Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress

Abstract

Members of the family of calcium dependent protein kinases (CDPK’s) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii.

Author Summary

Toxoplasma gondii can cause severe disease and death in the immunocompromised and in those infected congenitally. Due to limitations of existing drugs there is a need for studying proteins that are unique and essential to the parasite. We recently established that TgCDPK3, a member of a family of calcium dependent protein kinase present in plants and some parasites but absent in human cells, regulates parasite egress from the host cell. While it has been hypothesized that TgCDPK3 controls rapid exit from the host by phosphorylating proteins needed for activating motility, the particular substrates of this kinase remained unknown. We have now applied an interaction trap system to identify the proteins that are modified by this kinase, which include a parasite motor protein Myosin A (TgMyoA). We show that TgCDPK3 specifically phosphorylates TgMyoA and this phosphorylation is important for parasite egress and motility.