The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells

Abstract

Many members of the phylum of Apicomplexa have adopted an obligate intracellular life style and critically depend on active invasion and egress from the infected cells to complete their lytic cycle. Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa, and as such, the invasive tachyzoite contains an organelle termed the conoid at its extreme apex. This motile organelle consists of a unique polymer of tubulin fibres and protrudes in both gliding and invading parasites. The class XIV myosin A, which is conserved across the Apicomplexa phylum, is known to critically contribute to motility, invasion and egress from infected cells. The MyoA-glideosome is anchored to the inner membrane complex (IMC) and is assumed to translocate the components of the circular junction secreted by the micronemes and rhoptries, to the rear of the parasite. Here we comprehensively characterise the class XIV myosin H (MyoH) and its associated light chains. We show that the 3 alpha-tubulin suppressor domains, located in MyoH tail, are necessary to anchor this motor to the conoid. Despite the presence of an intact MyoA-glideosome, conditional disruption of TgMyoH severely compromises parasite motility, invasion and egress from infected cells. We demonstrate that MyoH is necessary for the translocation of the circular junction from the tip of the parasite, where secretory organelles exocytosis occurs, to the apical position where the IMC starts. This study attributes for the first time a direct function of the conoid in motility and invasion, and establishes the indispensable role of MyoH in initiating the first step of motility along this unique organelle, which is subsequently relayed by MyoA to enact effective gliding and invasion.

Fig 1. T. gondii myosin H appears during IMC formation and localizes close to the pre-conoidal rings.

(A) TgMyoH-3Ty is found at the predicted molecular weight by western blot (174 kDa). T. gondii catalase (CAT) was used as loading control. (B) MyoH-3Ty localizes at the extremities of the conoid in intra- (upper panel) and extracellular parasites (lower panel) (arrowhead). GAP45 stains the periphery of the parasites. Scale bar 2 μm. (C) MyoH-Ty localization during parasite division using respectively the IMC sub-compartment protein 1 (ISP1) and the inner membrane complex 1 (IMC1) markers. MyoH (arrowhead) appears early during division and accumulates at the apical end of the newly formed parasites. Scale bar 2 μm. (D) The precise localization of MycMyoH relatively to the conoid markers: RING1 (RNG1) at the apical polar ring (APR); calmodulin 1 (CAM1) in the middle part of the conoid and centrin 2 (CEN2) at the pre-conoidal rings (PCR). Co-localizations are assessed by the RGB profile plots determined using ImageJ along the arrows. Conoid protrusion in extracellular parasites was induced with A23187. IMC: inner membrane complex, SPM: subpellicular microtubules. Scale bar 1 μm.

Fig 2. T. gondii myosin H is involved in gliding motility, invasion and egress.

(A) Inducible MyoH (Myc-MyoH) migrates at the predicted molecular weight (171 kDa) by western blot and is down-regulated to undetectable level upon 48 h of ATc treatment. Catalase was used as a loading control. (B) Myc-MyoH localizes at the conoid (arrowhead) and is not detectable after 48 h of ATc treatment. Scale bar 2 μm. (C) Intracellular growth assay performed on myoH-iKD and Δku80 strains upon 48 h incubation ± ATc revealed no altered phenotype. Data are presented as mean ± SD. (D) Calcium ionophore-induced egress assay of myoH-iKD and parental (Δku80) strains performed by treating the parasites with DMSO or A23187 for 7 min after 48 h ± ATc. Results are expressed as percentage of ruptured vacuoles and represented as mean ± SD. (E) The invasion capacity of myoH-iKD and parental (Δku80) parasites was evaluated after 48 h ± ATc. Results are expressed as percentage of invading parasites and represented as mean ± SD. (F) Gliding assays performed with myoH-iKD strain after 48 h ± ATc. The trails are revealed by indirect fluorescence microscopy using anti-SAG1 antibodies. (G) Quantification of the gliding assay. Results are expressed as percentage of trails detected per field and normalized to the total number of parasites per field. Data are presented as mean ± SD. CD: Cytochalasin D. For (D), (E) and (G), the significance of the results was assessed using a parametric paired t-test and the two-tailed p-values are presented on the graphs, ns: non-significant.

Fig 3. T. gondii myosin H is not involved in conoid protrusion, microneme or rhoptry secretion, host cell attachment, AKMT relocalization, or glideosome assembly.

(A) Conoid protrusion assay performed on wt (Δku80) and myoH-iKO lines ± ATc for 48 h. DIC pictures are representative of each condition with the protruded conoid indicated by an arrow. Scale bar 1 μm. The graph represents the percentage of conoid protrusion induced by 3 μM of A23187, or 0.5 M ethanol (ETOH) and their respective controls. Data are represented as mean ± SD. The significance of the results was assessed using a parametric paired t-test, ns: non-significant. Microneme secretion assay performed on wt (Δku80) and myoH-iKO lines ± ATc for 48 h and analyzed by western blot using the following micronemal proteins (MICs); apical membrane antigen 1 AMA1, MIC4, MIC6 (B) or MIC2 (C). “p” correspond to the predicted cleaved forms of the different micronemal proteins analyzed. Catalase (CAT) was used as cytosolic control and dense granule 1 (GRA1) as control for constitutive secretion. ESA: excreted/secreted antigens, ESA-induced: induction with 0.5 M ethanol. (D) Attachment assay performed with a mixed population of wt (RH-GFP) and myoH-iKD ± ATc for 48 h. Controls represent the initial percentage of each line prior to the attachment assay. Data are represented as mean ± SD. (E) E-vacuole assay performed on the myoH-iKD line treated ± ATc for 48 h. CD: cytochalasin D. Scale bar 2 μm. (F) Cytosolic re-localization of the apical complex lysine methyltransferase (AKMT) upon induction with 3 μM of A23187 in presence or absence of MyoH. Scale bar 2 μm. (G) Co-IP experiments performed with anti-GAP45 antibodies on myoH-iKD parasites treated for 48 h ± ATc and metabolically labeled with [S³⁵]-methionine/cysteine.

Fig 4. The ATS1 domains of T. gondii myosin H are necessary for its conoid localization.

(A) MyoH-3Ty solubility was evaluated by fractionation after extraction in PBS, PBS/NaCl, PBS/Na2CO3 or PBS/Triton X-100. Their distribution in different fractions was assessed by western blot using anti-Ty antibodies. The profilin (PRF) was used as soluble control. (B) Deoxycholate (DOC) extraction shows that MycMyoH (upper panels) and DD-MyoH-NT-ΔATS1 (middle panels) remain bound to the conoid (arrowhead). Actin polymerization using jasplakinolide at 1 μM (JAS) showed MycMyoH strongly associated to the conoid (arrowhead) and not to the actin-containing apical extensions (lower panels). Scale bar 2 μm. (C) Schematic representation of the TgMyoH and FKBP destabilization domain (DD) constructs. MyoH contains eight IQ motifs and three ATS1/RCC1 domains. NT: Neck and Tail, T: Tail. (D) Western blot analysis using anti-Myc antibodies shows stabilization of DD-MyoH-NT (100 kDa), DD-MyoH-T (64 kDa), and DD-MyoH-NT-ΔATS1 (76 kDa) after 24 h of Shield-1 (Shld-1) treatment. Catalase (CAT) serves as loading control. (E) Only DD-MyoH-NT is present at the conoid. (F) Plaque assays performed with DD-MyoH-NT, DD-MyoH-T, and DD-MyoH-NT-ΔATS1 were fixed after 7 days. A strong impairment in the lytic cycle was observed for DD-MyoH-NT and DD-MyoH-NT-ΔATS1 in the presence of Shld-1.

Fig 5. T. gondii myosin H is associated with three myosin light chains.

(A) Endogenously tagged myosin light chains (MLC) 3, 5 and 7 localized to the conoid. Scale bar 2 μm. (B) MLC5 localization at the conoid is dependent upon the presence of TgMyoH. Scale bar 2 μm. (C) MLC7 localization at the conoid is independent on the presence of TgMyoH. Scale bar 2 μm. (D) No decrease of the MLC5 level was observed by western blot upon depletion of TgMyoH. Profilin (PRF) serves as loading control. (E) A decrease of the MLC7 level was observed by western blot upon depletion of TgMyoH. Profilin (PRF) serves as loading control. (F) Plaque assays performed with RH (parental strain), mlc5-KO, mlc7-KO and mlc5&7-KO lines and fixed after 7 days showed no defect in the lytic cycle. (G) TgMyoH localization at the conoid is independent on the presence of MLC5 and MLC7. (H) Metabolic labeling followed by Co-IP experiments performed with anti-Ty antibodies on MyoH-3Ty and MyoH-3Ty-mlc5&7-KO strains. A band corresponding to the size of MLC1 was detected.

Fig 6. T. gondii myosin H is part of a conoidal glideosome and triggers the first step of invasion.

(A) Invasion pulse of 7 min using myoH-iKD + 48 h ATc, myoA-KO and a parental strain. The surface antigen 1 (SAG1) and the rhoptry neck secreted protein (RON4) were used to visualize respectively the parasite surface and the circular junction formed by invading parasites. The host cell plasma membrane is illustrated by dashed white lines. Depletion of MyoH blocked most of the parasites at the attachment step, prior to circular junction translocation (RON4 is detectable as a dot). The myoA-KO are blocked predominantly during conoid penetration, probably at the IMC level (RON4 is visible as a ring below the conoid). Most of the parental parasites completed penetration but some of them were captured at any step of the entry process (RON4 is detectable as ring at various position along the parasite). Scale bar 1 μm. (B) Quantification of the invasion pulse described in (a). Parasites were scored as attached, conoid invaded up to the IMC or post conoid invaded. Results are expressed as mean ± SD. (C) Schematic representation of the MyoH and MyoA relay at the conoid–IMC interface delineated by ISP1 (IMC sub-compartment protein 1). The scheme represents a parasite in the myoA-KO situation as described in (a). HPM: host plasma membrane. (D) Co-localization of SAG1 with the apical cap marker ISP1 after pulse invasion in the myoA-KO cell line. Scale bar 2 μm.

Fig 7. Model.

(A) Electron micrographs depicting the conoid in non-protruded and protruded parasites. (B) Schematic representation of the MyoH and MyoA relay at the conoid-IMC interphase. MyoH (red hoops) localized at the upper part of the conoid, close to the pre-conoidal rings (PCR). The translocation of the adhesin complexes by MyoH (blue arrow), probably via actin, are further relayed at the level of the IMC by MyoA-GAP70 in the cap region and then by MyoA-GAP45 along the rest of the parasites (see Fig 6C). APR, apical polar ring; SPM, subpellicular microtubules; PPM, parasite plasma membrane. (C) Upper view of the conoid with MyoH initiating the translocation of the adhesin complexes in a possible corkscrew-like trajectory which is likely relayed at the level of the subpellicular microtubules indirectly connected to the MyoA-glideosome.