Figure 4: Differential Gene Expression (DGE) analyses and effects of inhibition of cytochrome bc1.

(a) DGE analysis of bradyzoite- and tachyzoite-specific markers during EGS infections of HFF cultures at 2, 18 and 48 hours (top panel), MM6 cells at 18 hours (middle panel) or NSC cultures at 18 hours (bottom panel) versus infections of same host cells with canonical strains GT1, ME49 or VEG at 18 hours (averaged across the three canonical strains for HFF infections). Genes reported as being over- or under-expressed during bradyzoite differentiation is indicated with red or green arrows respectively. “*”, q-value ≤ 0.05; LogFC, logarithm of the fold change in gene expression. CST1, SAG-related sequence SRS44^s114; LDH2, lactate dehydrogenase 2^S115; LDH1, lactate dehydrogenase 1^S115; ENO2, enolase 2; ENO1, enolase 1^S116; SAG1, SAG-related sequence SRS29B; BAG1, bradyzoite antigen BAG1^S115. (b) DGE analysis of genes encoding AP2 family of transcription factor during the same infection experiments as described in (a). Red and green arrows denote AP2 genes found to be over- or under-expressed during bradyzoite development^S117. “*”, q-value ≤ 0.05. (c) T. gondii cytochrome family genes differentially expressed during same experimental infections as described in (a). “*”, q-value ≤ 0.05. (d) Effect of known cytochrome b inhibitors on EGS. Morpholino conjugated to a Vivoporter (called PPMO) designed to knock down cytochrome b compared with off target control has a significant effect in reducing replication of YFP RH strain tachyzoites at 5 and 10 μM (p < 0.05) but only a very small effect on size and number of EGS cysts in HFF. As a poorly soluble inhibitor of cytochrome b, ELQ271 was reported to partially reduce cyst numbers in mice²⁷ and is shown herein also to reduce the EGS cysts in vitro at 10 μM in this novel model. This demonstrates the utility of this novel in vitro model by indicating that inhibition of cytochrome b Qi is associated with reduction of cysts in vivo in a mouse model, even when there are serious limitations caused by insolubility of this inhibitory compound. This poor solubility significantly limits ELQ271 as a candidate for progression to a medicine. Increasing selectivity for the parasite enzyme with our new scaffold is another critical challenge.