Figure 4.

Conditional mutagenesis enables a tetracycline-regulated knockdown of TgPEPCK_mt in glycolysis-impaired tachyzoites. A, scheme for generating a tetracycline-inducible mutant of TgPEPCK_mt (iΔtgpepck_mt) in a GT1 knock-out strain with impaired glycolysis (RHΔku80-TaTi-Δtggt1). In the first step, an ATc-repressible ORF of TgPEPCK_mt-HA was integrated at the TgUPRT locus by FUdR selection, and then the TgPEPCK_mt gene was replaced by DHFR-TS. The eventual Δtggt1/iΔtgpepck_mt mutant was identified by genomic PCR using 5′ and 3′-crossover-specific primers (TgPEPCK_mt-KO-5′Scr-F1/R1 and TgPEPCK_mt-KO-3′Scr-F1/R1). B, PCR confirming the regulation of TgPEPCK_mt transcript by ATc in the Δtggt1/iΔtgpepck_mt mutant. Total parasite RNA was used to amplify TgPEPCK_mt-HA and TgFBP2 (control for RNA integrity) using ORF-specific primers. C, immunostaining of the Δtggt1/iΔtgpepck_mt strain showing ATc regulation of TgPEPCK_mt-HA protein. The untreated control and drug-treated parasites were stained using α-HA and α-TgGap45 (a marker of inner membrane complex) antibodies. D, immunoblot depicting the ATc-mediated repression of TgPEPCK_mt-HA in the conditional mutant. Parasites (10⁷) were subjected to immunoblot analyses using α-HA and TgHsp90 (loading control) antibodies.