Serine protease inhibitors block invasion of host cells by Toxoplasma gondii

Abstract

We investigated the effect of protease inhibitors on the asexual development of the protozoan parasite Toxoplasma gondii. Among the inhibitors tested only two irreversible serine protease inhibitors, 3,4-dichloroisocoumarin and 4-(2-aminoethyl)-benzenesulfonyl fluoride, clearly prevented invasion of the host cells by specifically affecting parasite targets in a dose-dependent manner, with 50% inhibitory concentrations between 1 and 5 and 50 and 100 microM, respectively. Neither compound significantly affected parasite morphology, basic metabolism, or gliding motility within the range of the experimental conditions in which inhibition of invasion was demonstrated. No partial invasion was observed, meaning that inhibition occurred at an early stage of the interaction. These results suggest that at least one serine protease of the parasite is involved in the invasive process of T. gondii.

FIG. 1

Inhibition of T. gondii growth by various concentrations of 3,4-DCI and AEBSF measured by β-galactosidase assay. Extracellular tachyzoites were first pretreated for 1 h with inhibitors, and then cell infection was permitted for 1 h in the presence of inhibitors. Control values (C) were obtained after treatments with medium supplemented with dimethyl sulfoxide or water for DIC and AEBSF, respectively. Each value is the mean of three experiments done in triplicate + the standard deviation. The asterisks indicate the significant inhibitions compared with the control: ∗, P < 0.05; ∗∗, P < 0.01.

FIG. 2

Effect of 3,4-DCI and AEBSF concentrations on T. gondii growth as measured by β-galactosidase assay after a 1-h treatment of host cells or extracellular tachyzoites before infection in the absence of inhibitors. Each value is the mean of three experiments done in triplicate + the standard deviation, expressed as percentages of values (optical density [O.D.]) for control treatment. The asterisks indicate the significant inhibitions compared with the control: ∗, P < 0.05; ∗∗, P < 0.01.

FIG. 3

Comparison of the effect of various concentrations of 3,4-DCI (A) and AEBSF (B) on T. gondii growth and host cell invasion. Extracellular tachyzoites were pretreated for 1 h, and cell infection was performed for 1 h. Each value is the mean of three experiments (performed in triplicate) + the standard deviation, expressed as percentages of values (optical density [O.D.]) for control treatment. The asterisks indicate the significant inhibitions compared with the control: ∗, P < 0.05; ∗∗, P < 0.01.

FIG. 4

Time dependency effect of 3,4-DCI on tachyzoite gliding: average trail distance (A) and percentage of tachyzoites associated with trails (B). A gliding assay was performed with the inhibitor either for 30 min with no pretreatment or for 1 h after 1 h of pretreatment (2 h of treatment). The data presented are from a representative experiment with a 3,4-DCI concentration (10 μM) leading to 93.4% inhibition of invasion after a 30-min treatment.