Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step

Abstract

Toxoplasma gondii CDPK1 (TgCDPK1) was found to be the target of the toxoplasmocidal compound 1NM-PP1. When TgCDPK1 was mutated at position 128 from glycine to methionine, resistance was gained. Inhibition of gliding motility without inhibition of micronemal secretion by 1NM-PP1 suggests a function for TgCDPK1 in gliding motility.

Fig. 1.

Toxoplasmocidal properties of 1NM-PP1 and its putative targets. (A) The reduction in OD₆₀₀ values from mock-infected wells was calculated as the monolayer disruption capacity. Capacity in the absence of 1NM-PP1 was estimated to be 100%. The structure of 1NM-PP1 is also illustrated. The standard errors of the means from triplicate experiments are shown. (B) Alignment of subdomain V of 12 predicted as-kinases and the human protein kinase A (PKA) catalytic subunit alpha are shown. The gatekeeper residues are shown in the red box. Predicted secondary structures are indicated with an S (β-sheet) or H (α-helix) on the first line. (C) Kinetics properties of GST-TgCDPK1 with substrate peptide syntide-2 and effects of 1NM-PP1 on GST-TgCDPK1 and GST-TgCDPK1G128M. Reactions were performed with 1.0 ng of kinase in 30 μl of reaction buffer (20 mM HEPES [pH 7.5], 10 mM MgCl₂, 1 mg/ml bovine serum albumin, 100 μM CaCl₂, 2 μM dithiothreitol, 2 μM ATP, and 5.0 μCi [γ-³²P]ATP). For the inhibitory assay, 100 μM syntide-2 was used.

Fig. 2.

1NM-PP1-resistant strain RH/ai-C1. (A) Confirmation of Flag-tagged TgCDPK1G128M expression in RH/TgCDPK1_G128MFlag based on Western blotting. Vero cells infected with RH/ht⁻, RH/TgCDPK1_G128MFlag, or mock infected were subjected to a Western blot assay with the anti-M2 Flag antibody (α-M2Flag; Sigma, St. Louis, MO) (upper panel) or anti-T. gondii aldolase rabbit antibody newly raised as described previously (15) against myelin basic protein-TgALD1 expressed in Escherichia coli (24), in order to load adequate amounts of parasites. (B) Effects of 1NM-PP1 on the overall life cycle with RH/ht⁻ or RH/TgCDPK1_G128MFlag in the host monolayer disruption assay. OD₆₀₀ values in the absence of infection and without 1NM-PP1 were estimated as 100%. (C) Effects of 1NM-PP1 on RH/ht⁻ and RH/TgCDPK1_G128MFlag during parasite invasion. Invasion rates were calculated from the number of completely invaded parasites per number of whole parasites counted. Invasion rates of RH/ht⁻ in the absence of 1NM-PP1 were estimated as 100%. (D) Effects of 1NM-PP1 on gliding motility with RH/ht⁻ or RH/TgCDPK1_G128MFlag. Gliding trails were visualized with anti-SAG1 antibody. (E) Effects of 1NM-PP1 on secretions. The 35-kDa bands of TgM2AP are shown. In panels C to E, parasites were pretreated with 1NM-PP1, 1 μM staurosporine (S), or DMSO (D) at the listed concentrations. In panels B and C, the standard errors of the means from triplicate experiments are shown. **, P < 0.01, two-tailed Student's t test.