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Single TaqMan-qPCR for determining the copy number of integrated <i>mdhfr-ts</i> selectable marker.

Standard curves were made through a triplicate test of 10-fold serial dilutions of (A) P972 or (B) T. gondii RH DNA. (C) For each WT or KO clone, the number of existing dhfr-ts in the genome was determined according to the plasmid based standard curve (black bars) and the T. gondii RH DNA-based calibrator (grey bars). Since in the T. gondii genome the wtdhfr-ts is a single copy gene, the following equation was used: one WT tachyzoite = one-copy dhfr-ts, for the calculation based on T. gondii RH DNA based calibrator curve (grey bars). Error bars indicate standard deviation of triplicates for each sample. In (D), the number of inserted mdhfr-ts in each KO clone is defined by subtracting the dhfr-ts copy number found in the WT from the dhfr-ts copy number in the KO (black bars) or by subtracting the tachyzoite numbers determined for the WT from tachyzoite numbers corresponding the KO clone (grey bars). The optimal result of 1 indicates a single integration event of the mdhfr-ts into sag1.</p

Principle and potential outcome of the duplex TaqMan-qPCR.

Principle and potential outcome of the duplex TaqMan-qPCR.</p

Principle and potential outcomes of the single TaqMan-qPCR.

Principle and potential outcomes of the single TaqMan-qPCR.</p

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Proteasome activity in <i>E. multilocularis</i> cell extracts can be inhibited by BTZ.

<p>Chymotrypsin-like activity of the <i>E. multilocularis</i> proteasome was shown in cell extracts from in vitro-cultured metacestodes by applying the fluorogenic substrate SLLVT-AMC in solution (A) and in gel (B). Addition of BTZ led to dose-dependent inhibition of this activity. A, in solution proteasome assay with and without Halt protease inhibitor (to inhibit other proteases). As a control DMSO was added to same amounts as with the drug and the activity calculated as percentage from the DMSO control. This experiment was performed in technical quadruplicates and was repeated three times, which led to similar results each time. B, in gel assay to detect chymotrypsin-like activity in <i>E. multilocularis</i> cell extracts. Bovine chymotrypsin was used as positive control (C, 5 ng per lane), <i>E. multilocularis</i> extract was loaded at 25 µg. On the left the gel was incubated with DMSO, on the right with BTZ at 10 µM. C, Western blot was performed to detect the <i>E. multilocularis</i> proteasome subunit beta 5 (EmPSMB5). Note that this protein migrates at the same apparent molecular mass as the active band in B that could be inhibited by BTZ. Experiments B and C were repeated both twice leading to the same results.</p

Activity of the most active drugs of the FDA library against <i>E. multilocularis</i> metacestodes in vitro.

<p>The seven most active drugs (more than 50% damage of the control DB1127 at 20 µM after 5 days; BTZ, sorafenib tosylate, crystal violet, candesartan cilexetil, nitazoxanide, amlodipine besylate and axitinib) of the FDA drug library screen were further tested in triplicates by PGI assay at different concentrations (20, 10, 5, 1, 0.1 µM) and for their anti-metacestode activity in vitro. As a positive control, DB1127 was applied at 20 µM and the different drug activities are expressed as percentage of the positive control. DMSO served as a negative control at same dilutions as the drugs and was subtracted from all values. Note the high activity of BTZ down to the concentration of 0.1 µM.</p

Loss of <i>sag1</i> expression in <i>T</i>. <i>gondii</i> RH SAG1 knockouts by (A) Western blot analysis and (B) immunofluorescence.

Loss of sag1 expression in T. gondii RH SAG1 knockouts by (A) Western blot analysis and (B) immunofluorescence.</p

SAG1 gene disruption in <i>T</i>. <i>gondii</i> RH by CRISPR-Cas9 technology.

(A) Schematic representation of the strategy used to disrupt sag1 by inserting the pyrimethamine-resistance gene MDHFR-TS. (B) Diagnostic PCR revealing integration of a complete mdhfr-ts sequence into sag1 in four clones (C18, C23, C30 and C33) compared with the parental strain RH. The KO clone C31 showed a smaller band, clones C6 and 7 exhibited a band ≤ 1000 bp. The WT locus produced the expected PCR product (~ 216 bp).</p

Duplex TaqMan-qPCR for determining copy numbers of integrated <i>mdhfr-ts</i> selectable marker.

(A) Standard curve was made by using a 10-fold serial dilution of T. gondii RH DNA, with tachyzoites numbers ranging from 75 to 7.5 x 105 parasites. (B) For each WT or KO clone, the numbers of tachyzoites in the 3 ng DNA was determined according to amplification of dhfr-ts (black bars) and to the T. gondii 529 bp repeat element (grey bars). In (C), the number of inserted mdhfr-ts is given by the ratio of the number of tachyzoites as determined by dhfr-ts amplification and the number of tachyzoites determined by using the T. gondii 529 bp repeat element. A ratio equal to 2 indicates a single integration event of the mdhfr-ts in sag1. Error bars indicate standard deviations of triplicates for each sample.</p