Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii

Abstract

Microneme secretion is essential for motility, invasion, and egress in apicomplexan parasites. Although previous studies indicate that Ca(2+) and cGMP control microneme secretion, little is known about how these pathways are naturally activated. Here we have developed genetically encoded indicators for Ca(2+) and microneme secretion to better define the signaling pathways that regulate these processes in Toxoplasma gondii We found that microneme secretion was triggered in vitro by exposure to a single host protein, serum albumin. The natural agonist serum albumin induced microneme secretion in a protein kinase G-dependent manner that correlated with increased cGMP levels. Surprisingly, serum albumin acted independently of elevated Ca(2+) and yet it was augmented by artificial agonists that raise Ca(2+), such as ethanol. Furthermore, although ethanol elevated intracellular Ca(2+), it alone was unable to trigger secretion without the presence of serum or serum albumin. This dichotomy was recapitulated by zaprinast, a phosphodiesterase inhibitor that elevated cGMP and separately increased Ca(2+) in a protein kinase G-independent manner leading to microneme secretion. Taken together, these findings reveal that microneme secretion is centrally controlled by protein kinase G and that this pathway is further augmented by elevation of intracellular Ca(2.)

Keywords: calcium; calcium imaging; calcium intracellular release; cyclic GMP (cGMP); protein kinase G (PKG); protein secretion; second messenger.

FIGURE 1.

Generation and validation of MIC2-GLuc-C-myc: a novel reporter of microneme secretion. A, schematic representation of MIC2-GLuc-C-myc fusion reporter that was introduced into the genome by cotransfection with a chloramphenicol selection cassette (CAT-CDS). Microneme secretion from this epitope-tagged line can be detected by traditional Western blotting or luciferase assay. B, comparison of total MIC2 and MIC2-GLuc-C-myc expression in wild-type (RH) and luciferase expressing (RH-MIC2-GLuc-C-myc) parasites. Extracellular tachyzoites were fixed, permeabilized, and immunolabeled with rabbit anti-SAG1, mouse anti-C-myc, or mouse anti-MIC2 followed by their respective Alexa Fluor-conjugated secondary antibodies and visualized by flow cytometry. Histograms were gated on SAG1⁺ parasites. C, subcellular localization of MIC2-GLuc-C-myc. Intracellular tachyzoites grown in human foreskin fibroblasts were fixed, permeabilized, and immunolabeled to detect MIC2-GLuc-C-myc (mouse anti-C-myc) and markers for micronemes (rabbit anti-M2AP, upper panel), dense granules (rabbit anti-GRA2, middle panel), rhoptries (rabbit anti-ROP5, lower panel) by indirect immunofluorescence microscopy. Scale bar = 5 μm. D, comparison of methods to detect microneme secretion. Serial dilutions of purified RH-MIC2-GLuc-C-myc tachyzoites were incubated in the presence or absence of 1% serum, 1% EtOH. After incubating for 10 min at 37 °C, ESA was collected and equivalent volumes were subjected in parallel to Western blotting (upper panel) and luciferase (lower panel) assays. For Western blotting, membranes were probed with mouse anti-c-Myc and rabbit anti-GRA2 antibodies and visualized using IR dye-conjugated secondary antibodies.

FIGURE 2.

Serum albumin stimulates microneme secretion and is enhanced by ethanol. A, MIC2-GLuc-C-myc secretion assay. Purified RH-MIC2-GLuc-C-myc parasites were stimulated with serial dilutions of ethanol alone, serum alone, or ethanol in the presence of 1% serum for 10 min at 37 °C. Release of MIC2-GLuc-C-myc in ESA was determined by luciferase assay. The graph indicates the average and S.D. of triplicate wells for each treatment dilution and is representative of two independent experiments with similar outcomes. B, MIC2-GLuc-C-myc secretion assay comparing whole serum to filtered serum. Purified RH-MIC2-GLuc-C-myc parasites were incubated for 10 min at 37 °C with buffer alone (mock), 1% serum (whole), 30-kDa MWCO filtered 1% serum (<30 kDa), 30-kDa MWCO filtered 1% serum + 0.18% bovine serum albumin (BSA) add back (<30 kDa + BSA), or 0.18% serum albumin alone (BSA). Release of MIC2-GLuc-C-myc in ESA was determined by luciferase assay. The graph indicates the average and S.D. of triplicate wells for each treatment and is representative of two independent experiments with similar outcomes. *, p ≤ 0.05 versus mock by one-way analysis of variance with Tukey's multiple comparison test. C, MIC2-GLuc-C-myc secretion assay. Purified RH-MIC2-GLuc-C-myc parasites were stimulated with serial dilutions of serum or equivalent amounts of serum albumin alone for 10 min at 37 °C. Release of MIC2-GLuc-C-myc in ESA was determined by luciferase assay. The graph indicates the average and S.D. of triplicate wells for each treatment dilution and is representative of two independent experiments with similar outcomes. D, MIC10-GLuc-C-myc secretion assay. Purified RH-MIC10-Gluc parasites were treated with or without 1% BSA for 10 min at 37 °C. ESA was collected and subjected to a luciferase assay. *, p ≤ 0.05, unpaired Student's t test. E, MIC2-GLuc-C-myc secretion assay. Purified RH-MIC2-GLuc-C-myc parasites were stimulated with serial dilutions of ethanol with or without 1% serum albumin for 10 min at 37 °C. Release of MIC2-GLuc-C-myc in ESA was determined by luciferase assay. The graph indicates the average and S.D. of triplicate wells for each treatment dilution and is representative of two independent experiments with similar outcomes. F, MIC2-GLuc-C-myc secretion assay comparing IC versus EC buffer in the presence or absence of BAPTA-AM. Purified RH-MIC2-GLuc-C-myc parasites resuspended in IC buffer or EC buffer were pretreated with BAPTA-AM or vehicle control and incubated for 10 min at 37 °C with buffer alone (mock), 1% BSA, 1% ethanol, or 1% BSA + 1% ethanol. Release of MIC2-GLuc-C-myc in ESA was determined by luciferase assay. The graph indicates the average and S.D. of two independent experiments consisting of triplicate wells for each treatment. *, p ≤ 0.05 versus mock by two-way analysis of variance with Tukey's multiple comparison test. All BAPTA-AM treatments were significantly lower compared with their corresponding dimethyl sulfoxide (DMSO) controls.

FIGURE 3.

Monitoring intracellular Ca²⁺ dynamics with GCaMP6f expressing Toxoplasma. A, schematic of transgenic RH-GCaMP6f parasites that express a fluorescent reporter that is sensitive to cytosolic Ca²⁺ levels. B, time-lapse microscopy of A23187-treated RH-GCaMP6f. RH-GCaMP6f parasites were imaged before and after addition of 2 μm A12387 using bright field phase microscopy and epifluorescence in the GFP channel. Times are indicated in seconds (s). Scale bar = 5 μm. C, quantification of data in B. Fluorescence intensity (F) in the GFP channel was quantified for each parasite in B using ImageJ at each time slice and compared with their initial fluorescence intensity (F₀). F/F₀ was plotted for each parasite and their mean. D and E, kinetic analysis of intracellular Ca²⁺ in RH-GCaMP6f parasites. Purified RH-GCaMP6f parasites were treated with secretagogues as indicated and monitored by flow cytometry. Fluorescent output in the FL-1 channel was collected at each second for before and after addition of secretagogues. Traces indicate the mean fluorescent intensities of GCaMP6f versus time and are representative of at least two independent experiments with similar outcomes. F, microneme secretion assay. Purified RH-MIC2-GLuc-C-myc parasites were treated with serial dilutions of A23187 for 10 min at 37 °C and relative MIC2-GLuc-C-myc levels in ESA were determined by luciferase assay. The graph indicates the average and S.D. of duplicate wells for each treatment dilution and is representative of two independent experiments with similar outcomes. *, p ≤ 0.05 versus mock by one-way analysis of variance with Tukey's multiple comparison test. ns, not significant.

FIGURE 4.

Role of cGMP pathway on stimulated microneme secretion. A, determination of cyclic GMP kinetics in Toxoplasma. Purified RH-MIC2-GLuc parasites were mock-treated or treated with secretagogues for different time intervals at 37 °C and lysates were prepared. Cyclic GMP levels were determined by a competition HRP-linked immunoassay. Relative cGMP levels for each time point were normalized to the mock treated control. The graph indicates the average and S.D. of four independent experiments consisting of duplicate wells for each treatment. *, p ≤ 0.05, two-way analysis of variance with Tukey's multiple comparison test. ns, not significant. B, MIC2-GLuc-C-myc secretion assay. Purified RH-MIC2-GLuc-C-myc parasites were stimulated with serial dilutions of zaprinast alone or in the presence of 1% BSA for 10 min at 37 °C. Relative MIC2-GLuc-C-myc levels in ESA were determined by luciferase assay. The graph indicates the average and S.D. of triplicate wells for each treatment dilution and is representative of two independent experiments. C, MIC2-GLuc-C-myc secretion assay. Transgenic parasites expressing PKG with gatekeeper T or M alleles and expressing the MIC2-GLuc-C-myc reporter were pretreated with 2 μm Compound 1 or vehicle control for 10 min followed by stimulation with the indicated secretagogues. The graph indicates the average and S.D. of duplicate wells for each treatment from four independent experiments with similar outcomes. *, p ≤ 0.05, two-way analysis of variance with Tukey's multiple comparison test. ns, not significant. D, kinetic analysis of intracellular Ca²⁺ in RH-GCaMP6f. Purified RH-GCaMP6f parasites were treated with secretagogues as indicated and monitored by flow cytometry. Fluorescent output in the FL-1 channel was collected at each second before and after addition of secretagogues. Traces indicate the mean fluorescent intensities of GCaMP6f versus time and are representative of two independent experiments with similar outcomes. E, role of PKG on zaprinast-induced Ca²⁺. RH-PKG^T/M expressing strains expressing GCaMP6f were pre-treated for 10 min with 2 μm Compound 1 or vehicle control and Ca²⁺ levels were monitored by flow cytometry by acquiring data in the FL-1 channel before and after addition of 0.5 mm zaprinast. Traces from a representative experiment indicate the percent of parasites that mobilized Ca²⁺ in response to zaprinast (left graph) and the mean fluorescent intensities of GCaMP6f (right graph) versus time.