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  • Research article
  • Open Access

InsP3R-SEC5 interaction on phagosomes modulates innate immunity to Candida albicans by promoting cytosolic Ca2+ elevation and TBK1 activity

†Contributed equally
BMC Biology201816:46

https://doi.org/10.1186/s12915-018-0507-6

©  Yang et al. 2018

Received: 29 November 2017

Accepted: 20 March 2018

Published: 27 April 2018

Abstract

Background

Candida albicans (C. albicans) invasion triggers antifungal innate immunity, and the elevation of cytoplasmic Ca2+ levels via the inositol 1,4,5-trisphosphate receptor (InsP3R) plays a critical role in this process. However, the molecular pathways linking the InsP3R-mediated increase in Ca2+ and immune responses remain elusive.

Results

In the present study, we find that during C. albicans phagocytosis in macrophages, exocyst complex component 2 (SEC5) promotes InsP3R channel activity by binding to its C-terminal α-helix (H1), increasing cytosolic Ca2+ concentrations ([Ca2+]c). Immunofluorescence reveals enriched InsP3R-SEC5 complex formation on phagosomes, while disruption of the InsP3R-SEC5 interaction by recombinant H1 peptides attenuates the InsP3R-mediated Ca2+ elevation, leading to impaired phagocytosis. Furthermore, we show that C. albicans infection promotes the recruitment of Tank-binding kinase 1 (TBK1) by the InsP3R-SEC5 interacting complex, leading to the activation of TBK1. Subsequently, activated TBK1 phosphorylates interferon regulatory factor 3 (IRF-3) and mediates type I interferon responses, suggesting that the InsP3R-SEC5 interaction may regulate antifungal innate immune responses not only by elevating cytoplasmic Ca2+ but also by activating the TBK1-IRF-3 pathway.

Conclusions

Our data have revealed an important role of the InsP3R-SEC5 interaction in innate immune responses against C. albicans.

Keywords

  • Inositol 1,4,5-trisphosphate receptors (InsP3R)
  • Exocyst complex component 2 (SEC5)
  • Tank-binding kinase 1 (TBK1)
  • Antifungal innate immune response

Background

Candida albicans (C. albicans) is the most common type of infectious fungus and is usually considered to be harmless in healthy individuals. However, systemic fungal infection by C. albicans may be life-threatening in immunocompromised patients who have undergone surgery, chemotherapy, or organ or bone marrow transplantation [1, 2]. The body defends against systemic C. albicans infection by recruiting innate immune cells, especially macrophages and neutrophils. Early recognition of C. albicans invasion by immune cells is mediated by opsonic receptors, such as the Fcγ receptor, and dedicated pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and C-type lectins [3]. PRRs recognize microbe-specific pathogen-associated molecular patterns (PAMPs) and trigger multiple intracellular signaling pathways to orchestrate a pathogen-specific host immune response [3]. Previous studies have suggested that phospholipase Cγ2 (PLCγ2) is a key component in the C-type lectin-mediated immune response against fungal infection [4, 5]. Activation of PLCγ2 cleaves the membrane phospholipid PI(4,5)P2 to generate inositol 1,4,5-trisphosphate (InsP3), which then triggers the activation of the inositol 1,4,5-trisphosphate receptor (InsP3R) signaling pathway [6]. InsP3R is a major endoplasmic reticulum (ER) Ca2+ channel, and its activation promotes the release of Ca2+ from intracellular Ca2+ stores, resulting in an elevation of cytosolic Ca2+ concentrations ([Ca2+]c) [7, 8]. Vaeth et al. reported that such intracellular Ca2+ elevation in macrophages and dendritic cells is required for the activation of key downstream antifungal functions such as phagocytosis, cytokine production, and inflammasome activation [9]. These studies suggest that InsP3Rs may play an indispensable role in regulating antifungal immunity. However, the molecular mechanisms linking InsP3R to antifungal innate immunity have yet to be determined.

Three homologous isoforms of InsP3R are ubiquitously expressed in mammalian tissues, namely InsP3R1, InsP3R2, and InsP3R3 [7]. Their amino acid sequences are 60–80% identical, showing particularly strong homology in the C-terminal tails of the receptors [7]. Despite the fact that the InsP3-binding properties, ion permeation, and gating behavior of each isoform are distinct [7], it appears in general that the receptors require Ca2+ binding and that the bound Ca2+ modulates the temporal and spatial Ca2+ release from intracellular Ca2+ stores [10]. Each InsP3R monomer consists of ~ 2600 amino acids divided into three functional domains: the N-terminal InsP3-binding domain, the “regulatory”/“coupling” domain, and the C-terminal channel domain. The C-terminal tail is referred to as the gatekeeper region of the receptor [11–13]; although it is at some distance from the InsP3-binding domain, its interactions with several proteins have been shown to affect the sensitivity of the channel to InsP3. For example, proteins such as Bcl-2 and Bcl-XL have been shown to interact with the carboxyl terminus of InsP3R to regulate InsP3R-mediated Ca2+ signals that play important regulatory roles in lymphocyte (T cell and B cell) development and selection [14, 15]. To identify novel InsP3R-binding partners involved in antifungal innate immunity, we performed yeast two-hybrid screens of a human brain complementary DNA (cDNA) library using a bait corresponding to the cytoplasmic tail of the type 1 InsP3R carboxy terminus, identifying exocyst complex component 2 (SEC5) as a protein that binds to the InsP3R C-terminus.

SEC5, a member of the hetero-octameric exocyst complex, regulates the targeting and tethering of selective secretory vesicles to specialized dynamic plasma membrane domains [15]. Moreover, SEC5 modulates a number of physiological processes in addition to, but separate from, its role as a component of the exocyst [16]. It has been shown that SEC5 associates with phagosomes involved in the uptake of Staphylococcus aureus [17], and it is also involved in Tank-binding kinase 1 (TBK1)-dependent type I interferon innate immune responses against viral infections [18, 19]. Although type I interferons (IFN-α/β) have traditionally been the hallmark of immune response to bacteria and viruses, their importance in host defense against C. albicans was recently reported [20–24]. However, the underlying molecular mechanisms are not fully defined.

To this end, we hypothesized that the interaction between SEC5 and InsP3R may be involved in the regulation of antifungal innate immunity by activating the InsP3R-mediated Ca2+ signal and/or the TBK1-dependent immune response. Here, we found that the binding of SEC5 to InsP3R promoted its channel activity, increasing cytosolic [Ca2+]c in macrophages during the phagocytosis of C. albicans. We also showed that the InsP3R-SEC5 interaction promoted the recruitment of TBK1 by SEC5, thus forming an InsP3R-SEC5-TBK1 complex to modulate C. albicans-induced type I interferon innate immune responses.

Results

InsP3R-modulated Ca2+ is required for C. albicans phagocytosis in mouse macrophages

Innate immune cells eliminate pathogens by phagocytosis, a receptor-mediated, actin-dependent process that requires calcium (Ca2+) [6, 25]. To evaluate the role of InsP3R-modulated Ca2+ signals in phagocytosis, we first monitored changes in cytoplasmic [Ca2+]c during phagocytosis by bone marrow-derived macrophages (BMDMs). C. albicans were added to BMDM, and [Ca2+]c levels were recorded in individual macrophages for 10–20 min during the course of phagocytosis. We found that C. albicans transiently triggered [Ca2+]c elevation, which was followed by the uptake of C. albicans (Fig. 1a, Additional file 1: Movie S1). We examined BMDMs internalizing C. albicans for 15 min through immunofluorescence microscopy and found that InsP3R was localized to phagosomes engulfing C. albicans (Fig. 1b). It has been demonstrated that the uptake of large particles is a Ca2+-dependent process [26]. To determine if the InsP3R-mediated Ca2+ signal is involved in phagocytosis, we treated BMDMs with the membrane-permeable Ca2+ chelator BAPTA-AM. Pretreatment with BAPTA-AM inhibited C. albicans uptake (Fig. 1c). Consistently, inhibition of InsP3R activity by preincubation with araguspongin B (ARB, a specific InsP3R inhibitor) [27, 28] significantly inhibited C. albicans-induced phagocytosis (Fig. 1c). Together, these results suggest that phagocytosis of C. albicans triggered Ca2+ release through InsP3R, and InsP3R-modulated Ca2+ release is essential for the phagocytosis of C. albicans in BMDMs.
Figure 1
Fig. 1

InsP3R-modulated Ca2+ is required for C. albicans phagocytosis. a Confocal images depicting the changes of cytosolic Ca2+ concentrations ([Ca2+]c) in BMDMs incubated with C. albicans. The upper line depicts individual cellular [Ca2+]c values. The upper histogram depicts the summary of peak [Ca2+]c changes during phagocytosis (n = 32). Representative snapshots of cytoplasmic Ca2+ images were taken at different times during the Ca2+ imaging experiments. Red arrows indicate internalized C. albicans. b Confocal images showing the localization of InsP3R in BMDMs incubated with C. albicans. Asterisks indicate C. albicans. c Effect of InsP3R-modulated Ca2+ release on the phagocytosis of BMDM induced by C. albicans. Macrophages were co-cultured with C. albicans for 30 min. Representative live-cell video microscopy still images showing variation in the number of C. albicans engulfed by individual macrophages at 30 min. Arrows indicate the BMDMs that took up C. albicans. The phagocytosis index was calculated as the average number of C. albicans ingested by BMDMs during 30 min. Phagocytosis percentages represent the percentage of BMDMs undergoing C. albicans internalization during 30 min. Data are summarized as the mean ± SEM from three experiments (***p < 0.001)

Additional file 1:

Movie S1. Live-cell video microscopy movie showing the elevation of [Ca2+]c in BMDM cells during phagocytosis of C. albicans. BMDM cells were stained using Fura-2 AM. BMDMs and C. albicans were co-cultured for 30 min at 37°C in complete CO2-independent medium. The video shows that cytosolic Ca2+ levels in BMDM cells were transiently increased during phagocytosis of C. albicans. (MP4 2454 kb)

InsP3R interacts with SEC5 in native cells

InsP3R is a huge protein whose channel activity can be regulated by numerous interacting proteins [7, 8]. To determine if phagocytosis is regulated by protein interaction with InsP3R, we performed yeast two-hybrid screening with a human brain cDNA library. Using the type 1 InsP3R carboxy terminus as the bait, we identified the exocyst complex component SEC5 as an InsP3R-interacting protein (Additional file 2: Figure S1). Because SEC5 had previously been reported to associate with phagosomes [17], we speculated that the interaction between SEC5 and InsP3R may be involved in BMDM phagocytosis against C. albicans infection. We then validated the interaction between SEC5 and InsP3R in macrophages using co-immunoprecipitation. As shown in Fig. 2a, endogenous SEC5 was successfully co-precipitated from murine macrophage RAW264.7 cell lysates using antibodies against InsP3R3. Conversely, InsP3R3 was found in the immunoprecipitates with the SEC5 antibodies. Using immunofluorescence staining, we detected that endogenous SEC5 and InsP3R were partially co-localized in the cytoplasm of fixed resting BMDM cells (Fig. 2b).
Figure 2
Fig. 2

SEC5 interacts with InsP3R. a Interaction between SEC5 and InsP3R3 in RAW264.7 cell lysates. The cell lysates were immunoprecipitated (IP) with control rabbit IgG, anti-SEC5, or anti-InsP3R3 antibody. The lysate (Input) and IP samples were analyzed by western blotting with the indicated antibodies. b Confocal images showing SEC5 and InsP3R co-localization in BMDM cells (Pearson coefficient = 0.71). A representative resting BMDM cell was immunostained with anti-SEC5 (red) and anti-InsP3R3 antibodies (green). c Representative images of FRET donor (Alexa Fluor 488) and acceptor (Cy3). FRET pair intensities before and after Cy3 was photobleached are shown (left and middle columns). The histogram summarizes FRET efficiency in multiple regions of interest (data are summarized as the mean ± SEM; n = 16 for each pair of samples; ***p < 0.001). d Schematic illustration of the functional domains of rat InsP3R type 1 and GST fusion proteins (H1 to H4) of the InsP3R C-terminus (aa 2573–2749) used in SEC5 pull-down assays. A representative western blot depicting the GST pull-downs of SEC5 from RAW267.4 cell extracts is shown. The Coomassie blue-stained gel shows input GST-tagged H1 to H4 fragments. e Schematic diagram of recombinant SEC5 fragments used for testing interactions with InsP3R H1 helices (upper panel). Representative in vitro pull-down assays depict the interaction of different His-tagged SEC5 fragments with GST-InsP3R-H1

To demonstrate their physical interaction in native BMDM cells, an acceptor photobleaching fluorescence resonance energy transfer (FRET) approach was adopted [29, 30], which measures the dequenching of donor (fluorescein isothiocyanate (FITC)) emission by acceptor (Cy3) photobleaching. After photobleaching with light of the excitation wavelength for the acceptor, the fluorescence intensity in the donor channel increased in co-localized SEC5-InsP3R regions, but not in non-co-localized control pairs (Fig. 2c), indicating that SEC5 and InsP3R are within 10 nm of each other in those interacting regions. These results provide evidence that SEC5 interacts with InsP3R in intact cells.

The C-terminus of InsP3R contains four α-helices, denoted H1 to H4, which are of higher homology in all three InsP3R isoforms and have been shown to interact with Bcl-2 family proteins [14, 15, 31]. To verify that the InsP3R C-terminal helices interact with SEC5, we constructed glutathione S-transferase (GST)-tagged type 1 InsP3R C-terminal fragments and tested if they could pull down SEC5 from crude cellular extracts. These in vitro pull-down experiments showed that SEC5 was successfully pulled down by GST-H1 only from RAW264.7 cell extracts (Fig. 2d). To delineate the sequences in SEC5 that interact with GST-H1, we divided the full-length SEC5 protein into four His-tagged fragments (SEC5-1 to SEC5-4 in Fig. 2e). Recombinant GST-H1 peptides pulled down His-tagged SEC5-2 and SEC5-4 fragments, as detected with anti-His antibodies (Fig. 2e). This finding was further confirmed by in vitro direct protein interaction assays (Additional file 3: Figure S2). Our data demonstrated that SEC5 interacted with the C-terminus of InsP3R in native cells, and revealed their respective interacting sequences.

SEC5 modulates InsP3R channel gating

Because Bcl-2 and Bcl-XL have been shown to modulate InsP3R channel activity by binding to its C-terminal α-helices [14, 15, 32], we speculated that SEC5 might regulate InsP3R channel gating by binding to the H1 helix. To test this, we performed single-cell Ca2+ imaging with human embryonic kidney (HEK) 293 cells transfected with an enhanced green fluorescent protein (EGFP)-tagged SEC5 plasmid. Cells transfected with EGFP plasmids were used as a control. Twenty-four hours after transfection, HEK293 cells were loaded with Fura-2 AM, and then [Ca2+]c was recorded using a computer-controlled imaging system. We stimulated the cells with carbachol, an acetylcholine analog, to trigger InsP3R-mediated Ca2+ release [33, 34]. The SEC5-transfected HEK293 cells showed enhanced carbachol-induced Ca2+ release compared to that of cells expressing EGFP (Fig. 3a). In contrast, when endogenous SEC5 was knocked down by SEC5-specific small interfering RNA (siRNA) (siRNA-SEC5 in Fig. 3b), the carbachol-induced elevation of [Ca2+]c was significantly decreased compared with that of cells transfected with scramble siRNA (Fig. 3b). To confirm that carbachol induces Ca2+ release via InsP3R, we pretreated cells with the InsP3R inhibitor ARB. Carbachol-induced Ca2+ release was blocked by ARB when compared with that of untreated control cells (Additional file 4: Figure S3). These results suggested that SEC5 enhances the InsP3R-mediated elevation of [Ca2+]c.
Figure 3
Fig. 3

Effects of SEC5 on InsP3R channel activity. a, b Representative Ca2+ transients in HEK293 cells stimulated by Ca2+-free Hank’s balanced salt solution (white bar), followed by addition of 50 μM carbachol to the extracellular solution (black bar). a Cells were transfected with pEGFP-SEC5 or pEGFP-N1 vectors. Representative western blot depicting the expression of SEC5 in cells transfected with pEGFP-SEC5 or pEGFP-N1 vectors. Actin was used as a loading control. b Cells were transfected with scramble siRNA or SEC5-siRNA. A western blot depicting the knock down of SEC5 by siRNA is shown. Data are summarized as the mean ± SEM from three experiments with more than 100 GFP-positive cells analyzed. c Co-immunoprecipitation assays in RAW264.7 lysates demonstrated that recombinant H1 peptide competed with endogenous InsP3R3 for binding to SEC5. The bar chart summarizes the amount of InsP3R3 co-immunoprecipitated by anti-SEC5 antibodies. Data represent the mean ± SEM intensities of the co-immunoprecipitated InsP3R3 band from three experiments. d Representative Ca2+ traces depicting carbachol-induced ER Ca2+ release (black bar) under the influence of recombinant H1 peptide or H1 scramble peptide. Data are summarized as the mean ± SEM from three experiments with at least 100 cells analyzed. e Representative single-channel current traces activated by 500 nM InsP3 and 2 μM free Ca2+ in the pipette solution. Arrow indicates zero-current level, whereas downward deflection represents channel openings at pipette voltage = 40 mV. The channel open probability was enhanced by the addition of SEC52 peptide in the pipette solution. Data represent the mean ± SEM for at least three individual nuclear patches. (*p < 0.05, **p < 0.005, ***p < 0.001)

The H1 sequences showed higher homology (more than 90%) in all three InsP3R isoforms of different species (Additional file 5: Figure S4). To verify that the effect of SEC5 on InsP3R is due to their direct binding, we designed a cell-permeable recombinant peptide, H1-TAT, composed of the HIV-1 Tat protein transduction domain fused with 25 amino acids derived from the H1 motif of three different InsP3R subtypes (Additional file 5: Figure S4). A scrambled H1-TAT peptide (H1S-TAT) was synthesized and used as a control (Additional file 5: Figure S4). The effect of the peptides on the SEC5-InsP3R interaction was tested in a competitive co-immunoprecipitation assay by monitoring the formation of the endogenous SEC5-InsP3R complex using the SEC5 antibodies. We found that in the presence of H1-TAT, the formation of the endogenous SEC5-InsP3R complex was inhibited (Fig. 3c). Furthermore, H1-TAT treatment significantly attenuated the carbachol-induced Ca2+ release in SEC5- and EGFP-overexpressing HEK293 cells compared to the control or cells treated with the H1S-TAT scramble peptide (Fig. 3d). These results indicated that the SEC5-InsP3R interaction plays a role in increasing the agonist-induced Ca2+ release from the ER.

To provide direct evidence that SEC5-2 enhances InsP3R channel activity, nuclear membrane electrophysiology was performed in a chicken B-lymphoid cell line DT40 expressing recombinant homotetrameric rat type 3 InsP3R, in which the endogenous InsP3Rs have been genetically knocked out (DT40KO-rInsP3R3) [35–37]. With 500 nM InsP3 and 2 μM Ca2+ in the pipette solution, the InsP3R channel open probability (Po) was 0.07±0.024. Using the same ligand-stimulating conditions and in the presence of purified SEC5-2 recombinant peptides, the channel Po was significantly enhanced in a dose-dependent manner (Fig. 3e; 1 μM, 0.17±0.029 and 2 μM, 0.38±0.051). Taken together, these results demonstrated that SEC5 modulates InsP3R Ca2+ channel gating through direct interaction.

The SEC5-InsP3R interaction modulates macrophage phagocytosis

SEC5 has been reported to associate with phagosomes [17], and we speculated that the interaction between SEC5 and InsP3R may regulate BMDM phagocytosis against C. albicans infection. Thus, we used live-cell video microscopy to record BMDM behavior in real time [38]. Enhancing SEC5 expression in BMDM cells increased the phagocytosis index (average C. albicans uptake per BMDM cell) and the ratio of active BMDMs (Fig. 4a). In contrast, knocking down SEC5 expression in BMDMs significantly decreased their phagocytosis activity (Fig. 4a). These results indicated that SEC5 regulates the phagocytosis activity of BMDM cells. In addition, super-resolution structured illumination microscopy (SIM) images indicated that the co-localization of SEC5 and InsP3R was enriched in phagosomes when compared with that of resting BMDM and RAW264.7 cells (Fig. 4b, Additional file 6: Figure S5, and Additional file 7: Movie S2). Using type 1 InsP3R antibodies to precipitate SEC5 from RAW264.7 cells, we found that C. albicans stimulation increased the quantity of endogenous SEC5-InsP3R complexes (Fig. 4c), indicating that C. albicans stimulation promoted the association of SEC5 and InsP3R. In addition, we found that disrupting the SEC5-InsP3R interaction with H1-TAT peptides significantly inhibited BMDM phagocytosis when compared with that of cells treated with H1S-TAT (Fig. 4d), suggesting that the interaction between SEC5 and InsP3R contributes significantly to the phagocytosis of C. albicans in BMDM cells.
Figure 4
Fig. 4

SEC5-InsP3R interaction facilitates macrophage phagocytosis. a Effect of SEC5 expression on the phagocytosis of C. albicans by BMDMs. Cells were transfected with vectors encoding SEC5-EGFP, EGFP, SEC5-siRNA, or scramble siRNA, and cells were incubated with C. albicans for 30 min (MOI = 10). Phagocytosis index was calculated as the average number of C. albicans ingested by BMDM cells during 30 min. Phagocytosis percentage represents the percentage of BMDMs undergoing C. albicans internalization during 30 min. Data are summarized as the mean ± SEM from three experiments. b Confocal images depicting the localization of SEC5 and InsP3R in resting (upper panel) or activated (C. albicans-infected; lower panel) BMDM cells. c Enhanced co-immunoprecipitation of endogenous SEC5 using antibodies against InsP3R1 in RAW264.7 cell lysates treated with C. albicans (MOI = 10) for 1 h. Lysates from cells not infected with C. albicans were used as a negative control. Quantification of SEC5 immunoreactive bands, normalized to their respective InsP3R1 bands, is shown in the right panel. Bars represent fold change in SEC5 intensity (mean ± SEM from three experiments). d Effect of recombinant cell-permeable H1-TAT peptide on BMDM phagocytosis. BMDM cells were pretreated with H1-TAT or scramble H1S-TAT (2 μM) for 6 h using the same protocol as described in a. e Infected mouse kidney CFU assay. Groups of C57B/L6 female mice were injected via lateral tail vein with 1 × 105 CFU of C. albicans (SC5314) and treated with the indicated peptides. Kidney CFU assays (n = 3 × 3) were performed 24 h after infection. In all panels in a and d, unless otherwise noted, data represent the mean ± SEM from at least three independent experiments (*p < 0.05, **p < 0.005, ***p < 0.001)

Additional file 7:

Movie S2. Z stack of confocal images depicting the relocalization of SEC5 and InsP3R in activated BMDM cells. Related to Fig. 4b. (AVI 1355 kb)

To validate our in vitro findings that the SEC5-InsP3R interaction is involved in antifungal innate immunity, we examined the effects of the H1-TAT peptide in a murine model of systemic candidiasis. C57BL/6 mice were injected via the lateral tail vein with 1 × 105 colony-forming units (CFUs) of C. albicans, followed by intraperitoneal (i.p.) injection with the indicated cell-permeable peptides. Compared to the controls, the kidney fungal burdens were significantly higher in the groups treated with H1-TAT or SEC5-2 peptides, while co-injection of SEC5-2 and H1-TAT neutralized their respective toxic effects (Fig. 4e). These data indicated that the SEC5-InsP3R interaction promotes innate immunity against C. albicans infection in vivo, while disturbing their interaction weakens innate immunity against C. albicans.

SEC5 and TBK1 regulate IFN-β production in response to C. albicans

Type I interferon (IFNα/β) is crucial for responding to infections through its antiviral properties and by its function in coordinating the immunocompetent cells involved in antiviral or antibacterial immunity [20]. Recent studies have shown that IFN-β produced in response to C. albicans infection is crucial for defending against candidiasis [21–24]. SEC5 has been shown to modulate the TBK1-IRF-3 signaling-dependent type I interferon responses against viral infections [18, 19]. To determine if the SEC5-TBK1-IRF-3 signaling cascade is involved in C. albicans infection, we analyzed the phosphorylation of TBK1 (p-TBK1) and IRF-3 nuclear translocation during fungal infection, the results of which showed that C. albicans stimulation increased p-TBK1 levels in RAW264.7 cells (Fig. 5a). It has been shown that activated TBK1 phosphorylates IRF-3 and promotes its nuclear translocation [17]. In agreement with this, we found enhanced IRF-3 levels in the nuclear fraction of RAW264.7 lysates (Fig. 5a). Conversely, when SEC5 expression was knocked down by small hairpin RNA (shRNA), TBK1 phosphorylation and the nuclear fraction of IRF-3 remained unchanged during C. albicans stimulation (Fig. 5a). We also assessed the production of IFN-β upon C. albicans stimulation by quantitative real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) (Fig. 5b and c). The production of IFN-β transcripts and cytokines was increased upon fungal infection, while knocking down SEC5 inhibited the production of INF-β (Fig. 5b and c). Furthermore, overexpression of SEC5 in RAW264.7 significantly enhanced the production of INF-β (Fig. 5d), while treating RAW264.7 cells with the TBK1 inhibitor MRT67307 markedly inhibited the production of IFN- β (Fig. 5e). Altogether, these data demonstrated that C. albicans infection activates TBK1, and the subsequent nuclear translocation of IRF-3 promotes INF-β production in a SEC5-dependent manner.
Figure 5
Fig. 5

SEC5 and TBK1 modulate C. albicans-induced IFN-β production. a Representative immunoblots showing the amount of phosphorylated TBK1 (p-TBK1) in the cytosolic fraction and IRF-3 in the nuclear fraction from RAW264.7 cells at different time points of C. albicans stimulation (MOI = 10). Cells were pretreated with SEC5-shRNA or scramble shRNA (NC-shRNA), and western blots for SEC5 after shRNA treatments are shown for comparison. b Expression of IFN-β in C. albicans-infected RAW264.7 cells pretreated with SEC5-shRNA or scramble shRNA was measured by quantitative real-time PCR at the indicated time points. Data were normalized to the expression of β-actin. c BMDMs were transfected with SEC5-EGFP or EGFP vectors for 48 h, stimulated with C. albicans for 4 h, and then IFN-β production was analyzed by ELISA. d BMDMs were treated with SEC5-siRNA or scramble siRNA for 48 h, stimulated with C. albicans for 4 h, and then IFN-β production was analyzed by ELISA. e BMDMs were treated with 20 nM MRT67307 for 2 h, stimulated with C. albicans for 4 h, and then IFN-β production was analyzed by ELISA. Data are expressed as the mean ± SEM from at least three independent experiments (***p < 0.001)

InsP3R regulates SEC5-TBK1-IRF-3 signaling

As C. albicans infection promoted the SEC5-InsP3R interaction (Fig. 4c), we examined the consequences of InsP3R regulation on the SEC5-TBK1 complex and TBK1 activity. We performed GST pull-down assays using a recombinant InsP3R1 H1 helix as the bait. The results indicated that C. albicans infection increased the amount of SEC5 and TBK1 pulled down by GST-H1 from RAW264.7 cell lysate (Fig. 6a). Furthermore, we examined BMDMs internalizing C. albicans for 15 min by immunofluorescence microscopy and found that InsP3R and TBK1 co-localized on phagosomes (Fig. 6b and Additional file 8: Figure S6). These results suggest that C. albicans infection promotes the SEC5-InsP3R-TBK1 interaction, which may subsequently activate the TBK1-IRF-3 signaling cascade.
Figure 6
Fig. 6

SEC5-InsP3R interaction influences TBK1-IRF-3 signaling cascade. a In vitro pull-down of SEC5 and TBK1 in C. albicans-stimulated (MOI = 10) RAW264.7 cell lysates by GST-H1 or GST. Quantification of SEC5 and TBK1 immunoblot signal intensities from GST-H1 pull-down experiments is shown. Data are summarized as the mean ± SEM from three separated experiments. b Confocal images depicting the co-localization of TBK1 and InsP3R on phagosomes (Pearson coefficient = 0.87). White arrow indicates phagosome. c Immunoblots showing TBK1 and p-TBK1 immunoprecipitated using V5 antibodies in the presence of H1-TAT or H1S-TAT recombinant peptides. d Immunoblots depicting the phosphorylation of recombinant IRF-3-C in the presence of H1 peptides. e Immunoblots of in vitro pull-down assays using GST-SEC5-rbd and purified His-SEC5-2. Purified SEC5-rbd inhibited the interaction of SEC5-2 peptides with InsP3R-H1. f Immunoprecipitation examining the interaction of V5-BTK1 and purified His-SEC5-N in the presence of H1-TAT or H1S-TAT recombinant peptides

We performed co-immunoprecipitation assays to further validate that the SEC5-InsP3R interaction is critical for SEC5-InsP3R-TBK1 complex formation. In COS-7 cells expressing V5-tagged SEC5, addition of recombinant H1 peptides dose-dependently enhanced the amount of co-immunoprecipitated TBK1 and p-TBK1, whereas H1 scramble peptides did not (Fig. 6c). Intriguingly, InsP3R-H1-enhanced formation of the TBK1-SEC5 complex correlated with enhanced TBK1 activity, as measured by in vitro phosphorylation of recombinant IRF-3-C, a direct TBK1 substrate [39, 40] (Fig. 6d). TBK1 was previously reported to interact directly with the Ral-binding domain (rbd) of SEC5 (SEC5-rbd, amino acids (aa) 1–120), which has been shown to bind to SEC5 [18], while the region of SEC5 responsible for the SEC-rbd interaction was unknown. Using GST-SEC5-rbd and purified His-SEC5-2 to perform in vitro pull-down assays, we found that SEC5-rbd bound to SEC5-2, and their interaction prevented InsP3R-H1 from binding to SEC5-2 (Fig. 6e). We then hypothesized that InsP3R-H1 competes with SEC5-rbd to bind to SEC5-2, which releases SEC5-rbd and allows it to bind to TBK1, thereby increasing TBK1 enzyme activity to subsequently activate the transcription factor IRF-3. To test this hypothesis, we purified the N-terminus of SEC5 (aa 1–440, containing the rbd and the InsP3R-binding domain) and V5-TBK1. Immunoprecipitation assays showed that peptide H1 promoted the binding of TBK1 to SEC5-N, unlike the H1 scramble peptide. These data indicate that InsP3R regulates the interaction of SEC5 and TBK1. However, neither the binding of SEC5 with InsP3R-H1 nor the enzyme activity of TBK1 was Ca2+ dependent (Additional file 9: Figure S7).

Discussion

Invasive candidiasis is the most common cause of systemic fungal infection in hospitalized immunocompromised patients [1, 2]. C. albicans infections are rare in healthy individuals, as the infection triggers human host immune mechanisms when it interacts with macrophages, neutrophils, or dendritic cells that subsequently eliminate the invading fungus by phagocytosis [3]. Although the underlying mechanisms for phagocytosis are elusive, it is believed that the kinetics and efficiency of the formation and maturation of phagosomes are promoted by both localized and global cytoplasmic Ca2+ elevations that drive the remodeling of F-actins [25, 41, 42]. It has been shown that C. albicans infection promotes the activation of PLCγ, which transiently mobilizes Ca2+ from the endoplasmic reticulum (ER) and subsequently opens the store-operated Ca2+ entry (SOCE) channel to provide a sustained Ca2+ signal for initiating host innate immunity [43, 44]. Intriguingly, Vaeth et al. demonstrated that deletion of SOCE did not significantly affect the innate immunity of macrophages and dendritic cells [9]. We found that InsP3R localized on phagosomes and mediated Ca2+ release from the ER, and these processes were necessary for macrophage phagocytosis (Fig. 1c). In addition to functioning as a Ca2+ storage organelle, it has been reported that the ER also provides membranes to phagosomes for the internalization of pathogens by macrophages during phagocytosis [45, 46]. Although the fusion of ER membranes to phagosomes is controversial [47], ER has been observed in close proximity to the base of the phagocytic cup by electron microscopy [45, 48], indicating that the ER or the associated InsP3R plays an indispensable role in fungus-induced phagocytosis.

Here, we have proposed a unique molecular mechanism that regulates the host innate immune response against C. albicans fungal infections. We showed that the N-terminal region of SEC5 interacts with the α-helix of the InsP3R C-terminus (H1) and their interaction regulates both ER Ca2+ release and type I interferon formation.

The three mammalian InsP3R isoforms are 60–70% identical in sequence and share a common domain structure [7, 8]. It has been proposed that the C-terminal cytoplasmic region of the InsP3R channel functions as the gatekeeper [11–13]. Several proteins, such as Bcl2, Bcl-XL, polyglutamine expanded huntingtin (Httexp), huntingtin-associated protein 1A, and protein kinase C substrate 80 K-H (80 K-H), have been shown to interact with this region and regulate the channel activity of InsP3R [8]. Here, we found that SEC5 directly binds to the α-helix H1 of InsP3R (rat InsP3R1: aa 2571–2606), which provides a mechanistic linkage between InsP3R-mediated Ca2+ levels and antifungal innate immunity. A high-resolution cryo-electron microscopy (cryo-EM) structure of rat type 1 InsP3R was recently solved [13], which demonstrates that the sixth transmembrane (TM6) helix, which we have referred to as H1 here, extends into the cytoplasm. In the structure, the phenylalanine at aa 2585 (F2585) appears to form the channel gate. It is not obvious how SEC5 could access this site. Nevertheless, interaction with such a critical site in the channel would be predicted to influence channel gating, as we observed.

Type I interferons (IFNs-I) are classical antiviral cytokines, which can also be induced by intracellular and extracellular bacterial invasions through the activation of the interferon regulatory factors (IRF-3, IRF-5, and IRF-7) [49–51]. It has been recently reported that fungal infections also induce IFNs-I production, and defective IFNs-I responses render both mice and humans susceptible to systemic candidiasis [21–24]. Several PRRs, such as the toll-like receptors (TLR7 and TLR9), Dectin-1, and the interferon regulatory factor family, namely, IRF-1, IRF-3, IRF-5 and IRF-7, have been reported to regulate IFN-β production upon C. albicans infection [21–23]. Although TBK1 can directly phosphorylate the IRF-3, IRF-5, and IRF-7 transcription factors, enhancing their transactivating potential, the role of TBK1 in antifungal innate immunity is elusive. Chien et al. found that the RalB-SEC5-TBK1 activation complex supported the host defense response, triggered by either extracellular double-stranded RNA (dsRNA) or exposure to Sendai virus [18]. Similarly, we found that the InsP3R-SEC5-TBK1 complex regulated the production of IFN-β in response to C. albicans stimulation, which was independent of RalB (Additional file 10: Figure S8). Although we focused on the ability of InsP3Rs to release Ca2+ from intracellular stores, InsP3Rs may also regulate associated proteins independently of their ability to release Ca2+. Here, we found that InsP3R-H1 and SEC5-rbd compete for a common binding site on SEC5 (here referred to as SEC5-2), suggesting that the direct interaction between InsP3Rs and SEC5 released the SEC5-rbd domain, thus forming an “open conformation” for TBK1 docking and facilitating the activation of TBK1. We also found that the enzymatic activity of TBK1 was not Ca2+ dependent. In agreement with this, ER stress has been shown to augment transcription of select IRF-3-regulated genes, but increasing cytoplasmic Ca2+ alone was insufficient to phosphorylate IRF-3 [52], indicating that the direct interaction of InsP3R and SEC5 may be involved in the activation of IRF-3. Interestingly, Bourgeois et al. reported that Candida-induced IFN-β release required phagocytosis [22]. In the present study, we have shown that the interaction of SEC5 with InsP3R modulates both C. albicans-induced phagocytosis and IFN-β production.

Conclusions

In summary, we have defined the mechanistic basis by which InsP3R-mediated Ca2+ release regulates innate immunity against C. albicans and revealed that the InsP3R-SEC5 effector complex is required for the proximal activation of TBK1 in response to C. albicans infection. The formation of the InsP3R-SEC5 complex not only promoted InsP3R-mediated [Ca2+]c elevation, which is necessary for macrophage phagocytosis, but also enhanced TBK1 activity to activate the IRF-3-dependent type I interferon innate immune response against fungal infection. Our investigation has suggested a novel molecular mechanism to regulate the innate immune response against C. albicans infection.

Methods

Yeast two-hybrid screening

The yeast two-hybrid experiment was performed by Jun Yang following the protocol as previously described [53] in Dr. J. Kevin Foskett’s laboratory at the University of Pennsylvania, Perelman School of Medicine. Briefly, a cDNA fragment encoding the carboxyl terminus of rat InsP3R1 aa 2590-C was cloned into the pLexA vector (Clontech) to be used as a bait to screen a human fetal brain cDNA library (Invitrogen). Library screening was performed following the company’s instructions. From 6 × 106 primary transformants we discovered 14 positive colonies. The library plasmids were recovered using a yeast plasmid extraction kit (Qiagen). The prey sequences were identified by the DNA sequence facility of the University of Pennsylvania. Positive plasmids were confirmed by co-transformation of the library and bait plasmids for comparison with non-specific interactions by co-transformation of the library plasmid and the empty pLexA vector.

Cell culture

Murine macrophage RAW264.7 and human embryonic kidney (HEK) 293 cells were purchased from ATCC, and grown in Dulbecco’s modified Eagle’s medium (DMEM) and minimum essential medium containing 10% fetal bovine serum (FBS), respectively. Chicken B-lymphoid cell line DT40 cells were derived from Dr. J. Kevin Foskett’s laboratory. They were maintained in RPMI 1640 containing 10% FBS, 1% chicken serum, 1% Antibiotic-Antimycotic at 37°C, 95% humidity, 5% CO2.

Mouse macrophage differentiation and culture

Primary cultures of BMDMs from C57BL/6 mice were prepared as previously described [54]. In brief, bone marrow cells were aseptically collected from the female mice aged 6–8 weeks by flushing the euthanized mice’s leg bones with phosphate-buffered saline (PBS). The cells were then incubated in red cell lysis buffer (155 mM NH4Cl, 10 mM NaHCO3, and 0.1 mM ethylenediaminetetraacetic acid (EDTA)) before centrifuging. The cells were cultured for 7 days in DMEM containing 20% FBS, 100 μg/ml streptomycin, 55 mM mercaptoethanol, 100 U/ml penicillin, and 30% conditioned media from L929 cells expressing macrophage colony-stimulating factor (M-CSF). Non-adherent cells were removed. Flow cytometry analysis indicated that the harvested cell population contained 86–95% CD11b + F4/80 + cells.

Plasmid construction

Human full-length SEC5 cDNA and IRF-3 cDNA were purchased from YR Gene. Plasmids were constructed according to standard protocols, i.e., by cloning the cDNA corresponding to the residues of interest into vectors including pET28a, pCDNA3.1-V5-His (Life Tech), pECFP-N1 (Clontech), and pGEX-6P-1 (GE). Restriction enzymes for cloning were purchased from New England Biolabs or Takara.

C. albicans infection model and fungal burden

For in vivo C. albicans infection, a group of C57B/L6 female mice aged 6–8 weeks were injected via lateral tail vein with 1 × 105 CFUs of C. albicans in 200 μl sterile saline. One hour later, the mice were injected i.p. with H1-Tat peptide or H1-scrambled-TAT peptide at 2 mg/kg, or with SEC5-2 peptide at 50 μg/kg. Kidney CFU assays were performed 2 days after infection. The kidneys were removed, weighed, and homogenized in sterile PBS in a tissue grinder. The number of viable Candida cells in the tissues was determined by plating serial dilutions on SD agar plates. The CFUs were counted after 24 h of incubation at 30°C and expressed as CFUs per gram of tissue.

Recombinant protein purification from E. coli and GST pull-down assays

Glutathione S-transferase (GST) fusion proteins were expressed in BL21 (DE3)pLysS E. coli and purified with Glutathione Sepharose 4B following the manufacturer’s protocol (GE). His-tagged proteins were expressed in BL21 (DE3)pLysS E. coli using pET28A vectors and purified using Ni-columns according to the manufacturer’s protocol (Qiagen). Purified proteins were dialyzed against 1 × Dulbecco’s PBS (DPBS) buffer. Unless specified, pull-down assays and western blotting were performed according to standard protocols. Cellular lysates, prepared using 1 × DPBS containing 1% Triton X-100, 0.5–1 mg/ml total cellular protein (dependent on expression), in a total volume of 1 ml, were mixed with 20 μg GST fusion protein bound to beads and incubated at 4°C while rotating for 2 h. The beads were spun down and washed thrice using lysate buffer. Bound proteins were eluted using 1 × loading buffer and detected by western blotting.

Co-immunoprecipitation

RAW264.7 cells, stimulated with C. albicans (multiplicity of infection (MOI) = 10) or not, were lysed using 1 × DPBS containing 1% Triton X-100. Ten micrograms of rabbit InsP3R1 or InsP3R3 antibodies, or an equivalent amount of rabbit immunoglobulin G (IgG) isotype control (Sigma; #18140), was conjugated with 30 μl of a 50% slurry of protein G agarose resin according to the manufacturer’s instructions (Yeasen; #36405ES08). Then, the antibody-conjugated agarose resin was incubated with protein lysates (500 mg of protein) with gentle agitation for 2 h. The beads were retained after three washes with PBS. Next, 1 × loading buffer was added to dissociate immunoprecipitates, and western blotting was performed accordingly.

Acceptor photobleaching fluorescence resonance energy transfer (FRET)

Acceptor photobleaching FRET was measured by confocal microscopy (A1R, Nikon, Japan) according to a previously described method [29, 30]. Energy transfer was detected as an increase in donor fluorescence (Alexa Fluor 488) following the complete photobleaching of the Cy3 acceptor molecules. The amount of energy transfer at co-localization areas was calculated as the percentage increase in donor fluorescence, and non-co-localization areas were selected as negative controls. The imaging sequence was as follows: First, donor and acceptor channels were sequentially scanned at low laser intensity before the acceptor was bleached with the 561-nm laser line at 70% intensity, and the donor and acceptor channels were scanned again at low laser intensity. Acquired images were exported to 8-bit TIFF format, and FRET efficiency was analyzed using Nikon NIS-Elements software.

Immunofluorescence staining

BMDM cells at 7 × 105 were placed on 12-mm coverslips (Fisher Scientific, UK) and incubated with C. albicans at 37°C in 5% CO2 for 60 min. The cells were fixed using 4% paraformaldehyde (PFA) in DPBS, followed by blocking and permeabilization with 0.1% IGEPAL (Sigma, St. Louis, MO, USA) in DPBS with 2% bovine serum albumin (BSA) (Amresco, Solon, OH, USA). Primary antibodies, diluted in 2% BSA, were applied at 4°C overnight. The cells were subsequently washed four times with DPBS before being incubated with the appropriate secondary antibodies (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) diluted in 2% DPBS. The coverslips were washed four times with DPBS before being mounted using Vectashield. Confocal images were acquired with a Nikon A1R confocal system. Pearson’s correlation coefficients were calculated using the NIS-Elements AR 4.5 software to show the overlap between Alexa Fluor 488 and Cy3 at each co-localization area.

Calcium imaging

HEK293 cells were seeded in 35-mm dishes and incubated with 2 μM Fura-2 AM (Invitrogen, Thermo Fisher Scientific, MA, USA) in Hank’s balanced salt solution (HBSS, Sigma, St. Louis, MO, USA) containing 0.04% Pluronic F-127 (Sigma) for 30 min, in normal culture media, at 37°C and 5% CO2. The cells were then washed and continuously perfused with HBSS containing 1.8 mM CaCl2 and 0.8 mM MgCl2, pH 7.4. In the experiments to measure calcium imaging, the cells were perfused with HBSS containing 50 μM carbachol. Fura-2 was alternately excited at 340 and 380 nm, and the emitted fluorescence, filtered at 510 nm, was recorded using Nikon NIS-Elements software. Individual cells were chosen as different regions of interest (ROIs), and the ratio (340/380) of each ROI in all time frames was obtained after background correction. Dye calibration was performed, and ratios were then converted to cytosolic Ca2+ concentrations by the Grynkiewicz equation: [Ca2+] = Kd*β*(R-Rmin)/(Rmax-R) [55].

Nuclear patch-clamp recording of InsP3R

Nuclear patch-clamp recording was performed as previously described [35–37]. Briefly, DT40TKO-InsP3R3 nuclei were isolated by homogenizing cells in nuclei isolation solution. Then, 80 μl of cell homogenate was added to 2 ml of bath solution. Single InsP3R channels were detected using a HEKA EPC-10 amplifier (HEKA Elektronik) and pipettes filled with pipette solution. Peptides were directly added to the patch pipette solution. Single-channel analysis was performed using QuB software (University of Buffalo).

Transfection with siRNA and plasmids

The cells were seeded in 6-well plates (1 × 106 cells/well) in 1 ml DMEM containing 10% FBS before being transfected with 120 pmol siRNA against SEC5, non-targeting control siRNA, or 2 μg SEC5-EGFP or pcDNA-EGFP control plasmids, using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. The cells were lysed 48 h after transfection, and the relative intensities of SEC5 protein were determined by western blotting.

Phagocytosis assays

Phagocytosis assays were performed using the standard protocol with modifications [38]. Briefly, C. albicans (SC5314) cells were added to 1 × 106 BMDM macrophages in glass-based imaging dishes at an MOI of 5. Video microscopy was performed using a Nikon ECLIPSE Ti microscope in a 37°C chamber, and images were captured at 20-ss intervals over a 1-h period using an EMCCD camera. Live-cell video microscopy enables internalization of C. albicans to be visualized without the need to stain external Candida. At least three independent experiments were performed for each group and at least three videos were analyzed from each experiment using ImageJ analysis software.

In vitro TBK1 kinase assays

TBK1 kinase assays were performed using the standard protocol with modifications [56]. Endogenous TBK1 from COS-1 cells was added to each kinase activity assay in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.6), 0.1 mM sodium vanadate, 20 mM β-glycerophosphate, 10 mM MgCl2, 50 mM NaCl, and 100 μM ATP. For each reaction, 20 μg of purified recombinant GST-tagged IRF-3-C was used as a substrate for TBK1, and the reactions were carried out for 30 min at 30°C. The reactions were terminated by adding sample buffer. The phosphorylation of recombinant GST-IRF3-C was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with phosphor-IRF-3 (Ser396) antibodies (Cell Signaling Technology, Cat#29047), and the equivalent protein concentrations were confirmed by immunoblotting with anti-TBK1 and anti-GST antibodies.

Cytokine measurements

The enzyme-linked immunosorbent assay kits for IFN-β were purchased from NeoBioscience. All samples were measured in triplicate according to the manufacturer’s protocol.

Statistical analysis

At least three biological replicates were performed for all experiments, unless otherwise indicated. Individual data for each experiment are provided as Additional file 11. Student’s t test was used for statistical analyses of paired observations. Differences between means were accepted as statistically significant at the 95% level (p < 0.05).

Antibodies

The primary antibodies used for immunoblotting were rabbit monoclonal anti-TBK1 (Cell Signaling Technology, Danvers, MA, USA, Cat# 3504S RRID:AB_225566,1:1000 dilution), rabbit monoclonal anti-phosphorylated TBK1 (phosphor S172) (Cell Signaling Technology Cat# 5483P RRID:AB_10693472,1:1000), rabbit monoclonal anti-β-actin (Cell Signaling Technology Cat# 4970S RRID:AB_2223172, 1:1000), rabbit monoclonal anti-PCNA (Cell Signaling Technology Cat# 13110 RRID:AB_2636979,1:1000), rabbit monoclonal anti-IRF3 (phosphor Ser396) (Cell Signaling Technology Cat# 4302S RRID:AB_1904036,1:1000), rabbit monoclonal anti-phosphorylated IRF-3 (Cell Signaling Technology Cat# 29047 RRID:AB_823547,1:1000), rabbit polyclonal anti-SEC5 (Sigma-Aldrich, Atlas Antibodies Cat# HPA032093 RRID:AB_10611964, 1:1000), mouse monoclonal anti-InsP3R-3 (BD Biosciences, Franklin, NJ, USA,BD Biosciences Cat# 610313 RRID:AB_397705,1:1000), rabbit polyclonal anti-GST (ABclonal Technology, Wuhan, China, ABclonal Technology Cat#AE006,1:1000), and mouse monoclonal anti-His tag (ABclonal Technology Cat#AE003,1:1000).

The antibodies used for immunofluorescence were rabbit monoclonal anti-TBK1 (Abcam, Cambridge, UK, Abcam Cat# ab40676 RRID:AB_776632,1:100 dilution), mouse monoclonal anti-InsP3R-3 (BD Biosciences Cat# 610313 RRID:AB_397705, 1:100), rabbit polyclonal anti-SEC5 (Atlas Antibodies Cat# HPA032093 RRID:AB_10611964, 1:100), Alexa Fluor 488-conjugated donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Life Technologies, Molecular Probes Cat# A-21202 RRID:AB_141607,1:400), and Cy3-conjugate donkey anti-rabbit IgG, species adsorbed, antibody (Millipore, Burlington, MA, USA, Millipore Cat# AP182C RRID:AB_92588,1:400).

Polyclonal antibody anti-Type 1 InsP3R was generated by Abgent Biotech. Co. Ltd. (Shanghai, China) against the peptides RIGLLGHPPHMNVNPQQPA, strictly following Akihiko Tanimura’s method [57]. Antigens were prepared by GL Biochem. Ltd. (Shanghai, China). The specificity of the InsP3R1 antibody has been previously validated [57, 58]. We also reconfirmed the specificity of the polyclonal antisera made against the peptides from the type 1 InsP3 receptors in chicken DT40 cells with all InsP3R isoforms genetically deleted (DT40-KO) and engineered to stably express InsP3R type 1 (KO-R1) or type 2 (KO-R2), which were provided by Dr. J. Kevin Foskett. The mouse monoclonal antibody against InsP3R3 was purchased from BD Biosciences. Its specificity was confirmed by western blot of DT40-KO stably expressing a mutant InsP3R3, which agreed with the results of immunocytochemistry and western blotting assay of InsP3R3 by other groups [58, 59].

Reagents

Inositol-1,4,5-trisphosphate (Cat#10008205) and araguspongin B (Cat#10006797) were from Cayman Chemical (Ann Arbor, MI, USA). The other reagents are indicated as follows:

  • SiRNA-SEC5 (Shanghai GenePharma Co., Ltd., Shanghai, China)

5’-GGUCGGAAAGACAAGGCAGTT-3’

  • SiRNA-Negative Control (Shanghai GenePharma Co., Ltd., Shanghai, China)

  • 5’-UUCUCCGAACGUGUC ACG UTT-3’

ShRNA-SEC5 (Shanghai Genechem Co., Ltd., Shanghai, China)

5’-CCGGGCCGAAGAGATAAAGAGATTACTCGAGTAATCTCTTTATCTCTTCGGCTTTTTT-3’

Animals

C57BL/6 mice were purchased from Shanghai BK Animal Model Inc. Ltd., China.

Abbreviations

InsP3R: 

inositol 1,4,5-trisphosphate receptors

TBK1: 

tank-binding kinase 1

IRF-3: 

interferon regulatory factor 3

SEC5: 

exocyst complex component 2; C. albicans: Candida albicans

PRRs: 

pattern recognition receptors

TLRs: 

Toll-like receptors

PAMPs: 

pathogen-associated molecular patterns

PLCγ2: 

phospholipase Cγ2

14: 

endoplasmic reticulum

Par100: 

inositol 1,4,5-trisphosphate

[Ca2+]c : 

cytosolic Ca2+ concentrations

BMDMs: 

bone marrow-derived macrophages

ARB: 

araguspongin B

FRET: 

fluorescence resonance energy transfer

SIM: 

super-resolution structured illumination microscopy

siRNA: 

small interfering RNA

IFNs-I: 

type I interferons

M-CSF: 

macrophage colony-stimulating factor

GST: 

Glutathione S-transferase

Declarations

Acknowledgements

We thank Mr. Qingfeng Xiao from Nikon and Miss Yan Wang at the National Center for Protein Science, Shanghai (NCPSS) for performing the SIM imaging. We thank Dr. J. Kevin Foskett for the DT40KO-InsP3R3 cells, plasmid, and yeast two-hybrid information.

Funding

This work was supported by the General Research Fund of Hong Kong 764113 & 17104514 to KHC, the Shanghai Institute of Planned Parenthood Research, Pandeng Planning Grant PD2012–4 to JY, and by the National Basic Research Program of China 2013CB531602 to YYJ.

Availability of data and materials

All the data on which the conclusions of the paper are based are presented in the paper and its additional files.

Authors’ contributions

LoY performed molecular biology, protein biochemistry, fungal infection, and phagocytosis assays; WWG, immunofluorescence and Ca2+ imaging; WWG, BCT, and KHC, patch-clamp studies; JY, yeast two-hybrid assays. YYJ and JY designed the experiments; JY, LoY, YYJ, KHC, and LaY wrote the manuscript; JY contributed ideas. All authors reviewed the results and approved the final version of the manuscript.

Ethics approval

The animal experimental protocol was approved by the Animal Ethics Committee of the Shanghai Institute of Planned Parenthood Research (SIPPR Regulation#2015-13), in accordance with the 588th regulation of animal experiments issued by the Chinese Government in 2011. All animal experiments were performed under the audit of the SIPPR Animal Ethics Committee.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
School of Pharmacy, Second Military Medical University, Shanghai, China
(2)
NHFPC Key Laboratory of Reproduction Regulation, Shanghai Institute of Planned Parenthood Research, Shanghai, China
(3)
School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China
(4)
Jinan Military General Hospital, Jinan, China
(5)
School of Medicine, Tongji University, Shanghai, China

References

  1. Brown GD, et al. Hidden killers: human fungal infections. Sci Transl Med. 2012;4:165rv13.View ArticlePubMedGoogle Scholar
  2. Kullberg BJ, Arendrup MC. Invasive candidiasis. N Engl J Med. 2015;373:1445–56.View ArticlePubMedGoogle Scholar
  3. Netea MG, Joosten LAB, van der Meer JWM, Kullberg B-J, van de Veerdonk FL. Immune defence against Candida fungal infections. Nat Rev Immunol. 2015;15:630–42.View ArticlePubMedGoogle Scholar
  4. Xu S, Huo J, Lee K-G, Kurosaki T, Lam K-P. Phospholipase Cγ2 is critical for Dectin-1-mediated Ca2+ flux and cytokine production in dendritic cells. J Biol Chem. 2009;284:7038–46.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Gorjestani S, et al. Phospholipase Cγ2 (PLCγ2) is key component in Dectin-2 signaling pathway, mediating anti-fungal innate immune responses. J Biol Chem. 2011;286:43651–9.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Nunes P, Demaurex N. The role of calcium signaling in phagocytosis. J Leukoc Biol. 2010;88:57–68.View ArticlePubMedGoogle Scholar
  7. Foskett JK, White C, Cheung K-H, Mak D-OD. Inositol trisphosphate receptor Ca2+ release channels. Physiol Rev. 2007;87:593–658.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Prole DL, Taylor CW. Inositol 1,4,5-trisphosphate receptors and their protein partners as signalling hubs. J Physiol Lond. 2016;594:2849–66.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Vaeth M, et al. Ca2+ signaling but not store-operated Ca2+ entry is required for the function of macrophages and dendritic cells. J Immunol. 2015;195:1202–17.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Patterson RL, Boehning D, Snyder SH. Inositol 1,4,5-trisphosphate receptors as signal integrators. Annu Rev Biochem. 2004;73:437–65.View ArticlePubMedGoogle Scholar
  11. Uchida K, Miyauchi H, Furuichi T, Michikawa T, Mikoshiba K. Critical regions for activation gating of the inositol 1,4,5-trisphosphate receptor. J Biol Chem. 2003;278:16551–60.View ArticlePubMedGoogle Scholar
  12. Taylor CW, da Fonseca PCA, Morris EP. IP(3) receptors: the search for structure. Trends Biochem Sci. 2004;29:210–9.View ArticlePubMedGoogle Scholar
  13. Fan G, et al. Gating machinery of InsP3R channels revealed by electron cryomicroscopy. Nature. 2015;527:336–41.View ArticlePubMedPubMed CentralGoogle Scholar
  14. White C, et al. The endoplasmic reticulum gateway to apoptosis by Bcl-XL modulation of the InsP3R. Nat Cell Biol. 2005;7:1021–8.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Chang M-J, et al. Feedback regulation mediated by Bcl-2 and DARPP-32 regulates inositol 1,4,5-trisphosphate receptor phosphorylation and promotes cell survival. Proc Natl Acad Sci U S A. 2014;111:1186–91.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Tanaka T, Goto K, Iino M. Diverse functions and signal transduction of the exocyst complex in tumor cells. J Cell Physiol. 2016; https://doi.org/10.1002/jcp.25619.
  17. Stuart LM, et al. A systems biology analysis of the Drosophila phagosome. Nature. 2007;445:95–101.View ArticlePubMedGoogle Scholar
  18. Chien Y, et al. RalB GTPase-mediated activation of the IκB family kinase TBK1 couples innate immune signaling to tumor cell survival. Cell. 2006;127:157–70.View ArticlePubMedGoogle Scholar
  19. Ishikawa H, Ma Z, Barber GN. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature. 2009;461:788–92.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Ishikawa H, Barber GN. The STING pathway and regulation of innate immune signaling in response to DNA pathogens. Cell Mol Life Sci. 2011;68:1157–65.View ArticlePubMedGoogle Scholar
  21. Biondo C, et al. Recognition of yeast nucleic acids triggers a host-protective type I interferon response. Eur J Immunol. 2011;41:1969–79.View ArticlePubMedGoogle Scholar
  22. Bourgeois C, et al. Conventional dendritic cells mount a type I IFN response against Candida spp. requiring novel phagosomal TLR7-mediated IFN-β signaling. J Immunol. 2011;186:3104–12.View ArticlePubMedGoogle Scholar
  23. del Fresno C, et al. Interferon-β production via Dectin-1-Syk-IRF5 signaling in dendritic cells is crucial for immunity to C. albicans. Immunity. 2013;38:1176–86.View ArticlePubMedGoogle Scholar
  24. Smeekens SP, et al. Functional genomics identifies type I interferon pathway as central for host defense against Candida albicans. Nat Commun. 2013;4:1342.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Groves E, Dart AE, Covarelli V, Caron E. Molecular mechanisms of phagocytic uptake in mammalian cells. Cell Mol Life Sci. 2008;65:1957–76.View ArticlePubMedGoogle Scholar
  26. Czibener C, et al. Ca2+ and synaptotagmin VII-dependent delivery of lysosomal membrane to nascent phagosomes. J Cell Biol. 2006;174:997–1007.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Gafni J, et al. Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron. 1997;19:723–33.View ArticlePubMedGoogle Scholar
  28. Adebiyi A. RGS2 regulates urotensin II-induced intracellular Ca2+ elevation and contraction in glomerular mesangial cells. J Cell Physiol. 2014;229:502–11.View ArticlePubMedGoogle Scholar
  29. Kinoshita A, et al. Demonstration by FRET of BACE interaction with the amyloid precursor protein at the cell surface and in early endosomes. J Cell Sci. 2003;116:3339–46.View ArticlePubMedGoogle Scholar
  30. Herman B, Krishnan RV, Centonze VE. Microscopic analysis of fluorescence resonance energy transfer (FRET). Methods Mol Biol. 2004;261:351–70.PubMedGoogle Scholar
  31. Eckenrode EF, Yang J, Velmurugan GV, Foskett JK, White C. Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca2+ signaling. J Biol Chem. 2010;285:13678–84.View ArticlePubMedPubMed CentralGoogle Scholar
  32. Yang J, Vais H, Gu W, Foskett JK. Biphasic regulation of InsP3 receptor gating by dual Ca2+ release channel BH3-like domains mediates Bcl-xL control of cell viability. Proc Natl Acad Sci U S A. 2016;113:E1953–62.View ArticlePubMedPubMed CentralGoogle Scholar
  33. Luo J, Busillo JM, Benovic JL. M3 muscarinic acetylcholine receptor-mediated signaling is regulated by distinct mechanisms. Mol Pharmacol. 2008;74:338–47.View ArticlePubMedGoogle Scholar
  34. Tovey SC, Taylor CW. Cyclic AMP directs inositol (1,4,5)-trisphosphate-evoked Ca2+ signalling to different intracellular Ca2+ stores. J Cell Sci. 2013;126:2305–13.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Mak D-OD, Vais H, Cheung K-H, Foskett JK. Isolating nuclei from cultured cells for patch-clamp electrophysiology of intracellular Ca2+ channels. Cold Spring Harb Protoc. 2013;2013:880–4.PubMedPubMed CentralGoogle Scholar
  36. Mak D-OD, Vais H, Cheung K-H, Foskett JK. Nuclear patch-clamp electrophysiology of Ca2+ channels. Cold Spring Harb Protoc. 2013;2013:885–91.PubMedPubMed CentralGoogle Scholar
  37. Mak D-OD, Vais H, Cheung K-H, Foskett JK. Patch-clamp electrophysiology of intracellular Ca2+ channels. Cold Spring Harb Protoc. 2013;2013:787–97.PubMedPubMed CentralGoogle Scholar
  38. Lewis LE, Bain JM, Okai B, Gow NAR, Erwig LP. Live-cell video microscopy of fungal pathogen phagocytosis. J Vis Exp. 2013; https://doi.org/10.3791/50196.
  39. Sharma S, et al. Triggering the interferon antiviral response through an IKK-related pathway. Science. 2003;300:1148–51.View ArticlePubMedGoogle Scholar
  40. Fitzgerald KA, et al. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol. 2003;4:491–6.View ArticlePubMedGoogle Scholar
  41. Huynh KK, Kay JG, Stow JL, Grinstein S. Fusion, fission, and secretion during phagocytosis. Physiology. 2007;22:366–72.View ArticlePubMedGoogle Scholar
  42. Melendez AJ, Tay HK. Phagocytosis: a repertoire of receptors and Ca2+ as a key second messenger. Biosci Rep. 2008;28:287–98.View ArticlePubMedGoogle Scholar
  43. Shaw PJ, Qu B, Hoth M, Feske S. Molecular regulation of CRAC channels and their role in lymphocyte function. Cell Mol Life Sci. 2013;70:2637–56.View ArticlePubMedGoogle Scholar
  44. Shaw PJ, Feske S. Regulation of lymphocyte function by ORAI and STIM proteins in infection and autoimmunity. J Physiol Lond. 2012;590:4157–67.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Gagnon E, et al. Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell. 2002;110:119–31.View ArticlePubMedGoogle Scholar
  46. Desjardins M. ER-mediated phagocytosis: a new membrane for new functions. Nat Rev Immunol. 2003;3:280–91.View ArticlePubMedGoogle Scholar
  47. Touret N, et al. Quantitative and dynamic assessment of the contribution of the ER to phagosome formation. Cell. 2005;123:157–70.View ArticlePubMedGoogle Scholar
  48. Nunes P, et al. STIM1 juxtaposes ER to phagosomes, generating Ca2+ hotspots that boost phagocytosis. Curr Biol. 2012;22:1990–7.View ArticlePubMedGoogle Scholar
  49. Trinchieri G. Type I interferon: friend or foe? J Exp Med. 2010;207:2053–63.View ArticlePubMedPubMed CentralGoogle Scholar
  50. Decker T, Müller M, Stockinger S. The yin and yang of type I interferon activity in bacterial infection. Nat Rev Immunol. 2005;5:675–87.View ArticlePubMedGoogle Scholar
  51. Bergstrøm B, et al. TLR8 senses Staphylococcus aureus RNA in human primary monocytes and macrophages and induces IFN-β production via a TAK1-IKKβ-IRF5 signaling pathway. J Immunol. 2015;195:1100–11.View ArticlePubMedGoogle Scholar
  52. Liu Y-P, et al. Endoplasmic reticulum stress regulates the innate immunity critical transcription factor IRF3. J Immunol. 2012;189:4630–9.View ArticlePubMedPubMed CentralGoogle Scholar
  53. Yang J, McBride S, Mak DO, Vardi N, Palczewski K, Haeseleer F, Foskett JK. Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca2+ release channels. Proc Natl Acad Sci U S A. 2002;99(11):7711–6.View ArticlePubMedPubMed CentralGoogle Scholar
  54. Jia X-M, et al. CARD9 mediates Dectin-1-induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity. J Exp Med. 2014;211:2307–21.View ArticlePubMedPubMed CentralGoogle Scholar
  55. Bootman MD, Rietdorf K, Collins T, Walker S, Sanderson M. Converting fluorescence data into Ca2+ concentration. Cold Spring Harb Protoc. 2013;2013:126–9.PubMedGoogle Scholar
  56. Chien Y, White MA. Characterization of RalB-Sec5-TBK1 function in human oncogenesis. Meth Enzymol. 2008;438:321–9.View ArticlePubMedGoogle Scholar
  57. Tanimura A, Tojyo Y, Turner RJ. Evidence that type I, II, and III inositol 1,4,5-trisphosphate receptors can occur as integral plasma membrane proteins. J Biol Chem. 2000;275(35):27488–93.PubMedGoogle Scholar
  58. Li C, et al. Apoptosis regulation by Bcl-xL modulation of mammalian inositol 1,4,5-trisphosphate receptor channel isoform gating. Proc Natl Acad Sci U S A. 2007;104:12565–70.View ArticlePubMedPubMed CentralGoogle Scholar
  59. Vazquez G, Wedel BJ, Bird GSJ, Joseph SK, Putney JW. An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes. EMBO J. 2002;21:4531–8.View ArticlePubMedPubMed CentralGoogle Scholar

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