytosis.We verified that C.albican uptake triggers[Ca²⁺]_c elevation in BMDM.Depleting cytosolic Ca²⁺using the cell permeable Ca²⁺chelator,BAPTA-AM,or inhibiting Ca²⁺release from InsP₃R channel using ARB significantly inhibited C.albican-induced phagocytosis.These data suggest that InsP₃R-modulated[Ca²⁺]_c elevations play an important role in BMDM phagocytosis during C.albican infection.Enhancing SEC5 expression in BMDM increased the phagocytosis.In contrast,knocking down SEC5 expression in BMDM significantly decreased the activity of BMDM phagocytosis.Immunofluorescence images of super-resolution structured illumination microscopy?SIM?indicated the enriched SEC5 and InsP₃R co-localization in phagosomes.Using the InsP₃R1 antibody to precipitate SEC5from RAW264.7cells,we found that C.albican infection promoted the SEC5 and InsP₃R association.In addition,we found that disruption the interaction of SEC5 with InsP₃R with H1-TAT peptide significantly inhibited BMDM phagocytosis compared with that of H1S-TAT peptide.These results indicate that the interaction between SEC5 and InsP₃R regulates phagocytosis in BMDM.Part IV,SEC5 and InsP₃R interaction modulates TBK1-IRF3-signaling.We found C.albican infection increased p-TBK1 levels in RAW264.7cells,which caused the activation of TBK1-IRF3 signaling pathway.When we knocked down SEC5 expression in RAW264.7 cells,p-TBK1 levels and the amount of nuclear IRF3 were decreased.Further results showed that C.albican infection prompted GST-H1 to pull down greater amounts of SEC5 and TBK1 from RAW264.7 cell lysate,which indicates that C.albican infection promoted the formation of InsP₃R-SEC5-TBK1 complexes.In verifying the mechanism of the interaction,we found that InsP₃R-H1 competes with SEC5-RBD binding to SEC5-2and releasing SEC5-rbd bounding to TBK1,thus increasing TBK1 enzyme activity to subsequently activate IRF3 signaling pathway.Taking together,we identified a novel InsP₃R-interacting protein,SEC5,which can directly bind to the COOH terminal of InsP₃R and modulate InsP₃R channel gating.The interaction of InsP₃R with SEC5 not only promotes InsP₃R channel gating to elevate[Ca²⁺]_c,which is necessary for Ca²⁺dependent macrophage phagocytosis,but also enhanced TBK1 activity to activate the IRF3 dependent type I interferon innate immune response against fungal infection.Our findings provide an unidentified modulatory mechanism linking InsP₃Rto antifungal innate immune responses.