Abstract
ABSTRACTIn the protozoan parasite Toxoplasma gondii, protein kinases have been shown to play key roles in regulating parasite motility, invasion, replication, egress, and survival within the host. The tyrosine kinase-like (TKL) family of proteins are an unexplored set of kinases in Toxoplasma. Of the eight annotated TKLs in the Toxoplasma genome, a recent genome-wide loss-of-function screen showed that six are important for tachyzoite fitness. By utilizing an endogenous tagging approach, we showed that these six T. gondii TKLs (TgTKLs) localize to various subcellular compartments, including the nucleus, the cytosol, the inner membrane complex, and the Golgi apparatus. To gain insight into the function of TKLs in Toxoplasma, we first characterized TgTKL1, which contains the plant-like enhanced disease resistance 1 (EDR1) domain and localizes to the nucleus. TgTKL1 knockout parasites displayed significant defects in progression through the lytic cycle; we show that the defects were due to specific impairment of host cell attachment. Transcriptomics analysis identified over 200 genes of diverse functions that were differentially expressed in TgTKL1 knockout parasites. Importantly, numerous genes implicated in host cell attachment and invasion were among those most significantly downregulated, resulting in defects in microneme secretion and processing. Significantly, all of the mice inoculated intraperitoneally with TgTKL1 knockout parasites survived the infection, suggesting that TgTKL1 plays an essential role in acute toxoplasmosis. Together, these findings suggest that TgTKL1 mediates a signaling pathway that regulates the expression of multiple factors required for parasite virulence, underscoring the potential of this kinase as a novel therapeutic target.
Highlights
In the protozoan parasite Toxoplasma gondii, protein kinases have been shown to play key roles in regulating parasite motility, invasion, replication, egress, and survival within the host
Four of the eight T. gondii TKLs (TgTKLs) are differentially expressed between the acute and chronic stages of murine infection; TgTKL8 expression is increased during chronic infection, while the expression of TgTKL1, TgTKL2, and TgTKL5 is decreased during chronic infection [39]
Endogenous tagging of the six TgTKLs that have recently been shown to be important for tachyzoite fitness in vitro showed that members of this group localize to multiple subcellular compartments, including the nucleus, the cytoplasm, the inner membrane complex (IMC), and the Golgi apparatus
Summary
In the protozoan parasite Toxoplasma gondii, protein kinases have been shown to play key roles in regulating parasite motility, invasion, replication, egress, and survival within the host. All of the mice inoculated intraperitoneally with TgTKL1 knockout parasites survived the infection, suggesting that TgTKL1 plays an essential role in acute toxoplasmosis Together, these findings suggest that TgTKL1 mediates a signaling pathway that regulates the expression of multiple factors required for parasite virulence, underscoring the potential of this kinase as a novel therapeutic target. Members of seven of the nine groups of eukaryotic protein kinases (ePK) [26, 27] are present; Toxoplasma lacks members of the receptor guanylate cyclase (RGC) and tyrosine kinase (TK) groups In addition to these standard ePKs, Toxoplasma contains an expanded group of coccidian-specific secreted rhoptry kinases (ROPK) that includes important virulence factors [22, 26, 28,29,30,31]. Functional characterization of Toxoplasma kinases has shown that many of these proteins have potential as drug targets, and identification and characterization of novel inhibitors of parasite kinases are active areas of research [32,33,34]; while representatives of many of the ePK groups have been functionally characterized in Toxoplasma, members of the tyrosine kinase-like (TKL) kinase group have remained completely unstudied
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