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1.  Echinococcus P29 Antigen: Molecular Characterization and Implication on Post-Surgery Follow-Up of CE Patients Infected with Different Species of the Echinococcus granulosus Complex 
PLoS ONE  2014;9(5):e98357.
The protein P29 is a potential serological marker for post-treatment monitoring of cystic echinococcosis (CE) especially in young patients. We now have demonstrated that P29 is encoded in the Echinococcus genus by a single gene consisting of 7 exons spanning 1.2 kb of DNA. Variability of the p29 gene at inter- and intra-species level was assessed with 50 cDNA and 280 genomic DNA clones isolated from different E. granulosus s.l. isolates (E. granulosus sensu stricto (G1), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6), E. canadensis (G7) and E. canadensis (G10)) as well as four E. multilocularis isolates. Scarce interspecies polymorphism at the p29 locus was observed and affected predominantly E. granulosus s.s. (G1), where we identified two alleles (A1 and A2) coding for identical P29 proteins and yielding in three genotypes (A1/A1, A2/A2 and A1/A2). Genotypic frequencies expected under Hardy-Weinberg equilibrium revealed a high rate of heterozygosity (47%) that strongly supports the hypothesis that E. granulosus s.s. (G1) is predominantly outbreeding. Comparative sequence analyses of the complete p29 gene showed that phylogenetic relationships within the genus Echinococcus were in agreement with those of previous nuclear gene studies. At the protein level, the deduced P29 amino acid (AA) sequences exhibited a high level of conservation, ranging from 97.9% AA sequence identity among the whole E. granulosus s.l. group to 99.58% identity among E. multilocularis isolates. We showed that P29 proteins of these two species differ by three AA substitutions without implication for antigenicity. In Western-blot analyses, serum antibodies from a human CE patient infected with E. canadensis (G6) strongly reacted with recombinant P29 from E. granulosus s.s. (G1) (recEg(G1)P29). In the same line, human anti-Eg(G1)P29 antibodies bound to recEcnd(G6)P29. Thus, minor AA sequence variations appear not to impair the prognostic serological use of P29.
PMCID: PMC4031130  PMID: 24851904
2.  In Vitro Effects of Novel Ruthenium Complexes in Neospora caninum and Toxoplasma gondii Tachyzoites 
Antimicrobial Agents and Chemotherapy  2013;57(11):5747-5754.
Upon the screening of 16 antiproliferative compounds against Toxoplasma gondii and Neospora caninum, two hydrolytically stable ruthenium complexes (compounds 16 and 18) exhibited 50% inhibitory concentrations of 18.7 and 41.1 nM (T. gondii) and 6.7 and 11.3 nM (N. caninum). To achieve parasiticidal activity with compound 16, long-term treatment (22 to 27 days at 80 to 160 nM) was required. Transmission electron microscopy demonstrated the rapid impact on and ultrastructural alterations in both parasites. These preliminary findings suggest that the potential of ruthenium-based compounds should thus be further exploited.
PMCID: PMC3811262  PMID: 23979747
3.  Neospora caninum Calcium-Dependent Protein Kinase 1 Is an Effective Drug Target for Neosporosis Therapy 
PLoS ONE  2014;9(3):e92929.
Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.
PMCID: PMC3969379  PMID: 24681759
4.  In Vitro and In Vivo Activities of Dicationic Diguanidino Compounds against Echinococcus multilocularis Metacestodes 
Alveolar echinococcosis (AE) is a disease predominantly affecting the liver, with metacestodes (larvae) of the tapeworm Echinococcus multilocularis proliferating and exhibiting tumor-like infiltrative growth. For many years, chemotherapeutical treatment against alveolar echinococcosis has relied on the benzimidazoles albendazole and mebendazole, which require long treatment durations and exhibit parasitostatic rather than parasiticidal efficacy. Although benzimidazoles have been and still are beneficial for the patients, there is clearly a demand for alternative and more efficient treatment options. Aromatic dications, more precisely a small panel of di-N-aryl-diguanidino compounds, were screened for efficacy against E. multilocularis metacestodes in vitro. Only those with a thiophene core group were active against metacestodes, while furans were not. The most active compound, DB1127, was further investigated in terms of in vivo efficacy in mice experimentally infected with E. multilocularis metacestodes. This diguanidino compound was effective against AE when administered intraperitoneally but not when applied orally. Thus, thiophene-diguanidino derivatives with improved bioavailability when administered orally could lead to treatment options against AE.
PMCID: PMC3719769  PMID: 23716058
5.  Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development 
BMC Biology  2014;12:5.
The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host’s liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood.
Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite’s glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin.
Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.
PMCID: PMC3923246  PMID: 24468049
Cestode; Tapeworm; Echinococcus; Echinococcosis; Insulin; Receptor kinase; Kinase inhibitor; Host-parasite interaction
6.  Subcutaneous Infection Model Facilitates Treatment Assessment of Secondary Alveolar Echinococcosis in Mice 
Alveolar echinococcosis (AE) in humans is a parasitic disease characterized by severe damage to the liver and occasionally other organs. AE is caused by infection with the metacestode (larval) stage of the fox tapeworm Echinococcus multilocularis, usually infecting small rodents as natural intermediate hosts. Conventionally, human AE is chemotherapeutically treated with mebendazole or albendazole. There is, however still the need for improved chemotherapeutical options. Primary in vivo studies on drugs of interest are commonly performed in small laboratory animals such as mice and Mongolian jirds, and in most cases, a secondary infection model is used, whereby E. multilocularis metacestodes are directly injected into the peritoneal cavity or into the liver. Disadvantages of this methodological approach include risk of injury to organs during the inoculation and, most notably, a limitation in the macroscopic (visible) assessment of treatment efficacy. Thus, in order to monitor the efficacy of chemotherapeutical treatment, animals have to be euthanized and the parasite tissue dissected. In the present study, mice were infected with E. multilocularis metacestodes through the subcutaneous route and were then subjected to chemotherapy employing albendazole. Serological responses to infection were comparatively assessed in mice infected by the conventional intraperitoneal route. We demonstrate that the subcutaneous infection model for secondary AE facilitates the assessment of the progress of infection and drug treatment in the live animal.
Author Summary
Alveolar echinococcosis is a disease which affects humans and inflicts severe damage to the liver and other organs. It is caused by a parasite whose definitive host is the fox. Despite being a relatively rare disease, an increasing number of new cases has been reported in central and eastern European countries more recently. The current therapy in human AE patients consists of benzimidazoles. The treatment has to be taken on a daily basis for very long periods of time, or even lifelong. New options are currently being searched for, mainly based on compounds that show efficacy in experimental animal infection models. The infection is commonly done by injecting parasites directly into the peritoneal cavity of the animals, with risk of damage to the surrounding organs. The efficacy of applied treatments can only be evaluated at the end of the studies by dissection of the animals. In this study we show that the subcutaneous infection model can be applied for drug treatment trials and enables the direct monitoring of treatment effects during the entire study period.
PMCID: PMC3662659  PMID: 23717701
7.  Di-cationic arylimidamides act against Neospora caninum tachyzoites by interference in membrane structure and nucleolar integrity and are active against challenge infection in mice 
Graphical abstract
► The dicationic arylimidamide DB745 is highly active against Neospora caninum tachyzoites in vitro. ► The drug inhibits host cell invasion and intracellular proliferation. ► DB745 interferes in the structural integrity of the parasite plasma membrane and the nucleolus. ► The two N. caninum isolates Nc-1 and Nc-Liverpool differ in their ability to adapt to DB745 in vitro. ► In vivo treatment of Nc-1 infected mice with DB745 reduces clinical signs and cerebral parasite load.
Neospora caninum is considered to be the main cause of bovine abortion in Europe and the USA, leading to considerable financial impact. Losses are caused directly by abortions or indirectly through breeding of calves with impaired viability. Due to the lack of effective chemotherapy against bovine neosporosis, there is a need to develop new anti-protozoal compounds, which would either eliminate the parasite or avoid its transmission. In order to identify compounds of interest, the in vitro activities of 41 di-cationic pentamidine derivatives were studied employing a transgenic N. caninum clone expressing beta-galactosidase as a reporter gene. The arylimidamide DB745, previously shown to be highly active against Leishmania donovani in vitro and in vivo, appeared as the most promising compound, with an IC50 of 80 nM in 3-day growth assays and severely affecting both host cell invasion as well as intracellular proliferation. TEM of intracellular tachyzoites identified distinct alterations related to the nucleolus and the nuclear and cellular membrane. Long-term growth assays showed that DB745 acted parasiticidal upon the Nc-Liv isolate, but not against the Nc-1 isolate of N. caninum. In vivo studies in N. caninum (Nc-1 isolate) infected mice showed that daily intraperitoneal application of DB745 for a period of 14 days resulted in a decreased number of clinically affected animals, and lower cerebral parasite burdens in DB745-treated mice compared to non-treated mice. These results illustrate the potential of dicationic arylimidamides for the treatment of N. caninum infections.
PMCID: PMC3862485  PMID: 24533272
Neospora caninum; Arylimidamides; Tachyzoites; Proliferation; Invasion; Nucleolus; Membrane integrity; In vivo activity
8.  In Vitro Efficacy of Dicationic Compounds and Mefloquine Enantiomers against Echinococcus multilocularis Metacestodes▿ 
Antimicrobial Agents and Chemotherapy  2011;55(10):4866-4872.
The current chemotherapy of alveolar echinococcosis (AE) is based on benzimidazoles such as albendazole and has been shown to be parasitostatic rather than parasiticidal, requiring lifelong duration. Thus, new and more efficient treatment options are urgently needed. By employing a recently validated assay based on the release of functional phosphoglucose isomerase (PGI) from dying parasites, the activities of 26 dicationic compounds and of the (+)- and (−)-erythro-enantiomers of mefloquine were investigated. Initial screening of compounds was performed at 40 μM, and those compounds exhibiting considerable antiparasitic activities were also assessed at lower concentrations. Of the dicationic drugs, DB1127 (a diguanidino compound) with activities comparable to nitazoxanide was further studied. The activity of DB1127 was dose dependent and led to severe structural alterations, as visualized by electron microscopy. The (+)- and (−)-erythro-enantiomers of mefloquine showed similar dose-dependent effects, although higher concentrations of these compounds than of DB1127 were required for metacestode damage. In conclusion, of the drugs investigated here, the diguanidino compound DB1127 represents the most promising compound for further study in appropriate in vivo models for Echinococcus multilocularis infection.
PMCID: PMC3186988  PMID: 21768518
9.  In Vitro and In Vivo Efficacies of Mefloquine-Based Treatment against Alveolar Echinococcosis▿  
Alveolar echinococcosis (AE) is caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis and causes severe disease in the human liver, and occasionally in other organs, that is fatal when treatment is unsuccessful. The present chemotherapy against AE is based on mebendazole and albendazole. Albendazole treatment has been found to be ineffective in some instances, is parasitostatic rather than parasiticidal, and usually involves the lifelong uptake of large doses of drugs. Thus, new treatment options are urgently needed. In this study we investigated the in vitro and in vivo efficacy of mefloquine against E. multilocularis metacestodes. Treatment using mefloquine (20 μM) against in vitro cultures of metacestodes resulted in rapid and complete detachment of large parts of the germinal layer from the inner surface of the laminated layer within a few hours. The in vitro activity of mefloquine was dependent on the dosage. In vitro culture of metacestodes in the presence of 24 μM mefloquine for a period of 10 days was parasiticidal, as determined by murine bioassays, while treatment with 12 μM was not. Oral application of mefloquine (25 mg/kg of body weight administered twice a week for a period of 8 weeks) in E. multilocularis-infected mice was ineffective in achieving any reduction of parasite weight, whereas treatment with albendazole (200 mg/kg/day) was highly effective. However, when the same mefloquine dosage was applied intraperitoneally, the reduction in parasite weight was similar to the reduction seen with oral albendazole application. Combined application of both drugs did not increase the treatment efficacy. In conclusion, mefloquine represents an interesting drug candidate for the treatment of AE, and these results should be followed up in appropriate in vivo studies.
PMCID: PMC3028781  PMID: 21135182
10.  In Vitro and In Vivo Treatments of Echinococcus Protoscoleces and Metacestodes with Artemisinin and Artemisinin Derivatives▿  
In vitro treatment of Echinococcus multilocularis and Echinococcus granulosus larval stages with the antimalarials dihydroartemisinin and artesunate (10 to 40 μM) exhibited promising results, while 6 weeks of in vivo treatment of mice infected with E. multilocularis metacestodes (200 mg/kg of body weight/day) had no effect. However, combination treatments of both drugs with albendazole led to a substantial but statistically not significant reduction in parasite weight compared to results with albendazole alone.
PMCID: PMC2533465  PMID: 18625777
11.  Major Surface Glycoproteins of Insect Forms of Trypanosoma brucei Are Not Essential for Cyclical Transmission by Tsetse 
PLoS ONE  2009;4(2):e4493.
Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.
PMCID: PMC2637416  PMID: 19223969
12.  Host Cells Participate in the In Vitro Effects of Novel Diamidine Analogues against Tachyzoites of the Intracellular Apicomplexan Parasites Neospora caninum and Toxoplasma gondii▿  
The in vitro effects of 19 dicationic diamidine derivatives against the proliferative tachyzoite stages of the apicomplexan parasites Neospora caninum and Toxoplasma gondii were investigated. Four compounds (DB811, DB786, DB750, and DB766) with similar structural properties exhibited profound inhibition of tachyzoite proliferation. The lowest 50% inhibitory concentrations were found for DB786 (0.21 μM against Neospora and 0.22 μM against Toxoplasma) and DB750 (0.23 μM against Neospora and 0.16 μM against Toxoplasma), with complete proliferation inhibition at 1.7 μM for both drugs against both species. DB750 and DB786 were chosen for further studies. Electron microscopy of N. caninum-infected human foreskin fibroblast (HFF) cultures revealed distinct alterations and damage of parasite ultrastructure upon drug treatment, while host cells remained unaffected. For true parasiticidal efficacy against N. caninum, a treatment duration of 3 h at 1.7 μM was sufficient for DB750, while a longer treatment period (24 h) was necessary for DB786. Pretreatment of tachyzoites for 1 h prior to host cell exposure had no effect on infectivity. However, pretreatment of uninfected host cells had a significant adverse effect on N. caninum proliferation: exposure of HFFs to 1.7 μM DB750 for 6, 12, or 24 h, followed by infection with N. caninum tachyzoites and subsequent culture in the absence of DB750, resulted in significantly delayed parasite proliferation. This suggests that either (i) these compounds or their respective active metabolites were still present after the removal of the drugs or (ii) the drug treatments reversibly impaired some functional activities in HFFs that were essential for parasite proliferation and/or survival.
PMCID: PMC2415759  PMID: 18362190
13.  Peroxide Bond-Dependent Antiplasmodial Specificity of Artemisinin and OZ277 (RBx11160)▿  
Using nonperoxidic analogs of artemisinin and OZ277 (RBx11160), the strong in vitro antiplasmodial activities of the latter two compounds were shown to be peroxide bond dependent. In contrast, the weak activities of artemisinin and OZ277 against six other protozoan parasites were peroxide bond independent. These data support the iron-dependent artemisinin alkylation hypothesis.
PMCID: PMC1932508  PMID: 17562801
14.  A Novel Giardia lamblia Nitroreductase, GlNR1, Interacts with Nitazoxanide and Other Thiazolides▿  
The nitrothiazole analogue nitazoxanide [NTZ; 2-acetolyloxy-N-(5-nitro-2-thiazolyl)benzamide] represents the parent compound of a class of drugs referred to as thiazolides and exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. NTZ and other thiazolides are active against a wide range of other intracellular and extracellular protozoan parasites in vitro and in vivo, but their mode of action and respective subcellular target(s) have only recently been investigated. In order to identify potential targets of NTZ and other thiazolides in Giardia lamblia trophozoites, we have developed an affinity chromatography system using the deacetylated derivative of NTZ, tizoxanide (TIZ), as a ligand. Affinity chromatography on TIZ-agarose using cell extracts of G. lamblia trophozoites resulted in the isolation of an approximately 35-kDa polypeptide, which was identified by mass spectrometry as a nitroreductase (NR) homologue (EAA43030.1). NR was overexpressed as a six-histidine-tagged recombinant protein in Escherichia coli, purified, and then characterized using an assay for oxygen-insensitive NRs with dinitrotoluene as a substrate. This demonstrated that the NR was functionally active, and the protein was designated GlNR1. In this assay system, NR activity was severely inhibited by NTZ and other thiazolides, demonstrating that the antigiardial activity of these drugs could be, at least partially, mediated through inhibition of GlNR1.
PMCID: PMC1891416  PMID: 17438059
15.  In Vitro Metacestodicidal Activities of Genistein and Other Isoflavones against Echinococcus multilocularis and Echinococcus granulosus▿  
Antimicrobial Agents and Chemotherapy  2006;50(11):3770-3778.
Echinococcus multilocularis and Echinococcus granulosus metacestode infections in humans cause alveolar echinococcosis and cystic echinococcosis, respectively, in which metacestode development in visceral organs often results in particular organ failure. Further, cystic hydatidosis in farm animals causes severe economic losses. Although benzimidazole derivatives such as mebendazole and albendazole are being used as therapeutic agents, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of a number of isoflavones against Echinococcus metacestodes and protoscoleces. The most prominent isoflavone, genistein, exhibits significant metacestodicidal activity in vitro. However, genistein binds to the estrogen receptor and can thus induce estrogenic effects, which is a major concern during long-term chemotherapy. We have therefore investigated the activities of a number of synthetic genistein derivatives carrying a modified estrogen receptor binding site. One of these, Rm6423, induced dramatic breakdown of the structural integrity of the metacestode germinal layer of both species within 5 to 7 days of in vitro treatment. Further, examination of the culture medium revealed increased leakage of parasite proteins into the medium during treatment, but zymography demonstrated a decrease in the activity of metalloproteases. Moreover, two of the genistein derivatives, Rm6423 and Rm6426, induced considerable damage in E. granulosus protoscoleces, rendering them nonviable. These findings demonstrate that synthetic isoflavones exhibit distinct in vitro effects on Echinococcus metacestodes and protoscoleces, which could potentially be exploited further for the development of novel chemotherapeutical tools against larval-stage Echinococcus infection.
PMCID: PMC1635224  PMID: 16954323
16.  In Vitro Effects of Thiazolides on Giardia lamblia WB Clone C6 Cultured Axenically and in Coculture with Caco2 Cells 
The thiazolides represent a novel class of anti-infective drugs, with the nitrothiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] (NTZ) as the parent compound. NTZ exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ), which exhibits similar activities. We have here comparatively investigated the in vitro effects of NTZ, TIZ, a number of other modified thiazolides, and metronidazole (MTZ) on Giardia lamblia trophozoites grown under axenic culture conditions and in coculture with the human cancer colon cell line Caco2. The modifications of the thiazolides included, on one hand, the replacement of the nitro group on the thiazole ring with a bromide, and, on the other hand, the differential positioning of methyl groups on the benzene ring. Of seven compounds with a bromo instead of a nitro group, only one, RM4820, showed moderate inhibition of Giardia proliferation in axenic culture, but not in coculture with Caco2 cells, with a 50% inhibitory concentration (IC50) of 18.8 μM; in comparison, NTZ and tizoxanide had IC50s of 2.4 μM, and MTZ had an IC50 of 7.8 μM. Moreover, the methylation or carboxylation of the benzene ring at position 3 resulted in a significant decrease of activity, and methylation at position 5 completely abrogated the antiparasitic effect of the nitrothiazole compound. Trophozoites treated with NTZ showed distinct lesions on the ventral disk as soon as 2 to 3 h after treatment, whereas treatment with metronidazole resulted in severe damage to the dorsal surface membrane at later time points.
PMCID: PMC1346829  PMID: 16377682
17.  In Vitro Efficacies of Nitazoxanide and Other Thiazolides against Neospora caninum Tachyzoites Reveal Antiparasitic Activity Independent of the Nitro Group 
The thiazolide nitazoxanide [2-acetolyloxy-N-(5-nitro-2-thiazolyl)benzamide] (NTZ) exhibits a broad spectrum of activities against a wide variety of intestinal and tissue-dwelling helminths, protozoa, and enteric bacteria infecting animals and humans. The drug has been postulated to act via reduction of its nitro group by nitroreductases, including pyruvate ferredoxin oxidoreductase. In this study, we investigated the efficacies of nitazoxanide and a number of other thiazolides against Neospora caninum tachyzoites in vitro. We employed real-time-PCR-based monitoring of tachyzoite adhesion, invasion, and intracellular proliferation, as well as electron microscopic visualization of the effects imposed by nitazoxanide. In addition, we investigated several modified versions of this drug. These modifications included on one hand the replacement of the nitro group on the thiazole ring with a bromide, thus removing the most reactive group, and on the other hand the differential positioning of methyl groups on the salicylate ring. We show that the thiazole-associated nitro group is not necessarily required for the action of the drug and that methylation of the salicylate ring can result in complete abrogation of the antiparasitic activity, depending on the positioning of the methyl group. These findings indicate that other mechanisms besides the proposed mode of action involving the pyruvate ferredoxin oxidoreductase enzyme could be responsible for the wide spectrum of antiparasitic activity of NTZ and that modifications in the benzene ring could be important in these alternative mechanisms.
PMCID: PMC1195425  PMID: 16127045
18.  Inhibitory Effect of Aureobasidin A on Toxoplasma gondii 
The apicomplexan parasite Toxoplasma gondii is a leading opportunistic pathogen associated with AIDS and congenital birth defects. Due to the need for identifying new parasite-specific treatments, the possibility of targeting sphingolipid biosynthesis in the parasite was investigated. Aureobasidin A, an inhibitor of the enzyme synthesizing the sphingolipid inositol phosphorylceramide, which is present in fungi, plants, and some protozoa but absent in mammalian cells, was found to block in vitro T. gondii replication without affecting host cell metabolism. Aureobasidin A treatment did not induce tachyzoite to bradyzoite stage conversion in T. gondii but resulted in a loss of intracellular structures and vacuolization within the parasite. In addition, aureobasidin A inhibited sphingolipid synthesis in T. gondii. Sphingolipid biosynthetic pathways may therefore be considered targets for the development of anti-T. gondii agents.
PMCID: PMC1087623  PMID: 15855498
19.  Identification and Characterization of a Neospora caninum Microneme-Associated Protein (NcMIC4) That Exhibits Unique Lactose-Binding Properties  
Infection and Immunity  2004;72(8):4791-4800.
Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by α-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37°C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced Mr but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.
PMCID: PMC470650  PMID: 15271941
20.  Isolation and Characterization of a Secretory Component of Echinococcus multilocularis Metacestodes Potentially Involved in Modulating the Host-Parasite Interface  
Infection and Immunity  2004;72(1):527-536.
Echinococcus multilocularis metacestodes are fluid-filled, vesicle-like organisms, which are characterized by continuous asexual proliferation via external budding of daughter vesicles, predominantly in the livers of infected individuals. Tumor-like growth eventually leads to the disease alveolar echinococcosis (AE). We employed the monoclonal antibody (MAb) E492/G1, previously shown to be directed against a carbohydrate-rich, immunomodulatory fraction of Echinococcus granulosus, to characterize potentially related components in E. multilocularis. Immunofluorescence studies demonstrated that MAb E492/G1-reactive epitopes were found predominantly on the laminated layer and in the periphery of developing brood capsules. The respective molecules were continuously released into the exterior medium and were also found in the parasite vesicle fluid. The MAb E492/G1-reactive fraction in E. multilocularis, named Em492 antigen, was isolated by immunoaffinity chromatography. Em492 antigen had a protein/carbohydrate ratio of 0.25, reacted with a series of lectins, and is related to the laminated layer-associated Em2(G11) antigen. The epitope recognized by MAb E492/G1 was sensitive to sodium periodate but was not affected by protease treatment. Anti-Em492 immunoglobulin G1 (IgG1) and IgG2 and, at lower levels, IgG3 were found in sera of mice suffering from experimentally induced secondary, but not primary, AE. However, with regard to cellular immunity, a suppressive effect on concanavalin A- or crude parasite extract-induced splenocyte proliferation in these mice was observed upon addition of Em492 antigen, but trypan blue exclusion tests and transmission electron microscopy failed to reveal any cytotoxic effect in Em492 antigen-treated spleen cells. This indicated that Em492 antigen could be modulating the periparasitic cellular environment during E. multilocularis infection through as yet unidentified mechanisms and could be one of the factors contributing to immunosuppressive events that occur at the host-parasite interface.
PMCID: PMC344003  PMID: 14688134
21.  In Vitro Induction of Neospora caninum Bradyzoites in Vero Cells Reveals Differential Antigen Expression, Localization, and Host-Cell Recognition of Tachyzoites and Bradyzoites  
Infection and Immunity  2004;72(1):576-583.
We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 μM sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites.
PMCID: PMC343979  PMID: 14688139
22.  In Vitro Parasiticidal Effect of Nitazoxanide against Echinococcus multilocularis Metacestodes 
When humans serve as inadvertent intermediate hosts for Echinococcus multilocularis, disease (alveolar echinococcosis [AE]) may result from the expanding parasite metacestode in visceral organs, mostly in the liver. Benzimidazole carbamate derivatives such as mebendazole and albendazole are used for chemotherapeutic treatment of AE. However, these treatments are, in most cases, parasitistatic rather than parasiticidal. As treatment is discontinued, a recurrence of parasite growth has been observed in many AE patients with nonradical resections. The only curative treatment for AE is radical surgical resection of the parasite tissue and support by chemotherapy. As there is a need for new treatment options for AE, the in vitro efficacy of nitazoxanide (NTZ), a broad-spectrum drug used against intestinal parasites and bacteria, was investigated. We showed that in vitro treatment of E. multilocularis metacestodes with NTZ induced high levels of alkaline phosphatase activity in the medium. Concurrently, distinct morphological and ultrastructural alterations were detected. Most significantly, two distinct types of alterations were observed as soon as after 3 h of NTZ treatment. At first, the drug induced a peripheral output of membranous vesicles from the tegumental membrane into the laminated layer. Simultaneously, germinal layer-associated undifferentiated cells produced large vacuoles filled with lipid-like and often electron-dense membranous segments. Other alterations were observed at later time points, including vacuolization of the germinal layer, accumulation of lipid droplets, and lastly, loss of microtriches and separation of the laminated and germinal layers. The pattern of damage induced by NTZ was different from the alterations earlier observed in albendazole sulfoxide-treated vesicles. The nonviability of NTZ-treated metacestodes was confirmed through bioassay, i.e., inoculation of treated and untreated parasites into mice. These experiments demonstrate the in vitro parasiticidal effect of NTZ on E. multilocularis metacestodes.
PMCID: PMC151752  PMID: 12543645
23.  Identification of a Neospora caninum Microneme Protein (NcMIC1) Which Interacts with Sulfated Host Cell Surface Glycosaminoglycans  
Infection and Immunity  2002;70(6):3187-3198.
The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37°C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.
PMCID: PMC127992  PMID: 12011014
24.  Application of Real-Time Fluorescent PCR for Quantitative Assessment of Neospora caninum Infections in Organotypic Slice Cultures of Rat Central Nervous System Tissue 
Journal of Clinical Microbiology  2002;40(1):252-255.
The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection.
PMCID: PMC120097  PMID: 11773124
25.  Neospora caninum Microneme Protein NcMIC3: Secretion, Subcellular Localization, and Functional Involvement in Host Cell Interaction 
Infection and Immunity  2001;69(10):6483-6494.
In apicomplexan parasites, host cell adhesion and subsequent invasion involve the sequential release of molecules originating from secretory organelles named micronemes, rhoptries, and dense granules. Microneme proteins have been shown to be released at the onset of the initial contact between the parasite and the host cell and thus mediate and establish the physical interaction between the parasite and the host cell surface. This interaction most likely involves adhesive domains found within the polypeptide sequences of most microneme proteins identified to date. NcMIC3 is a microneme-associated protein found in Neospora caninum tachyzoites and bradyzoites, and a large portion of this protein is comprised of a stretch of four consecutive epidermal growth factor (EGF)-like domains. We determined the subcellular localization of NcMIC3 prior to and following host cell invasion and found that NcMIC3 was secreted onto the tachyzoite surface immediately following host cell lysis in a temperature-dependent manner. Surface-exposed NcMIC3 could be detected up to 2 to 3 h following host cell invasion, and at later time points the distribution of the protein was again restricted to the micronemes. In vitro secretion assays using purified tachyzoites showed that following secretion onto the surface, NcMIC3 was largely translocated towards the posterior end of the parasite, employing a mechanism which requires a functional actin microfilament system. Following this, the protein remained bound to the parasite surface, since it could not be detected in a soluble form in respective culture supernatants. Secretion of NcMIC3 onto the surface resulted in an outward exposure of the EGF-like domains and coincided with an increased capacity of N. caninum tachyzoites to adhere to Vero cell monolayers in vitro, a capacity which could be inhibited by addition of antibodies directed against the EGF-like domains. NcMIC3 is a prominent component of Triton X-100 lysates of tachyzoites, and cosedimentation assays employing prefixed Vero cells showed that the protein binds to the Vero cell surface. In addition, the EGF-like domains, expressed as recombinant proteins in Escherichia coli, also interacted with the Vero cell surface, while binding of NcSRS2 and NcSAG1, the major immunodominant surface antigens, was not as efficient. Our data are indicative of a functional role of NcMIC3 in host cell infection.
PMCID: PMC98784  PMID: 11553593

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