Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9.

Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics

Comparative Proteomic Analysis of Toxoplasma gondii RH Wild-Type and Four SRS29B (SAG1) Knock-Out Clones Reveals Significant Differences between Individual Strains.

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 generated knock-out (KO) clones of TgRH88_077450 (SRS29B; SAG1) which differed in the numbers of the 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 shapes, dimensions, and vitality. This prompted us to investigate the impact of genetic engineering 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. In addition to this protein, 20 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

In Vitro versus in Mice: Efficacy and Safety of Decoquinate and Quinoline-O-Carbamate Derivatives against Experimental Infection with Neospora caninum Tachyzoites.

The effects of decoquinate (DCQ) and three O-quinoline-carbamate-derivatives were investigated using human foreskin fibroblasts (HFF) infected with Neospora caninum tachyzoites. These compounds exhibited half-maximal proliferation inhibition (IC50s) from 1.7 (RMB060) to 60 nM (RMB055). Conversely, when applied at 5 (DCQ, RMB054) or 10µM (RMB055, RMB060), HFF viability was not affected. Treatments of infected cell cultures at 0.5µM altered the ultrastructure of the parasite mitochondrion and cytoplasm within 24 h, most pronounced for RMB060, and DCQ, RMB054 and RMB060 did not impair the viability of splenocytes from naïve mice. Long-term treatments of N. caninum-infected HFF monolayers with 0.5µM of each compound showed that only exposure to RMB060 over a period of six consecutive days had a parasiticidal effect, while the other compounds were not able to kill all tachyzoites in vitro. Thus, DCQ and RMB060 were comparatively assessed in the pregnant neosporosis mouse model. The oral application of these compounds suspended in corn oil at 10 mg/kg/day for 5 d resulted in a decreased fertility rate and litter size in the DCQ group, whereas reproductive parameters were not altered by RMB060 treatment. However, both compounds failed to protect mice from cerebral infection and did not prevent vertical transmission/pup mortality. Thus, despite the promising in vitro efficacy and safety characteristics of DCQ and DCQ-derivatives, proof of concept for activity against neosporosis could not be demonstrated in the murine model

Differential Affinity Chromatography Coupled to Mass Spectrometry: A Suitable Tool to Identify Common Binding Proteins of a Broad-Range Antimicrobial Peptide Derived from Leucinostatin.

Leucinostatins are antimicrobial peptides with a broad range of activities against infectious agents as well as mammalian cells. The leucinostatin-derivative peptide ZHAWOC_6027 (peptide 6027) was tested in vitro and in vivo for activity against the intracellular apicomplexan parasite Toxoplasma gondii. While highly efficacious in vitro (EC50 = 2 nM), subcutaneous application of peptide 6027 (3 mg/kg/day for 5 days) in mice experimentally infected with T. gondii oocysts exacerbated the infection, caused mild clinical signs and elevated cerebral parasite load. Peptide 6027 also impaired the proliferation and viability of mouse splenocytes, most notably LPS-stimulated B cells, in vitro. To identify common potential targets in Toxoplasma and murine splenocytes, we performed differential affinity chromatography (DAC) with cell-free extracts from T. gondii tachyzoites and mouse spleens using peptide 6027 or an ineffective analogue (peptide 21,358) coupled to N-hydroxy-succinimide sepharose, followed by mass spectrometry. Proteins specifically binding to peptide 6027 were identified in eluates from the peptide 6027 column but not in peptide 21,358 nor the mock column eluates. In T. gondii eluates, 269 proteins binding specifically to peptide 6027 were identified, while in eluates from mouse spleen extracts 645 proteins specifically binding to this peptide were detected. Both datasets contained proteins involved in mitochondrial energy metabolism and in protein processing and secretion. These results suggest that peptide 6027 interacts with common targets in eukaryotes involved in essential pathways. Since this methodology can be applied to various compounds as well as target cell lines or organs, DAC combined with mass spectrometry and proteomic analysis should be considered a smart and 3R-relevant way to identify drug targets in pathogens and hosts, thereby eliminating compounds with potential side effects before performing tedious and costly safety and efficacy assessments in animals or humans

In vitro and in vivo activities of a trithiolato-diRuthenium complex conjugated with sulfadoxine against the apicomplexan parasite Toxoplasma gondii.

Organometallic compounds, including Ruthenium complexes, have been widely developed as anti-cancer chemotherapeutics, but have also attracted much interest as potential anti-parasitic drugs. Recently hybrid drugs composed of organometallic Ruthenium moieties that were complexed to different antimicrobial agents were synthesized. One of these compounds, a trithiolato-diRuthenium complex (RU) conjugated to sulfadoxine (SDX), inhibited proliferation of Toxoplasma gondii tachyzoites grown in human foreskin fibroblast (HFF) monolayers with an IC50 < 150 nM, while SDX and the non-modified RU complex applied either individually or as an equimolar mixture were much less potent. In addition, conjugation of SDX to RU lead to decreased HFF cytotoxicity. RU-SDX did not impair the in vitro proliferation of murine splenocytes at concentrations ranging from 0.1 to 0.5 μM but had an impact at 2 μM, and induced zebrafish embryotoxicity at 20 μM, but not at 2 or 0.2 μM. RU-SDX acted parasitostatic but not parasiticidal, and induced transient ultrastructural changes in the mitochondrial matrix of tachyzoites early during treatment. While other compounds that target the mitochondrion such as the uncouplers FCCP and CCCP and another trithiolato-Ruthenium complex conjugated to adenine affected the mitochondrial membrane potential, no such effect was detected for RU-SDX. Evaluation of the in vivo efficacy of RU-SDX in a murine T. gondii oocyst infection model comprised of non-pregnant outbred CD1 mice showed no effects on the cerebral parasite burden, but reduced parasite load in the eyes and in heart tissue

Common Molecular Targets of a Quinolone Based Bumped Kinase Inhibitor in Neospora caninum and Danio rerio.

Neospora caninum is an apicomplexan parasite closely related to Toxoplasma gondii, and causes abortions, stillbirths and/or fetal malformations in livestock. Target-based drug development has led to the synthesis of calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs). Previous studies have shown that several BKIs have excellent efficacy against neosporosis in vitro and in vivo. However, several members of this class of compounds impair fertility in pregnant mouse models and cause embryonic malformation in a zebrafish (Danio rerio) model. Similar to the first-generation antiprotozoal drug quinine, some BKIs have a quinoline core structure. To identify common targets in both organisms, we performed differential affinity chromatography with cell-free extracts from N. caninum tachyzoites and D. rerio embryos using the 5-aminopyrazole-4-carboxamide (AC) compound BKI-1748 and quinine columns coupled to epoxy-activated sepharose followed by mass spectrometry. BKI-binding proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from BKI-1748 as well as quinine columns. In N. caninum, 12 proteins were bound specifically to BKI-1748 alone, and 105 proteins, including NcCDPK1, were bound to both BKI-1748 and quinine. For D. rerio, the corresponding numbers were 13 and 98 binding proteins, respectively. In both organisms, a majority of BKI-1748 binding proteins was involved in RNA binding and modification, in particular, splicing. Moreover, both datasets contained proteins involved in DNA binding or modification and key steps of intermediate metabolism. These results suggest that BKI-1748 interacts with not only specific targets in apicomplexans, such as CDPK1, but also with targets in other eukaryotes, which are involved in common, essential pathways

Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii In Vitro and in Pregnant Mice Infected with T. gondii Oocysts

Abstract: The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, 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 selectivity 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, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC50 of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The 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

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

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

S1 Raw data -

(XLSX)</p