Optimization of Antitrypanosomatid Agents: Identification of Nonmutagenic Drug Candidates with in Vivo Activity

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Paranoplocephala sciuri (Cestoda: Anoplocephalidae), a Parasite of the Northern Flying Squirrel (Glaucomys sabrinus), with a Discussion of Its Systematic Status

This study redescribes Andrya sciuri Rausch, 1947 (Anoplocephalidae) from the northern flying squirrel, Glaucomys sabrinus (Shaw), in North America, to redefine the morphology and generic position of this poorly known cestode. Andrya sciuri is shown to belong unambiguously to the genus Paranoplocephala Lühe, 1910 sensu Haukisalmi and Wickström (2005). Paranoplocephala sciuri is compared with four species that resemble it morphologically, and features that can be used in its identification are presented. It is suggested that P. sciuri has speciated through a shift from arvicoline rodents (voles and lemmings) to G. sabrinus.

Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

Galactose Recognition by the Apicomplexan Parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.

Structural Investigation of Anti-Trypanosoma cruzi 2-Iminothiazolidin-4-ones Allows the Identification of Agents with Efficacy in Infected Mice

We modified the thiazolidinic ring at positions N3, C4, and C5, yielding compounds 6-24. Compounds with a phenyl at position N3, 15-19, 22-24, exhibited better inhibitory properties for cruzain and against the parasite than 2-iminothiazolidin-4-one S. We were able to identify one high-efficacy trypanocidal compound, 2-minothiazolidin-4-one 18, which inhibited the activity of cruzain and the proliferation of epirnastigotes and was cidal for trypomastigotes but was not toxic for splenocytes. Having located some of the structural determinants of the trypanocidal properties, we subsequently wished to determine if the exchange of the thiazolidine for a thiazole ring leaves the functional properties unaffected. We therefore tested thiazoles 26-45 and observed that they did not inhibit cruzain, but they exhibited trypanocidal effects. Parasite development was severely impaired when treated with 18, thus reinforcing the notion that this class of heterocycles can lead to useful cidal agents for Chagas disease.

Optimization of anti-Trypanosoma cruzi oxadiazoles leads to identification of compounds with efficacy in infected mice

We recently showed that oxadiazoles have anti-Trypanosoma cruzi activity at micromolar concentrations. These compounds are easy to synthesize and show a number of clear and interpretable structure-activity relationships (SAR), features that make them attractive to pursue potency enhancement. We present here the structural design, synthesis, and anti-T. cruzi evaluation of new oxadiazoles denoted 5a-h and 6a-h. The design of these compounds was based on a previous model of computational docking of oxadiazoles on the T. cruzi protease cruzain. We tested the ability of these compounds to inhibit catalytic activity of cruzain, but we found no correlation between the enzyme inhibition and the antiparasitic activity of the compounds. However, we found reliable SAR data when we tested these compounds against the whole parasite. While none of these oxadiazoles showed toxicity for mammalian cells, oxadiazoles 6c (fluorine), 6d (chlorine), and 6e (bromine) reduced epimastigote proliferation and were cidal for trypomastigotes of T. cruzi Y strain. Oxadiazoles 6c and 6d have IC50 of 9.5 +/- 2.8 and 3.5 +/- 1.8 mu M for trypomastigotes, while Benznidazole, which is the currently used drug for Chagas disease treatment, showed an IC50 of 11.3 +/- 2.8 mu M. Compounds 6c and 6d impair trypomastigote development and invasion in macrophages, and also induce ultrastructural alterations in trypomastigotes. Finally, compound 6d given orally at 50 mg/kg substantially reduces the parasitemia in T. cruzi-infected BALB/c mice. Our drug design resulted in potency enhancement of oxadiazoles as anti-Chagas disease agents, and culminated with the identification of oxadiazole 6d, a trypanosomicidal compound in an animal model of infection. (C) 2012 Elsevier Ltd. All rights reserved.

Paracoccidoides brasiliensis 30 kDa adhesin: identification as a 14-3-3 protein, cloning and subcellular localization in infection models

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis.

Identification of novel rhoptry proteins in Neospora caninum by LC/MS-MS analysis of subcellular fractions

Apicomplexan parasites possess an apical complex that is composed of two secretory organelles recognized as micronemes and rhoptries. Rhoptry contents are secreted into the parasitophorous vacuole during the host cell invasion process. Several rhoptry proteins have been identified in Toxoplasma gondii and seem to be involved in host-pathogen interactions and some of them are considered to be important virulence factors. Only one rhoptry protein, NcROP2, has been identified and extensively characterized in the closely related parasite Neospora caninum, and this has showed immunoprotective properties. Thus, with the aim of increasing knowledge of the rhoptry protein repertoire in N. caninum, a subcellular fractionation of tachyzoites was performed to obtain fractions enriched for this secretory organelle. 2-D SDS-PAGE followed by MS and LC/MS-MS were applied for fraction analysis and 8 potential novel rhoptry components (NcROP1, 5, 8, 30 and NcRON2, 3, 4, 8) and several kinases, proteases and phosphatases proteins were identified with a high homology to those previously found in T. gondii. Their existence in N. caninum tachyzoites suggests their involvement in similar events or pathways that occur in T. gondii. These novel proteins may be considered as targets that could be useful in the future development of immunoprophylactic measures.

Drug target identification in intracellular and extracellular protozoan parasites

The increasing demand for novel anti-parasitic drugs due to resistance formation to well-established chemotherapeutically important compounds has increased the demands for a better understanding of the mechanism(s) of action of existing drugs and of drugs in development. While different approaches have been developed to identify the targets and thus mode of action of anti-parasitic compounds, it has become clear that many drugs act not only on one, but possibly several parasite molecules or even pathways. Ideally, these targets are not present in any cells of the host. In the case of apicomplexan parasites, the unique apicoplast, provides a suitable target for compounds binding to DNA or ribosomal RNA of prokaryotic origin. In the case of intracellular pathogens, a given drug might not only affect the pathogen by directly acting on parasite-associated targets, but also indirectly, by altering the host cell physiology. This in turn could affect the parasite development and lead to parasite death. In this review, we provide an overview of strategies for target identification, and present examples of selected drug targets, ranging from proteins to nucleic acids to intermediary metabolism.

Assay of β-hematin formation by malaria parasite

Novel leads are urgently required for designing antimalarials due to the reduced efficacy of presently available drugs. The malaria parasite has a unique reaction of heme polymerization, which has attracted much attention in the recent past as a target for the design of antimalarial drugs. The process is hampered by non-availability of a proper assay method. Currently available methods are cumbersome and require advanced instrumentation or radioactive substrates. Here, we are describing an assay for hemozoin formation that is simple and reproducible. This assay has routinely been used by us for the identification of potential compounds with antimalarial activity.

Genetic identification of an oxyurid from a captive, black-handed spider monkey—implications for treatment and control

Parasites are of major clinical significance in captive primates in zoos, particularly those with direct life cycles. Oxyurid nematodes can be a persistent problem, as infection intensity and environmental contamination with infective eggs are usually high. Observations at the Basel Zoo in Switzerland have revealed that particularly black-handed spider monkeys (Ateles geoffroyi) exhibit continuous oxyurid nematode infection(s), despite regular deworming with anthelmintics. In the present study, using a molecular approach, we were able to identify the nematode (Trypanoxyuris atelis) causing this ongoing problem, and we are now evaluating a practical treatment and control regimen to tackle this parasite problem.

Babesia spp. in European wild ruminant species: parasite diversity and risk factors for infection.

Babesia are tick-borne parasites that are increasingly considered as a threat to animal and public health. We aimed to assess the role of European free-ranging wild ruminants as maintenance mammalian hosts for Babesia species and to determine risk factors for infection. EDTA blood was collected from 222 roe deer (Capreolus c. capreolus), 231 red deer (Cervus e. elaphus), 267 Alpine chamois (Rupicapra r. rupicapra) and 264 Alpine ibex (Capra i. ibex) from all over Switzerland and analysed by PCR with pan-Babesia primers targeting the 18S rRNA gene, primers specific for B. capreoli and Babesia sp. EU1, and by sequencing. Babesia species, including B. divergens, B. capreoli, Babesia sp. EU1, Babesia sp. CH1 and B. motasi, were detected in 10.7% of all samples. Five individuals were co-infected with two Babesia species. Infection with specific Babesia varied widely between host species. Cervidae were significantly more infected with Babesia spp. than Caprinae. Babesia capreoli and Babesia sp. EU1 were mostly found in roe deer (prevalences 17.1% and 7.7%, respectively) and B. divergens and Babesia sp. CH1 only in red deer. Factors significantly associated with infection were low altitude and young age. Identification of Babesia sp. CH1 in red deer, co-infection with multiple Babesia species and infection of wild Caprinae with B. motasi and Babesia sp. EU1 are novel findings. We propose wild Caprinae as spillover or accidental hosts for Babesia species but wild Cervidae as mammalian reservoir hosts for B. capreoli, possibly Babesia sp. EU1 and Babesia sp. CH1, whereas their role regarding B. divergens is more elusive.

Identification of Trypanosoma brucei components involved in trypanolysis by normal human serum

Normal human serum (NHS) confers human resistance to infection by the parasite Trypanosoma brucei owing to the trypanolytic activity of apolipoprotein L1 (APOL1), present in two serum complexes termed Trypanolytic Factors (TLF-1 and -2). In order to identify parasite components involved in the intracellular trafficking and activity of TLFs, an inducible RNA interference (RNAi) genomic DNA library constructed in bloodstream form T. brucei was subjected to RNAi induction and selection for resistant parasites under NHS conditions favouring either TLF-1 or TLF-2 uptake. While TLF-1 conditions readily selected the haptoglobin-haemoglobin (HP-HB) surface receptor TbHpHbR as expected, given its known ability to bind TLF-1, under TLF-2 conditions no specific receptor for TLF-2 was identified. Instead, the screen allowed the identification of five distinct factors expected to be involved in the assembly of the vacuolar proton pump V-ATPase and consecutive endosomal acidification. These data confirm that lowering the pH during endocytosis is required for APOL1 toxic activity.

What can a unicellular parasite tell us about mitochondrial biogenesis?

In the unicellular parasite Trypanosoma brucei, as in other eukaryotes, more than 95% of all mitochondrial proteins are imported from the cytosol. The recently characterized multisubunit ATOM complex, the functional analogue of the TOM complex of yeast, mediates import of essentially all proteins across the outer mitochondrial membrane in T. brucei. Moreover, an additional protein termed pATOM36, which is loosely associated with the ATOM complex, has been implicated in the import of only a subset of mitochondrial proteins. Here we have investigated more precisely which role pATOM36 plays in mitochondrial protein import. RNAi mediated ablation of pATOM36 specifically depletes a subset of outer mitochondrial membrane proteins including ATOM complex subunits and as a consequence results in the collapse of the ATOM complex as shown by Blue native PAGE. In addition, a SILAC-based global proteomic analysis of uninduced and induced pATOM36 RNAi cells together with in vitro import experiments suggest that pATOM36 might be a novel protein import factor acting on a subset of alpha-helically anchored mitochondrial outer membrane proteins. Identification of pATOM36 interaction partners by co-immunoprecipitation together with immunofluorescence analysis shows that unexpectedly a fraction of the protein is associated with the tripartite attachment complex (TAC). This complex is essential for proper inheritance of the mitochondrial DNA in T. brucei. It forms a physical connection between the single unit mitochondrial DNA and the basal body of the flagellum that is stable throughout the cell cycle. Thus, pATOM36 simultaneously mediates ATOM assembly, and thus protein import, as well as mitochondrial DNA inheritance since it is an essential component of the TAC.

Oral treatments of Echinococcus multilocularis-infected mice with the antimalarial drug mefloquine that potentially interacts with parasite ferritin and cystatin.

This study investigated the effects of oral treatments of Echinococcus multilocularis-infected mice with the antimalarial drug mefloquine (MEF) and identified proteins that bind to MEF in parasite extracts and human cells by affinity chromatography. In a pilot experiment, MEF treatment was applied 5 days per week and was intensified by increasing the dosage stepwise from 12.5 mg/kg to 200 mg/kg during 4 weeks followed by treatments of 100 mg/kg during the last 7 weeks. This resulted in a highly significant reduction of parasite weight in MEF-treated mice compared with mock-treated mice, but the reduction was significantly less efficacious compared with the standard treatment regimen of albendazole (ABZ). In a second experiment, MEF was applied orally in three different treatment groups at dosages of 25, 50 or 100 mg/kg, but only twice a week, for a period of 12 weeks. Treatment at 100 mg/kg had a profound impact on the parasite, similar to ABZ treatment at 200 mg/kg/day (5 days/week for 12 weeks). No adverse side effects were noted. To identify proteins in E. multilocularis metacestodes that physically interact with MEF, affinity chromatography of metacestode extracts was performed on MEF coupled to epoxy-activated Sepharose(®), followed by SDS-PAGE and in-gel digestion LC-MS/MS. This resulted in the identification of E. multilocularis ferritin and cystatin as MEF-binding proteins. In contrast, when human cells were exposed to MEF affinity chromatography, nicotinamide phosphoribosyltransferase was identified as a MEF-binding protein. This indicates that MEF could potentially interact with different proteins in parasites and human cells.