Differential Roles for Inner Membrane Complex Proteins across Toxoplasma gondii and Sarcocystis neurona Development

Abstract

The inner membrane complex (IMC) of apicomplexan parasites contains a network of intermediate filament-like proteins. The 14 alveolin domain-containing IMC proteins in Toxoplasma gondii fall into different groups defined by their distinct spatiotemporal dynamics during the internal budding process of tachyzoites. Here, we analyzed representatives of different IMC protein groups across all stages of the Toxoplasma life cycle and during Sarcocystis neurona asexual development. We found that across asexually dividing Toxoplasma stages, IMC7 is present exclusively in the mother's cytoskeleton, whereas IMC1 and IMC3 are both present in mother and daughter cytoskeletons (IMC3 is strongly enriched in daughter buds). In developing macro- and microgametocytes, IMC1 and -3 are absent, whereas IMC7 is lost in early microgametocytes but retained in macrogametocytes until late in their development. We found no roles for IMC proteins during meiosis and sporoblast formation. However, we observed that IMC1 and IMC3, but not IMC7, are present in sporozoites. Although the spatiotemporal pattern of IMC15 and IMC3 suggests orthologous functions in Sarcocystis, IMC7 may have functionally diverged in Sarcocystis merozoites. To functionally characterize IMC proteins, we knocked out IMC7, -12, -14, and -15 in Toxoplasma. IMC14 and -15 appear to be involved in switching between endodyogeny and endopolygeny. In addition, IMC7, -12, and -14, which are all recruited to the cytoskeleton outside cytokinesis, are critical for the structural integrity of extracellular tachyzoites. Altogether, stage- and development-specific roles for IMC proteins can be discerned, suggesting different niches for each IMC protein across the entire life cycle. IMPORTANCE The inner membrane complex (IMC) is a defining feature of apicomplexan parasites key to both their motility and unique cell division. To provide further insights into the IMC, we analyzed the dynamics and functions of representative alveolin domain-containing IMC proteins across developmental stages. Our work shows universal but distinct roles for IMC1, -3, and -7 during Toxoplasma asexual division but more specialized functions for these proteins during gametogenesis. In addition, we find that IMC15 is involved in daughter formation in both Toxoplasma and Sarcocystis. IMC14 and IMC15 function in limiting the number of Toxoplasma offspring per division. Furthermore, IMC7, -12, and -14, which are recruited in the G₁ cell cycle stage, are required for stress resistance of extracellular tachyzoites. Thus, although the roles of the different IMC proteins appear to overlap, stage- and development-specific behaviors indicate that their functions are uniquely tailored to each life stage requirement.

Keywords: IMC; Sarcocystis; Toxoplasma; endodyogeny; endopolygeny; gametocyte; merozoite; sporozoite.

FIG 1

Transcriptomic analysis and experimental overview of oocyst, sporocyst, tachyzoite, and bradyzoite development in Toxoplasma. (A) Bubble plot showing the relative mRNA expression levels of all IMC proteins collected by RNA-seq at the stages indicated (days indicate the times of induction and/or growth of the stages indicated). IMC proteins that belong to the same functional group as defined are grouped by their spatiotemporal localization pattern in tachyzoites as shown on the left (10). Bubble size matches the relative level of expression representing transcript levels in FPKM. At the top, the places where the particular development takes place are indicated by graphics, where the mouse represents any intermediate host, the cat any feline, and the sun the extracellular environment. Brad, bradyzoites; O, oocysts; M, merogony; G, gametogony. (B) Overview of the experiments reported in this paper. KO, knockout; KD, knockdown.

FIG 2

IMC proteins in bradyzoites. IFA of bradyzoites with IMC1, IMC3, and IMC7 antisera counterstained with D. biflorus agglutinin lectin to highlight the tissue cyst wall surrounding the bradyzoites and DAPI to label the nuclear material. Arrowheads mark dividing parasites (the bottom right inserts are 2.5-fold magnifications of the marked daughter buds), and arrows point to two parasite nuclei not surrounded by an IMC7 signal.

FIG 3

IMC proteins in merozoites and during schizogony. IFA with IMC1 (A), IMC3 (B), IMC7 (C) and costaining with IMC1 and IMC7 (D). Anti-ENO2 costaining was used to highlight the parasites (A to C). The marked stages in both the IFA and schematic are multinucleate (MN; this can be either an early schizont [ES] or early microgametocyte), LS, and MS. T and M mark trophozoites (early growth phase of schizont) and merozoites, respectively. IMC7 specifically stains the polyploid mother, whereas IMC1 and -3 are specific to (developing) merozoites. See Fig. S1 to S3 for single-color panels and additional images. Black lines in the schematics indicate the localization and presence of the IMC, whereas the colors represent the IMC proteins considered. Dotted lines indicate fragmented IMC and/or weak IMC protein localization. Red and green overlay is represented in yellow.

FIG 4

IMC proteins in gametocytes. IFA of Toxoplasma gametocyte stages where mature schizonts (MS), macrogametocytes (Ma), and microgametocytes (Mi) are marked. IMC1 and IMC3 were not detected in either gametocyte, whereas IMC7 stained the periphery of the macrogametocyte. See Fig. S1 to S3 for single-color panels and additional images. Scale bars, 1 μm. N, nuclei.

FIG 5

IMC development during sporogenesis and in sporozoites. (A) Sporogenesis tracked markers for the nuclear cycles and sporozoite development. Centrosomes (centrin) and kinetochores (Nuf2) control three nuclear divisions (ND1 to ND3) in sporulating Toxoplasma. Anti-β-tubulin staining marks developing sporozoites, while IMC3 shows up relatively late during sporozoite formation. Oocyst and sporocyst walls autofluoresce blue. Nuclei are labeled with DAPI. SD indicates sporozoite development. (B) Confocal laser scanning microscopy of in vitro excysted sporozoites stained with IMC1, IMC3, and IMC7 antisera. Parasites were costained with antiserum generated against Toxoplasma β-tubulin, which visualizes the subpellicular microtubules and identifies the apical end (top). IMC7 is not present in sporozoites, but IMC1 and -3 are.

FIG 6

IMC15 is not essential for the lytic cycle, but loss of IMC15 causes a multidaughter phenotype. (A) Representative images of plaque assays of parental (RHΔKu80) and IMC15 dKO lines after a 9-day incubation. (B) Quantification of plaque size (A.U., arbitrary units) of both lines by measurement of the area of 30 plaques. n = 3 ± the standard error of the mean. The differences are not statistically significant by unpaired t test. (C, D) IFA images of parental (ΔKu80) (C) and IMC15 dKO (D) parasite lines obtained with anti-IMC3 and anti-human centrin (hCentrin) antibodies after methanol fixation. The top row shows the vacuoles with initiating and early daughter buds, the middle row has budding daughters in midphase, and in the bottom row, the daughters are in the late phase of maturation. (E) Graph showing quantification of the multidaughter phenotype by counting 200 vacuoles in each experiment. n = 3 + the standard error of the mean. Statistical significance was calculated by using an unpaired two-tailed t test (***, P < 0.001).

FIG 7

Mature group of IMC proteins completely transitions to the cortex in late G₁ stage. (A, B) Schematic representations of the procedures used to arrest the parasite in mid G₁ stage by temperature restriction (A) and in late G₁ stage upon 3-MA treatment (B). (C to E) Quantification of FV-P6 parasite lines stably expressing mCherry fused to IMC7 (C), IMC12 (D), or IMC14 (E) in the cortex of the parasite after invasion at 35°C (permissive condition; random cycling parasites) and upon mid-G₁ arrest at 40°C. The percentage of parasites with a cortical IMC7 (C), -12 (D), or -14 (E) signal was quantified after 3-MA block in late G₁. All graphs were plotted by calculating the percentage of vacuoles with cortical IMC after counting >100 vacuoles. n = 3 + the standard deviation. Statistical significance was calculated by using an unpaired two-tailed t test. n.s, nonsignificant; **, P < 0.001; ***, P < 0.0001.

FIG 8

Localization of the mature group of IMC proteins in extracellular tachyzoites. (A) Examples of YFP signal localizing to the cytoplasm and cortex in extracellular parasites stably expressing YFP-IMC12. (B) Quantification of cortical localization of marked IMC signal in extracellular parasites at 0, 2, and 24 h after intracellular release. The graph was plotted from the average percentage of cortical localization of IMC in extracellular parasites determined by counting >200 parasites. n = 3 ± the standard deviation. One-way analysis of variance of IMC12 showed significant P values between 0 and 2 h (*, P < 0.01) and from 0 to 24 h (**, P < 0.001), as marked in the graph, suggesting that IMC12 translocates gradually to the IMC, while tachyzoites reside extracellularly. DIC, differential interference contrast.

FIG 9

IMC14 is required for synchronous division. (A) IFA images of parental RHΔKu80 and IMC14 dKO lines stained with anti-IMC3 antibody (red), anti-human centrin (hCentrin) antibody (green), and DAPI. The arrowhead indicates a parasite with four budding daughters as opposed to two in other parasites within the same vacuole. (B) Quantification of vacuoles harboring multiple daughters by blindly counting at least 100 vacuoles. n = 3 + the standard deviation.

FIG 10

IMC7 and IMC14 are required for osmotic stress resistance. (A) Percentages of permeabilized cells of the control parental line stained with PI after treatment with different concentrations of saponin. Permeabilization was measured by PI staining of the nuclear material by flow cytometry. Alexa 488-conjugated SAG1 was used to differentially stain parasites from debris, which were additionally gated by forward and side light scatter. (B to E) Percentages of permeabilized parasites positive for PI after exposure to hypoosmotic (0.5× or 0.25× HBSS) or hyperosmotic (1 M sorbitol) conditions, as well as a normoosmotic (1.0× HBSS) control. B, RHΔKu80 parental line; C, IMC7 cKD (48 h with ATc); D, IMC12 dKO; E, IMC14 dKO. n = 3 + the standard deviation. Two-way analysis of variance compared the hypotonic and hypertonic buffers with control 1× HBSS. *, P < 0.01; **, P < 0.001; ***, P < 0.0001.