In T. gondii, as well as in other model organisms, gene knock-out using CRISPR-Cas9 is a suitable tool to identify the role of specific genes. The general consensus implies that only the gene of interest is affected by the knock-out. Is this really the case? In a previous study, we have generated knock-out (KO) clones of TgRH88_077450 (SRS29B; SAG1) which differed in the numbers of integrated dihydrofolate-reductase-thymidylate-synthase (MDHFR-TS) drug-selectable marker. Clones 18 and 33 had a single insertion of MDHFR-TS within SRS29B. Clone 6 was disrupted by the insertion of a short unrelated DNA-sequence, but the marker was integrated elsewhere. In clone 30, the marker was inserted into SRS29B and several other MDHFR-TS copies were found in the genome. KO and wild-type (WT) tachyzoites had similar shape, dimensions and vitality. This prompted us to investigate the impact of the genetic engineering as such on the overall proteome patterns of the four clones as compared to the respective WT. Comparative shotgun proteomics of the five strains was performed. Overall, 3236 proteins were identified. Principal component analysis of the proteomes revealed five distinct clusters corresponding to the five strains by both iTop3 and iLFQ algorithms. Detailed analysis of the differentially expressed proteins revealed that the target of the KO, srs29B, was lacking in all KO clones. Besides this protein, twenty other proteins were differentially expressed between KO clones and WT or between different KO clones. The protein exhibiting the highest variation between the five strains was srs36D encoded by TgRH_016110. The deregulated expression of SRS36D was further validated by quantitative PCR. Moreover, the transcript levels of three other selected SRS genes, namely SRS36B, SRS46, and SRS57 exhibited significant differences between individual strains. These results indicate that knocking out a given gene may affect the expression of other genes. Therefore, care must be taken when specific phenotypes are regarded as a direct consequence of the KO of a given gene.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacte-rial and parasitic infections, most notably coccidiosis in poultry and in ruminants. We have in-vestigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. It induced distinct alterations in the parasite mitochondrion within 24h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selec-tivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ antici-pated to have better physicochemical properties than DCQ were assessed in vitro. One such com-pound RMB060 displayed an exceedingly low IC50 of 0.07 nM when applied concomitantly with infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6h after commencement of treatment. After 48h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features remi-niscent of bradyzoites. Exposure of infected cultures to 300 nM RMB060 for 52 days did not re-sult in complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. Treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.