Investigating Antiprotozoal Chemotherapies with Novel Proteomic Tools-Chances and Limitations: A Critical Review.

Identification of drug targets and biochemical investigations on mechanisms of action are major issues in modern drug development. The present article is a critical review of the classical "one drug"-"one target" paradigm. In fact, novel methods for target deconvolution and for investigation of resistant strains based on protein mass spectrometry have shown that multiple gene products and adaptation mechanisms are involved in the responses of pathogens to xenobiotics rather than one single gene or gene product. Resistance to drugs may be linked to differential expression of other proteins than those interacting with the drug in protein binding studies and result in complex cell physiological adaptation. Consequently, the unraveling of mechanisms of action needs approaches beyond proteomics. This review is focused on protozoan pathogens. The conclusions can, however, be extended to chemotherapies against other pathogens or cancer.

Transient Adaptation of Toxoplasma gondii to Exposure by Thiosemicarbazone Drugs That Target Ribosomal Proteins Is Associated with the Upregulated Expression of Tachyzoite Transmembrane Proteins and Transporters.

Thiosemicarbazones and their metal complexes have been studied for their biological activities against bacteria, cancer cells and protozoa. Short-term in vitro treatment with one gold (III) complex (C3) and its salicyl-thiosemicarbazone ligand (C4) selectively inhibited proliferation of T. gondii. Transmission Electron Microscopy (TEM) detected transient structural alterations in the parasitophorous vacuole membrane and the tachyzoite cytoplasm, but the mitochondrial membrane potential appeared unaffected by these compounds. Proteins potentially interacting with C3 and C4 were identified using differential affinity chromatography coupled with mass spectrometry (DAC-MS). Moreover, long-term in vitro treatment was performed to investigate parasitostatic or parasiticidal activity of the compounds. DAC-MS identified 50 ribosomal proteins binding both compounds, and continuous drug treatments for up to 6 days caused the loss of efficacy. Parasite tolerance to both compounds was, however, rapidly lost in their absence and regained shortly after re-exposure. Proteome analyses of six T. gondii ME49 clones adapted to C3 and C4 compared to the non-adapted wildtype revealed overexpression of ribosomal proteins, of two transmembrane proteins involved in exocytosis and of an alpha/beta hydrolase fold domain-containing protein. Results suggest that C3 and C4 may interfere with protein biosynthesis and that adaptation may be associated with the upregulated expression of tachyzoite transmembrane proteins and transporters, suggesting that the in vitro drug tolerance in T. gondii might be due to reversible, non-drug specific stress-responses mediated by phenotypic plasticity.

Single and combination treatment of Toxoplasma gondii infections with a bumped kinase inhibitor and artemisone in vitro and with artemiside in experimentally infected mice.

In previous studies, the artemisinin derivatives artemisone, its pro-drug artemiside and the bumped-kinase inhibitor BKI-1748 were effective against T. gondii via different modes of action. This suggests that they may act synergistically resulting in improved efficacies in vitro and in vivo. To test this hypothesis, the compounds were applied alone and in combination to T. gondii infected human fibroblast host cells in order to determine their inhibition constants and effects on cellular ultrastructure. In addition, the efficacy of either single- or combined treatments were assessed in an acute TgShSp1-oocyst infection model based on CD1 outbred mice. Whereas the IC50 of the compounds in combination (42 nM) was close to the IC50 of BKI-1748 alone (46 nM) and half of the IC50 of artemisone alone (92 nM), the IC90 of the combination was half of the values found with the single compounds (138 nM vs. ca. 270 nM). Another indication for synergistic effects in vitro were distinct alterations of the cellular ultrastructure of tachyzoites observed in combination, but not with the single compounds. These promising results could not be reproduced in vivo. There was no decrease in number of T. gondii positive brains by either treatment. However, the levels of infection in these brains, i. e. the number of tachyzoites, was significantly decreased upon BKI-1748 treatment alone, and the combination with artemiside did not produce any further decrease. The treatment with artemiside alone had no significant effects. A vertical transmission model could not be established since artemiside strongly interfered with pregnancy and caused abortion. These results show that is difficult to extrapolate from promising in vitro results to the situation in vivo.

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.

Synthesis and Antiparasitic Activity of New Trithiolato-Bridged Dinuclear Ruthenium(II)-arene-carbohydrate Conjugates.

Eight novel carbohydrate-tethered trithiolato dinuclear ruthenium(II)-arene complexes were synthesized using CuAAC 'click' (Cu(I)-catalyzed azide-alkyne cycloaddition) reactions, and there in vitro activity against transgenic T. gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) and in non-infected human foreskin fibroblasts, HFF, was determined at 0.1 and 1 µM. When evaluated at 1 µM, seven diruthenium-carbohydrate conjugates strongly impaired parasite proliferation by >90%, while HFF viability was retained at 50% or more, and they were further subjected to the half-maximal inhibitory concentration (IC50) measurement on T. gondii ß-gal. Results revealed that the biological activity of the hybrids was influenced both by the nature of the carbohydrate (glucose vs. galactose) appended on ruthenium complex and the type/length of the linker between the two units. 23 and 26, two galactose-based diruthenium conjugates, exhibited low IC50 values and reduced effect on HFF viability when applied at 2.5 µM (23: IC50 = 0.032 µM/HFF viability 92% and 26: IC50 = 0.153 µM/HFF viability 97%). Remarkably, compounds 23 and 26 performed significantly better than the corresponding carbohydrate non-modified diruthenium complexes, showing that this type of conjugates are a promising approach for obtaining new antiparasitic compounds with reduced toxicity.

Synthesis, Photophysical Properties and Biological Evaluation of New Conjugates BODIPY: Dinuclear Trithiolato-Bridged Ruthenium(II)-Arene Complexes.

The synthesis, photophysical properties and antiparasitic efficacy against Toxoplasma gondii ß-gal (RH strain tachyzoites expressing ß-galactosidase) grown in human foreskin fibroblast monolayers (HFF) of a series of 15 new conjugates BODIPY-trithiolato-bridged dinuclear ruthenium(II)-arene complexes are reported (BODIPY=4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, derivatives used as fluorescent markers). The influence of the bond type (amide vs. ester), as well as that of the length and nature (alkyl vs. aryl) of the spacer between the dye and the diruthenium(II) complex moiety, on fluorescence and biological activity were evaluated. The assessed photophysical properties revealed that despite an important fluorescence quenching effect observed after conjugating the BODIPY to the diruthenium unit, the hybrids could nevertheless be used as fluorescent tracers. Although the antiparasitic activity of this series of conjugates appears limited, the compounds demonstrate potential as fluorescent probes for investigating the intracellular trafficking of trithiolato-bridged dinuclear Ru(II)-arene complexes in vitro.

New Nucleic Base-Tethered Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Compounds: Synthesis and Antiparasitic Activity.

Aiming toward compounds with improved anti-Toxoplasma activity by exploiting the parasite auxotrophies, a library of nucleobase-tethered trithiolato-bridged dinuclear ruthenium(II)-arene conjugates was synthesized and evaluated. Structural features such as the type of nucleobase and linking unit were progressively modified. For comparison, diruthenium hybrids with other type of molecules were also synthesized and assessed. A total of 37 compounds (diruthenium conjugates and intermediates) were evaluated in a primary screening for in vitro activity against transgenic Toxoplasma gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in non-infected host cells (human foreskin fibroblasts, HFF) was determined by alamarBlue assay. Twenty compounds strongly impairing parasite proliferation with little effect on HFF viability were subjected to T. gondii ß-gal half maximal inhibitory concentration determination (IC50) and their toxicity for HFF was assessed at 2.5 µM. Two promising compounds were identified: 14, ester conjugate with 9-(2-oxyethyl)adenine, and 36, a click conjugate bearing a 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl substituent, with IC50 values of 0.059 and 0.111 µM respectively, significantly lower compared to pyrimethamine standard (IC50 = 0.326 µM). Both 14 and 36 exhibited low toxicity against HFF when applied at 2.5 µM and are candidates for potential treatment options in a suitable in vivo model.

Cellular and Molecular Targets of Nucleotide-Tagged Trithiolato-Bridged Arene Ruthenium Complexes in the Protozoan Parasites Toxoplasma gondii and Trypanosoma brucei.

Toxoplasma gondii is an apicomplexan parasite that infects and proliferates within many different types of host cells and infects virtually all warm-blooded animals and humans. Trypanosoma brucei is an extracellular kinetoplastid that causes human African trypanosomiasis and Nagana disease in cattle, primarily in rural sub-Saharan Africa. Current treatments against both parasites have limitations, e.g., suboptimal efficacy and adverse side effects. Here, we investigate the potential cellular and molecular targets of a trithiolato-bridged arene ruthenium complex conjugated to 9-(2-hydroxyethyl)-adenine (1), which inhibits both parasites with IC50s below 10-7 M. Proteins that bind to 1 were identified using differential affinity chromatography (DAC) followed by shotgun-mass spectrometry. A trithiolato-bridged ruthenium complex decorated with hypoxanthine (2) and 2-hydroxyethyl-adenine (3) were included as controls. Transmission electron microscopy (TEM) revealed distinct ultrastructural modifications in the mitochondrion induced by (1) but not by (2) and (3) in both species. DAC revealed 128 proteins in T. gondii and 46 proteins in T. brucei specifically binding to 1 but not 2 or 3. In T. gondii, the most abundant was a protein with unknown function annotated as YOU2. This protein is a homolog to the human mitochondrial inner membrane translocase subunit Tim10. In T. brucei, the most abundant proteins binding specifically to 1 were mitochondrial ATP-synthase subunits. Exposure of T. brucei bloodstream forms to 1 resulted in rapid breakdown of the ATP-synthase complex. Moreover, both datasets contained proteins involved in key steps of metabolism and nucleic acid binding proteins.

Coumarin-Tagged Dinuclear Trithiolato-Bridged Ruthenium(II)⋠Arene Complexes: Photophysical Properties and Antiparasitic Activity.

The synthesis, characterization, photophysical and biological properties of 13 new conjugate coumarin-diruthenium(II)⋅arene complexes against Toxoplasma gondii are presented. For all conjugate organometallic unit/coumarins, an almost complete loss of fluorescence efficacy was observed. However, the nature of the fluorophore, the type of bonding, the presence and length of a linker between the coumarin dye and the ruthenium(II) moiety, and the number of dye units influenced their biological properties. The in vitro activity against a transgenic T. gondii strain grown in human foreskin fibroblasts (HFF) leads to IC50 values for T. gondii ß-gal from 105 to 735 nM. Of note is that nine compounds displayed lower IC50 than the standard drug pyrimethamine. One compound applied at its IC50 did not affect B-cell proliferation but had an impact on T-cell proliferation in murine splenocyte cultures. Transmission electron microscopy of T. gondii ß-gal-infected HFF showed that treatment predominantly affected the parasites' mitochondrion.

Comparative analysis of excretory-secretory antigens of Anisakis simplex, Pseudoterranova decipiens and Contracaecum osculatum regarding their applicability for specific serodiagnosis of human anisakidosis based on IgG-ELISA.

Serodiagnosis of human anisakidosis is presently hampered by the current lack of standardised serological assays that allow sensitive and specific detection of Anisakidae-specific antibodies in human patients. In the present study, we comparatively evaluated the diagnostic value (by IgG-ELISA) of excretory-secretory antigens (ESAgs) of Anisakis simplex, Pseudoterranova decipiens and Contracaecum osculatum, representing the most frequently found genera responsible for human infection. In addition, we tested also a mix of the three ES preparations (Mix-ESAgs) as well as two recombinant allergens of A. simplex, rAni s 1 and rAni s 7. ES antigen from C. osculatum yielded the best diagnostic performance in IgG-ELISA-based serodiagnosis of the Spanish anisakidosis patients investigated in this study (relative serodiagnostic sensitivity 100%; specificity 89%) as compared to A. simplex ES-antigen (93% versus 57%) and P. decipiens (67% versus 93%) or a mix of the three ES antigens (100% versus 44%), respectively. Cross-reactions of C. osculatum ES antigen with serum-antibodies from patients suffering from other helminth infections were rare and were exclusively found with few sera from toxocariasis, ascariasis, and filariasis patients. The two recombinant allergens rAni s 1 and rAni s 7 did not prove sufficiently sensitive and specific in order to justify a further evaluation of these antigens regarding their suitability in IgG-ELISA-based serodiagnosis of human anisakidosis. In conclusion, the C. osculatum-ESAg-ELISA remains as key candidate to be further assessed for the serodiagnosis of symptomatic anisakidosis in different endemic regions.

Follow-up of surgically treated patients with cystic echinococcosis: can novel recombinant antigens compete with imaging? Analysis of a patient cohort.

OBJECTIVE: To compare the performance of two novel recombinant antigens (EgP29, 2B2t) with imaging in a well-defined cohort of surgically treated cystic echinococcosis (CE) patients to determine whether serology reflects surgical cure as defined by imaging. METHODS: From a cohort of 223 CE-confirmed patients of a national clinical center for echinococcosis, 36 surgically treated patients were eligible for analysis. Sera were tested by enzyme-linked immunosorbent assay (ELISA) for specific IgG and IgG4 antibodies against the EgP29 and 2B2t antigens. We used a hierarchical linear regression model to examine the course of antibody levels over time for each patient. A meta-analysis of the patient-specific estimates of the time to negativity was performed using the metan command in Stata. RESULTS: The range of positive serological results at the beginning of post-surgical monitoring was 34-60%: 2B2t 51%, 2B2t-IgG4 34%, EgP29 60% and EgP29-IgG4 40%. The pooled estimates of time to seronegativity were as follows: 2B2t-ELISA 3.92 (3.24, 4.61) years; 2B2t-IgG4-ELISA 4.60 (3.91, 5.29) years; EgP29-ELISA 3.94 (3.50, 4.39) years; EgP29-IgG4-ELISA 2.55 (1.93, 3.18) years. CONCLUSION: After surgical treatment, antibodies to the recombinant antigens 2B2t and EgP29 become negative in the majority of CE-confirmed, surgically cured patients. The major drawback is the fact that only around half of the CE-confirmed, surgically treated patients were at all responsive to the test antigens, so they are of limited benefit for documenting primary cure. Equally, these antigens do not appear to be sensitive to recurrences.

Molecular characterization of Echinococcus granulosus isolates from Bulgarian human cystic echinococcosis patients.

Although cystic echinococcosis (CE) is highly endemic in Bulgaria, there is still scarce information about species and/or genotypes of the Echinococcus granulosus complex that infect humans. Our study tackled the genetic diversity of E. granulosus complex in a cohort of 30 Bulgarian CE patients. Ten animal E. granulosus isolates from neighboring Greece were additionally included. Specimens were comparatively analyzed for partial sequences of five mitochondrial (mt) (cox I, nad I, rrnS, rrnL, and atp6) and three nuclear (nc) genes (act II, hbx 2, and ef-1α) using a PCR-sequencing approach. All 30 Bulgarian isolates were identified as E. granulosus sensu stricto (s.s.) and were showing identical sequences for each of the three examined partial nc gene markers. Based upon concatenated sequences from partial mtDNA markers, we detected 10 haplotypes: 6 haplotypes (H1-H6) clustering with E. granulosus s.s. (G1) and 4 haplotypes (H9-H13) grouping with E. granulosus s.s. (G3), with H1 and H10 being the most frequent in Bulgarian patients. The haplotypes H1, H4, and H11 were also present in Greek hydatid cyst samples of animal origin. In conclusion, E. granulosus s.s. (G1 and G3 genotypes) is the only causative agent found so far to cause human CE in Bulgaria. However, further studies including larger sample sizes and other additional geographic regions in Bulgaria will have to be performed to confirm our results.

A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa.

Reliable and rapid molecular tools for the genetic identification and differentiation of Echinococcus species and/or genotypes are crucial for studying spatial and temporal transmission dynamics. Here, we describe a novel dual PCR targeting regions in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rRNA) genes, which enables (i) the specific identification of species and genotypes of Echinococcus (rrnS + L-PCR) and/or (ii) the identification of a range of taeniid cestodes, including different species of Echinococcus, Taenia and some others (17 species of diphyllidean helminths). This dual PCR approach was highly sensitive, with an analytical detection limit of 1 pg for genomic DNA of Echinococcus. Using concatenated sequence data derived from the two gene markers (1225 bp), we identified five unique and geographically informative single nucleotide polymorphisms (SNPs) that allowed genotypes (G1 and G3) of Echinococcus granulosus sensu stricto to be distinguished, and 25 SNPs that allowed differentiation within Echinococcus canadensis (G6/7/8/10). In conclusion, we propose that this dual PCR-based sequencing approach can be used for molecular epidemiological studies of Echinococcus and other taeniid cestodes.

Comparison of ex vivo harvested and in vitro cultured materials from Echinococcus granulosus by measuring expression levels of five genes putatively involved in the development and maturation of adult worms.

Parts of the natural life cycle of Echinococcus granulosus can be retraced in vitro such as the development of protoscoleces into semiadult worms with three or more proglottids, or the redifferentiation of in vitro cultured protoscoleces into metacestode-like cystic structures. Most in vitro generated samples share-at the microscopical level-high similarities with those naturally grown, but developmental differences have also been documented, such as missing egg production in in vitro grown adults or unusual bladder/vesicle formation in protoscoleces cultured into the metacestode direction. The aim of the present study was to explore how far different in vitro generated stage-specific materials/structures match the natural situation on the transcriptome level, based on testing five exemplarily chosen different genes: the frizzled receptor eg-fz4 (posterior marker), the FGF receptor-like factor eg-fgfrl (anterior association), the cell differentiation protein eg-rcd1 (part of the CCR4-NOT complex, a key regulator of eukaryotic gene expression), the rapidly accelerated fibrosarcoma serin/threonin kinase eg-braf (part of the MAPK pathway involved, e.g., in EGF signaling) and the co-smad eg-smadD (downstream factor of TGFß/BMP2/activin signaling). These genes-tested via qPCR-were selected such as to allow a discussion on their potential role in the development of E. granulosus into the adult stage. Thus, testing took place with three ex vivo isolated samples, namely (i) egg-containing adult worms, (ii) invaginated protoscoleces, and (iii) protoscolex-free germinal layer tissue. Respective data were compared (a) with in vitro generated metacestode-like microcysts developed from protoscolices, and (b) different development stages of protoscoleces in vitro cultured toward adult maturation. As a finding, only eg-smadD and partially eg-fz4 showed high expression similarities between ex vivo harvested and in vitro cultured E. granulosus, thus suggesting a putative role in adult maturation. Conclusively, the fact of using "only" five genes did not allow answering the question if ex vivo and in vitro materials are similar on the transcriptome level. Another experimental restriction arises from different growth conditions of the in vitro cultured materials, and comparing these to the ex vivo harvested ones. Future experiments may solve the problems by using fully standardized E. granulosus sample collection and fully standardized culture conditions.

TaqMan-quantitative PCR assays applied in Neospora caninum knock-outs generated through CRISPR-Cas9 allow to determine the copy numbers of integrated dihydrofolate reductase-thymidylate synthase drug selectable markers.

As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.

Working towards the development of vaccines and chemotherapeutics against neosporosis-With all of its ups and downs-Looking ahead.

Neospora caninum is an apicomplexan and obligatory intracellular parasite, which is the leading cause of reproductive failure in cattle and affects other farm and domestic animals, but also induces neuromuscular disease in dogs of all ages. In cattle, neosporosis is an important health problem, and has a considerable economic impact. To date there is no protective vaccine or chemotherapeutic treatment on the market. Immuno-prophylaxis has long been considered as the best control measure. Proteins involved in host cell interaction and invasion, as well as antigens mediating inflammatory responses have been the most frequently assessed vaccine targets. However, despite considerable efforts no effective vaccine has been introduced to the market to date. The development of effective compounds to limit the effects of vertical transmission of N. caninum tachyzoites has emerged as an alternative or addition to vaccination, provided suitable targets and safe and efficacious drugs can be identified. Additionally, the combination of both treatment strategies might be interesting to further increase protectivity against N. caninum infections and to decrease the duration of treatment and the risk of potential drug resistance. Well-established and standardized animal infection models are key factors for the evaluation of promising vaccine and compound candidates. The vast majority of experimental animal experiments concerning neosporosis have been performed in mice, although in recent years the numbers of experimental studies in cattle and sheep have increased. In this review, we discuss the recent findings concerning the progress in drug and vaccine development against N. caninum infections in mice and ruminants.

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.

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.

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.

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.