Toxoplasma MIC2 is a major determinant of invasion and virulence

Abstract

Like its apicomplexan kin, the obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and uses a unique form of gliding motility. The recent identification of several transmembrane adhesive complexes, potentially capable of gripping external receptors and the sub-membrane actinomyosin motor, suggests that the parasite has multiple options for host-cell recognition and invasion. To test whether the transmembrane adhesin MIC2, together with its partner protein M2AP, participates in a major invasion pathway, we utilized a conditional expression system to introduce an anhydrotetracycline-responsive mic2 construct, allowing us to then knockout the endogenous mic2 gene. Conditional suppression of MIC2 provided the first opportunity to directly determine the role of this protein in infection. Reduced MIC2 expression resulted in mistrafficking of M2AP, markedly defective host-cell attachment and invasion, the loss of helical gliding motility, and the inability to support lethal infection in a murine model of acute toxoplasmosis. Survival of mice infected with MIC2-deficient parasites correlated with lower parasite burden in infected tissues, an attenuated inflammatory immune response, and induction of long-term protective immunity. Our findings demonstrate that the MIC2 protein complex is a major virulence determinant for Toxoplasma infection and that MIC2-deficient parasites constitute an effective live-attenuated vaccine for experimental toxoplasmosis.

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myc-tagged MIC2, which is secreted in mic2e/mic2i and Δmic2e/mic2i parasites based on probing with a myc antibody (unpublished data).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.

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.