Date Published: November 6, 2015
Publisher: Public Library of Science
Author(s): Rajshekhar Y. Gaji, Derrick E. Johnson, Moritz Treeck, Mu Wang, Andy Hudmon, Gustavo Arrizabalaga, Michael J Blackman.
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.
The phylum Apicomplexa encompasses numerous obligate intracellular parasites that pose a significant health risk to animals and humans. Among these, Toxoplasma gondii is one of the most widespread, infecting all warm-blooded animals including approximately one third of the human population. Humans become infected congenitally or by ingestion of either environmental oocysts, which are shed in the feces of cats, or tissue cysts in the undercooked meat of infected animals. Most infections are asymptomatic during the acute stage but as to evade the immune response the parasite converts to a latent encysted form, thus establishing a chronic infection. In immunocompromised individuals and lymphoma patients, new infections or rupture of pre-existing cysts can lead to life-threatening toxoplasmic encephalitis [1–3]. Additionally, in congenital infections, toxoplasmosis can lead to blindness, severe neurological problems, or even death, given the immature nature of the fetal immune system .
Although it is well established that TgCDPK3 is important for parasite egress, the particular mechanism by which this calcium-stimulated kinase regulates this key event of the lytic cycle is not known. A recent study revealed 156 phosphorylation sites out of more than 12,000 quantified that were differentially phosphorylated between wild type and Tgcdpk3 mutant parasites, with many of them related to motility, ion-homeostasis and metabolism . While some of these differentially phosphorylated sites might be direct substrates of TgCDPK3, one might expect that a number of these sites related to downstream effects and compensatory mechanisms related to a loss in TgCDPK3 signaling. In addition, TgCDPK3 is involved in several processes such as calcium homeostasis and parasite division, which might involve distinct substrates from those involved in egress. Therefore, additional efforts were needed to identify the specific protein(s) whose phosphorylation by TgCDPK3 is key for induced egress and initiation of motility. With this in mind we successfully adapted the BioID system to identify putative substrates and interactors of TgCDPK3. This approach, which is based on fusing the protein of interest to a promiscuous allele of the biotin ligase BirA, has the advantage that it can identify not only direct interactors but also proteins that are nearby or interact loosely or transiently, such as enzyme substrates. Identification by proximity labeling does not prove an enzyme/substrate relationship, and it is plausible that some of these interactions are structural in nature. However, in combination with our previous phosphoproteome analysis , which identified seven of the thirteen proteins identified through BioID as less phosphorylated in Tgcdpk3 mutant parasites, it provides important indirect evidence for a kinase/ substrate relationship. Having been linked to TgCDPK3 in two independent and distinct approaches makes these seven proteins strong candidates for being TgCDPK3 substrates during the events regulated by this kinase.