Figure 1.
Conditional Expression of MIC2
(A) Schematic diagram showing the strains in the study, including the parental strain (tTA-dhfr), and the mic2e/mic2i strain with both endogenous (mic2e) and induced (mic2i) copies of mic2. A knockout of mic2e leaves the regulatable mic2i in Δmic2e/mic2i.
(B) Illustration of intracellular structures including the micronemes, dense granules, Golgi, PV, PV membrane, and host and parasite nuclei in a four-parasite vacuole.
(C) Expression and localization of MIC2 (green) in intracellular parasites with another micronemal marker MIC5 (red). Arrows indicate the apical pole of parasites. Scale bar, 5 μm.
(D) Western blot analyses showing MIC2 steady-state expression (left blot) and secretion (right blot) levels. Asterisk indicates the full-length myc-tagged MIC2, which is secreted in mic2e/mic2i and Δmic2e/mic2i parasites based on probing with a myc antibody (unpublished data). Blots were probed with anti-MIC2 6D10 (top blots) and mouse anti-GRA1 (bottom blots), a dense granule protein, to normalize loading in all lanes. The bar graphs represent the relative percentages of MIC2 expressed in each strain and treatment compared to the reference tTA-dhfr level (100%) quantified from direct chemiluminescent imaging. Results are mean ± s.e.m, n = 3.
Figure 2.
Proteolytic Maturation and Localization of M2AP Is Dependent on Expression of MIC2
(A) Western blot of whole cell lysates showing steady-state expression levels. Blots were probed as indicated. Proform M2AP (proM2AP); mature M2AP (mM2AP).
(B) Expression and localization of M2AP in intracellular (IC) and extracellular (EC) parasites. Top panels: dual staining of M2AP (red) with AMA1 (green) in IC parasites showing mislocalization of M2AP in the PV of Δmic2e/mic2i + ATc parasites. Bottom panels: immunostaining of M2AP (red) and GRA4 (green) in EC parasites shows a progressive increase in M2AP expression in dense granules, particularly in Δmic2e/mic2i + ATc parasites. Arrows indicate the apical poles of parasites and an arrowhead indicates the PV.
Figure 3.
Invasion Phenotypes Associated with Reduced MIC2 Expression
(A) Illustration of the red-green invasion assay based on differential immunolabeling. Invading parasites (step 2) were counted as green.
(B) Quantification of the red-green invasion assay: red bars, attached extracellular parasites; green bars, invading and invaded parasites. A single asterisk indicates a statistically significant difference compared to tTA-dhfr; double asterisk indicates statistical difference compared to mic2e/mic2i + ATc (two-tailed Student's t-test). BAPTA-AM-treated parasites were included as a positive control for an attachment/invasion defect. Data are mean values ± s.e.m. of four separate experiments, each with three replicates and counting eight randomly selected fields per well.
(C) Correlation and linear regression of the percentage of MIC2 expression in cell lysates (left Y-axis and black line) and the percentage of MIC2 secretion (right Y-axis and red dashed line) with the numbers of invaded parasites.
(D) Attachment to glutaraldehyde-fixed host cells. An asterisk indicates that attachment was significantly lower than tTA-dhfr (p < 0.002, two-tailed Student's t-test). Data were compiled from three separate experiments, counting six fields per well per clone.
(E) Time-course invasion of tTA-dhfr and Δmic2e/mic2i parasites ± ATc over an 8 h period. Data represent five individual experiments with three replicates within each experiment.
Table 1.
Summary of Phenotypes Associated with MIC2-Depleted Parasites
Figure 4.
Gliding Phenotypes by Static Assay and Live Video Microscopy
(A) Assessment of gliding motility by trail deposition. Top panels: DMSO is used as a solvent control. Scale bar, 15 μm. Bottom panel: UVT153753 (Enh) enhancer-treated parasites. Arrowheads indicate non-circular trails and arrows denote circular trails.
(B) Quantification of non-circular and circular trails is presented on the left half of the graph, UVT153753- or DMSO-treated Δmic2e/mic2i ± ATc parasites are represented on the right half. Results are mean ± s.e.m of at least three experiments. Black bars, % non-circular gliding; grey hatched bars, % circular glide.
(C) Maximum projection images created from frames 1–60 (1 min videos taken at 1 frame per s). Red arrows, circular glide; red arrowheads, helical glide; closed black arrowhead, non-productive “gliding” parasite; open black arrowhead, twirling parasite.
(D) Quantification of types of movement in live gliding parasites; error bars represent standard deviation. An asterisk indicates a statistically significant difference between tTA-dhfr and Δmic2e/mic2i+ ATc parasites (p < 0.02).
(E) Immunofluorescent images of anti-tubulin-stained tachyzoites showing helical and straight cytoskeletons.
(F) Bar graph represents enumeration of at least 85 tachyzoite cytoskeletons. Enumeration was performed in a blinded fashion.
Figure 5.
MIC2-Depleted Parasites Are Avirulent in Mice and Confer Protective Immunity to Reinfection
(A) 5 × 104 tachyzoites of tTA-dhfr or Δmic2e/mic2i ± ATc were intraperitoneally injected into four BALB/c mice in each group.
(B) 5-fold increases in infection dosage with Δmic2e/mic2i +ATc; six mice were infected in each group.
(C) Six mice infected with Δmic2e/mic2i + ATc were challenged with 150 tachyzoites of RH at day 16 post-infection. A group of control mice were infected with RH at day 16.
Figure 6.
Δmic2e/mic2i + ATc Parasites Fail to Reach High Tissue Levels and Are Cleared
Parasite tissue burden of mice during infection with Δmic2e/mic2i treated with and without ATc. Organs from three mice were isolated at each time point and analyzed by rtqPCR using parasite specific primers. The normal time-till-death Δmic2e/mic2i without ATc is indicated by a “†.”
Figure 7.
Mice Infected with MIC2-Deficient Parasites Produce Lower Levels of Inflammatory Cytokines
Mice were infected with Δmic2e/mic2i ± ATc and treated with or without ATc in drinking water. Serum was collected from three mice on days 4 (A), 6 (B), and 8 (C) post-infection. Cytokine levels were determined by a quantitative cytokine protein microarray analysis.