QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 26 October 2004
doi: 10.1242/jcs.01495

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wetzel, D. M. Articles by Sibley, L. D. Search for Related Content PubMed PubMed Citation Articles by Wetzel, D. M. Articles by Sibley, L. D.

Calcium-mediated protein secretion potentiates motility in Toxoplasma gondii

¹ Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
² Laboratory of Molecular Parasitology, Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802, USA

View larger version (49K): [in a new window]   Fig. 1. Effects of CAL on gliding and invasion by Toxoplasma gondii. (A) Indirect immunofluorescence microscopy demonstrating that the average length of trails deposited during gliding increased with calmidazolium (CAL) treatment. Parasites were treated with DMSO (control) or 10 µM CAL and allowed to glide on serum-coated glass. Trails were visualized with anti-SAG1 mAb DG52 conjugated to Alexa488. Bar, 5 µm. (B) Quantification of trails deposited in assay shown in A demonstrated that trail length increased with 1 µM and 10 µM CAL treatment. Bars show average trail length in parasite body lengths (mean±s.e.m.). *A significant difference (P0.05) compared to control trail lengths, two-tailed Student's t-test. (C) Percentage of T. gondii invading host cells increased with CAL treatment. Parasites were treated with 1 µM or 10 µM CAL or DMSO and allowed to invade HFF cells. A two-color immunofluorescence assay was used to distinguish between intracellular and extracellular parasites as described previously (Carruthers et al., 1999a). 1 µM cytochalasin D (CD) was used as a negative control for invasion. Bars show the average percentage of intracellular parasites (mean±s.e.m.). *A significant difference (P<0.05) when compared to the percentage of control parasites that invaded host cells, two-tailed Student's t-test.

View larger version (30K): [in a new window]   Fig. 2. Effect of CAL on microneme secretion in T. gondii. (A) Western blot of parasite supernatants following stimulation of secretion at 37°C showed that secretion of MIC2 and MIC4 were increased by treatment with CAL. Stimulation with 1% ethanol was used as a positive control for secretion. Supernatants were compared to diluted lysates of cell standards (% pellets). Accidental parasite lysis was less than 1% as monitored by actin release. Identical blots were probed with monoclonal antibody 6D10 (cellular MIC2 and secreted MIC2; upper panel), rabbit polyclonal serum to MIC4 (middle panel) and rabbit polyclonal serum to actin (lower panel). (B) Phosphorimager analysis of western blots demonstrated that the amount of MIC2 and MIC4 secretion increased with increasing concentrations of CAL. Bars represent the average of four experiments (mean±s.e.m.). *A significant difference compared to DMSO levels, P<0.05; two-tailed Student's t-test.

View larger version (28K): [in a new window]   Fig. 3. Effect of CAL on m2apKO T. gondii. (A) Western blotting of microneme proteins released into supernatants indicated that m2apKO parasites were unable to secrete MIC2 even after CAL treatment, unlike wild-type parasites (RH strain). Stimulation with 1% ethanol was used as a positive control for secretion. Supernatants were compared to diluted lysates of cell standards (% pellets). Identical blots were probed with monoclonal antibody 6D10 (cellular MIC2 and secreted MIC2; upper panel) and rabbit polyclonal serum to MIC5 (lower panel). (B) Phosphorimager analysis of western blots demonstrated that the amount of MIC2 secretion by the m2apKO strain was less than 10% of wild-type (RH strain) secretion even after CAL treatment. However, secretion of the microneme protein MIC5 was normal. Bars represent the average of three experiments (mean±s.e.m.). (C) Quantification of SAG1-labeled trails deposited in gliding assays demonstrated that average trail length formed by the m2apKO strain did not increase with CAL treatment, unlike trail length in wild-type parasites (RH). 1 µM cytochalasin D (CD) was used as a negative control for gliding. Bars show average trail length in parasite body lengths (mean±s.e.m.). *A significant difference when compared to control trail lengths; P0.05, paired Student's t-test.

View larger version (22K): [in a new window]   Fig. 4. Effect of CAL on intracellular calcium levels. (A-C) Parasites were loaded with fura-2/AM as described in Materials and Methods, and suspended in buffer A containing 1 mM CaCl2 (A) or 1 mM EGTA (B,C). (A) Increasing the concentration of CAL (added where indicated by the arrow) stimulated progressively greater increases in the amount of intracellular calcium when parasites were in buffer containing calcium. (B) CAL was added where indicated by the arrow at the concentrations shown to the right. Incubating parasites in the buffer containing EGTA promoted increases in intracellular calcium at low drug concentrations but decreased intracellular calcium at higher concentrations of CAL. (C) 1 µM CAL (b) or 2.5 µl DMSO (a) were added where indicated by the first arrow and 2 mM CaCl2 was added where indicated by the second arrow. CAL-stimulated release of Ca2+ from intracellular stores led to Ca2+ entry from the extracellular medium. The increase in calcium detected with solvent alone was also observed in its absence and reflects the presence of some extracellular fura-2.

View larger version (34K): [in a new window]   Fig. 5. Effect of BAPTA on CAL-stimulated gliding and secretion. (A) Quantification of the length of SAG1-labeled trails deposited by wild-type parasites. Simultaneous BAPTA and CAL treatment did not stimulate increased trail length, unlike CAL treatment alone. 1 µM CD was used as a negative control for gliding. Bars show average trail length in parasite body lengths (mean±s.e.m.). *A significant difference when compared to control trail lengths; P<0.05, two-tailed Student's t test. (B) Western blotting of parasite supernatant proteins showed that CAL-stimulated secretion of MIC2 was blocked by BAPTA. Stimulation with 1% ethanol was used as a positive control for secretion. Supernatants were compared to diluted lysates of cell standards (% pellets). Blots were probed with monoclonal antibody 6D10 (cellular MIC2 (cMIC2) and secreted MIC2 (sMIC2). (C) Phosphorimager analysis of western blots demonstrated that the amount of MIC2 secretion no longer increased after BAPTA treatment, even with increasing concentrations of CAL. Bars represent the average of three experiments (mean±s.e.m.). (D) Caffeine treatment did not increase length of trails formed by parasites during a gliding assay. 1 µM CD was used as a negative control for gliding. Bars show average trail length in parasite body lengths (mean±s.e.m.).

View larger version (48K): [in a new window]   Fig. 6. Real-time calcium measurements in motile control and CAL-treated T. gondii. (A) Time-lapse images of calcium flux in DMSO, 1 µM CAL and 1 µM caffeine-treated parasites. Treated parasites were labeled with fluo-4 and allowed to glide. Parasites were observed by fluorescence and phase-contrast microscopy and recorded at 0.5 second intervals. Shown are selected merged images with the time elapsed between frames indicated in seconds. Bar, 5 µm. (B) Graph of absolute frame-by-frame fluorescence pixel intensity of one representative movie per condition showing fluo-4 fluorescence intensity oscillations during DMSO, CAL or caffeine-treated gliding. *The point at which the caffeine-treated parasite stopped gliding. (C) Normalized intensities of fluo-4 oscillations in control and CAL movies shown in B. Calcium oscillations (numbered) in CAL-treated parasites occurred at twice the frequency but half the amplitude of calcium oscillations in untreated parasites. Ft/Fo, temporal fluorescence intensity of fluo-4 divided by the fluorescence intensity at the start of each cycle.