Two Essential Light Chains Regulate the MyoA Lever Arm To Promote Toxoplasma Gliding Motility

Abstract

Key to the virulence of apicomplexan parasites is their ability to move through tissue and to invade and egress from host cells. Apicomplexan motility requires the activity of the glideosome, a multicomponent molecular motor composed of a type XIV myosin, MyoA. Here we identify a novel glideosome component, essential light chain 2 (ELC2), and functionally characterize the two essential light chains (ELC1 and ELC2) of MyoA in Toxoplasma. We show that these proteins are functionally redundant but are important for invasion, egress, and motility. Molecular simulations of the MyoA lever arm identify a role for Ca(2+) in promoting intermolecular contacts between the ELCs and the adjacent MLC1 light chain to stabilize this domain. Using point mutations predicted to ablate either the interaction with Ca(2+) or the interface between the two light chains, we demonstrate their contribution to the quality, displacement, and speed of gliding Toxoplasma parasites. Our work therefore delineates the importance of the MyoA lever arm and highlights a mechanism by which this domain could be stabilized in order to promote invasion, egress, and gliding motility in apicomplexan parasites.

Importance: Tissue dissemination and host cell invasion by apicomplexan parasites such as Toxoplasma are pivotal to their pathogenesis. Central to these processes is gliding motility, which is driven by an actomyosin motor, the MyoA glideosome. Others have demonstrated the importance of the MyoA glideosome for parasite motility and virulence in mice. Disruption of its function may therefore have therapeutic potential, and yet a deeper mechanistic understanding of how it works is required. Ca(2+)-dependent and -independent phosphorylation and the direct binding of Ca(2+) to the essential light chain have been implicated in the regulation of MyoA activity. Here we identify a second essential light chain of MyoA and demonstrate the importance of both to Toxoplasma motility. We also investigate the role of Ca(2+) and the MyoA regulatory site in parasite motility and identify a potential mechanism whereby binding of a divalent cation to the essential light chains could stabilize the myosin to allow productive movement.

FIG 1

ELC2 is a novel component of the MyoA glideosome. (A) Western blot analysis of lysate from ELC2-HA, ELC1-HA, and parental (RHΔHX) parasites probed with mouse monoclonal anti-HA antibodies (left panel) and rabbit polyclonal anti-ELC1 antibodies (right panel). (B) Immunofluorescence analysis of intracellular parasites expressing ELC1-HA and ELC2-HA with anti-HA antibodies (green). The pellicle was detected by costaining with anti-GAP45 antibodies (red). (C) Immunoprecipitation (IP) of lysate from parental parasites or parasites expressing ELC1-HA or ELC2-HA with mouse monoclonal anti-HA antibodies. Eluates were analyzed by Western blotting with rabbit anti-HA antibodies (left panel) and with anti-GAP45, anti-MyoA, anti-MLC1 (right panels), and anti-ELC1 (middle panel). (D) Co-IP of the same lysate as that described for panel C with anti-GAP45 antibodies and nonspecific serum (NS) as a negative control. Eluates were analyzed by Western blotting with anti-HA antibodies.

FIG 2

ELC1 and ELC2 interact specifically with the MyoA neck. (A) Amino acid sequence of the MyoA neck region containing two degenerate IQ motifs (core consensus sequences are underlined). IQ motif 2 is the binding site for MLC1. (B) Schematic representation of transgenic MyoA neck coding sequences fused to N-terminal DDTy tags. The full MyoA neck sequence is 61 amino acids in length (residues 770 to 831). Truncation of residues 770 to 799 removed the predicted ELC binding site. (C) Western blot analysis of mouse monoclonal anti-Ty antibodies and of lysate from RHΔHX parasites expressing the transgenes shown in panel B. (D) Immunofluorescence analysis of ΔMyoA parasites transiently expressing DDTyMyoA_770-831 and DDTyMyoA_800-831. MyoA transgenes were stabilized by overnight treatment with Shld-1 and detected using mouse anti-Ty antibodies. Localization of glideosome components was detected with rabbit anti-GAP45, anti-MLC1, and anti-ELC1 antibodies. (E) Size exclusion chromatography of recombinant ELC1, ELC2 (blue), and MyoA neck (black) regions either separately or together (red). (F) Homology model of the MyoA lever arm predicting that key residues of the MyoA neck (green) interact with ELC1 (orange). (G) Schematic representation of a transgenic version of MyoA. The full MyoA coding sequence was N-terminally fused to His-Flag-StrepII tags, and three key residues predicted to interact with ELC1 (highlighted in panel F) were mutated to alanine. (H) Immunofluorescence analysis of HFS-MyoA and HFS-MyoA-_LLY/AAA expressed transiently in ΔMyoA parasites (13) by the use of mouse monoclonal anti-Flag tag antibodies. The localization of ELC1 was examined using anti-ELC1 antibodies. Bars, 5 µm.

FIG 3

Conditional deletion of ELC1 and ELC2 and simultaneous removal of both essential light chains. (A) Schematic representing the genetic methods used to regulate endogenous essential-light-chain expression. (i) The genetic strategy used to create ELC1 and ELC2 conditional knockouts. The endogenous promoter of ELC1 and ELC2 was replaced with 7 Tet operating sequences and the SAG4 minimal promoter (T7S4) in ΔKu80:TATi parasites (29). A HA₃ epitope tag was inserted at the N terminus of ELC1 and ELC2, and transformants were selected for the dhfr gene. In the absence of anhydrotetracycline (ATc), the Tet transactivator (TATi) drives transcription of ELC1/2. In the presence of ATc, transcription is switched off. (ii) The genetic strategy used to create ELC1 and ELC2 double-knockout parasites. The elc1 ORF was replaced with cat to confer chloramphenicol resistance in cKO-ELC2 parasites. (B) Western blot analysis of lysate from ΔKu80:TATi, cKO-ELC1, cKO-ELC2, and cDKO-ELC parasites following treatment with 1.0 µg/ml ATc. Lysates were probed with mouse anti-HA, rabbit anti-ELC1, and anti-catalase as a loading control. (C) Immunofluorescence analysis of intracellular cKO-ELC1 and cKO-ELC2 parasites grown in the presence and absence of 1.0 µg/ml ATc for 96 h. DAPI, 4′,6-diamidino-2-phenylindole. (D) Two-color invasion assay performed on parasites grown with or without ATc for 96 h. DDTy-ELC1 and DDTy-ELC2 expression was stabilized by 1.0 µM Shld-1 at ~10 h prior to invasion. Numbers of invaded and total parasites were counted from >5 fields per slide. (E) Fixed-egress assay performed on intracellular vacuoles grown for 30 h prior to stimulation with 8 µM A23187. More than 100 vacuoles were counted for each slide. In panels D and E, duplicate or triplicate slides were counted per experiment. Column data represent mean percentage results from 3 independent experiments. Error bars represent ± SEM.

FIG 4

Assembly of the MyoA glideosome is disrupted in the absence of two essential light chains. (A) Immunofluorescence analysis of cDKO-ELC parasites grown with or without ATc. MyoA was detected using anti-Flag in parasites expressing His-Flag-StrepII-MyoA, and the pellicle was marked using anti-GAP45 antibodies. Expression of MLC1 was detected using anti-MLC1 antibodies. Bars, 5 µm. (B) Western blot analysis of ΔKu80:TATi, cKO-ELC1, cKO-ELC2, and cDKO-ELC parasites grown in the absence or presence of ATc for 7 days and probed with anti-MyoA, anti-HA, and anti-catalase.

FIG 5

Ca²⁺ binds to ELC1 at EFI, and homology modeling predicts key interactions important for essential-light-chain function. (A) Structural model of MyoA, ELC1, and MLC1 based on the crystal structures of scallop myosin, with ADP simulated at the nucleotide-binding site of MyoA. A predicted interaction with Ca²⁺ is shown at EFI, coordinated by the side chains of ELC1 residues. Potential backbone interactions exist between ELC1 D17 and the side chain of MLC1 E179. (B) The interaction between ELC1 and MLC1 is Ca²⁺ dependent. The distances between the interacting residues shown in panel A were measured during simulations performed in the presence and absence of Ca²⁺. (C) A structural model of MyoA, MLC1, and ELC2 superimposed on the model shown in panel A. (D) The ATP-bound state of the homology model shown in panel A shows a conformational change of ELC1 EFI. (E) Thermal shift assays on recombinant wild-type ELC1 (i) and mutant ELC1 (ii and iii) detect interactions with Ca²⁺ by a shift in the temperature of peak fluorescence in the presence of Ca²⁺ compared to EGTA. (F) The Ca²⁺ dissociation constants (K_m) of the recombinant ELC1 proteins shown in panel E ± standard deviations (SD).

FIG 6

Molecular dissection of predicted interactions at the MyoA lever arm. (A) Schematic representation of ectopic versions of ELC1. N-terminal DDTy tags were fused to the ELC1 coding sequence, and mutations of key residues were introduced. (B) Immunofluorescence microscopy of intracellular cDKO-ELC parasites stably expressing ectopic wild-type and mutant DDTyELC1 proteins by the use of anti-Ty antibodies (green) and anti-GAP45 antibodies (red). (C and D) Parasites grown with or without 1.0 µg/ml ATc for 96 h and with 1.0 µM Shld-1 for at least 8 h prior to experiments. (C) Fixed-egress assay performed on intracellular vacuoles grown for 30 h prior to stimulation with 8 µM A23187 for 2.5 min. At least 100 vacuoles were counted per slide. WT, wild type. (D) Two-color invasion assay performed to determine percentages of invaded parasites after a 30-min infection period. Numbers of invaded and total parasites were counted from >5 fields per slide. (E) Schematic representation of ectopic MLC1. N-terminal DDTy tags were fused to the MLC1 coding sequence, and mutations of key residues (Fig. 5A) were introduced. (F) Immunofluorescence microscopy of complemented MLC1 proteins with anti-Ty demonstrates peripheral localization and costaining with glideosome marker anti-GAP45. Bars, 5 µm. (G) Egress assay of MLC1 KO parasites (11) expressing wild-type and mutant MLC1 as shown in panel E. Parasites were treated with 50 µM rapamycin for 4 h and then grown without rapamycin for ~96 h prior to egress. DDTyMLC1 proteins were stabilized by 1.0 µM Shld-1 for >8 h prior to egress. The experiments represented in panels C, D, and G used duplicate (C and D) or triplicate (G) slides per experiment. Columns represent mean percentages of the results from 3 to 4 independent experiments. Error bars represent ± SEM.

FIG 7

Intermolecular interactions at the MyoA lever arm are important for Toxoplasma gliding motility. Tachyzoite motility assays were performed by live video microscopy. (A and B) Mean percentages of motile parasites counted for 2 to 3 videos/experiment and for 3 separate experiments. Error bars represent ± SEM. (C and D) Mean values were determined by automatic tracking of individual parasites using the MTrackJ plugin for ImageJ. Values were determined from 2 to 3 videos/experiment, where n = 3 independent experiments. Error bars represent ± SEM. (E) Instantaneous speed of representative individual parasites tracked as described for panels C and D.

FIG 8

Model of the Toxoplasma MyoA glideosome. The composition of the MyoA glideosome in Toxoplasma tachyzoites, localized at the parasite periphery in the space between the plasma membrane (PM) and inner membrane complex (IMC), is shown.