A step towards development of promising trypanocidal agents: Synthesis, characterization and in vitro biological evaluation of ferrocenyl Mannich base-type derivatives.

Chagas disease is a neglected chronical parasitosis caused by the parasite Trypanosoma cruzi (T. cruzi). Nine ferrocenyl Mannich base derivatives were synthetized and characterized to explore their in vitro activity on three T. cruzi strains of the parasite and their cytotoxicity on Vero cells to calculate the selectivity index (SI). Compound 2, 1-ferrocenyl-3-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)propan-1-one, stood out as the most promising derivative showing a half maximal inhibitory concentration (IC50) value around 5 µM in both amastigote and trypomastigote forms of T. cruzi and SI values higher than 13, being the best value on the trypomastigote forms of the Arequipa strain (SI = 41.7). Moreover, 2 decreased the number of infected cells and was not genotoxic. Furthermore, its possible mechanism of action was studied through the alteration of the metabolites excreted by the parasite during glucose metabolism, the detection of mitochondrial alterations and the inhibition of superoxide dismutase (SOD). Finally, docking studies were executed to analyze the binding mode of the studied compounds to Fe-SOD enzyme.

Genotyping of Toxoplasma gondii: The advantages of variable number tandem repeats within coding regions.

Toxoplasma gondii is an intracellular protozoan which is widely distributed. Infection occurs as a result of ingestion of uncooked meat and exposure to cat feces. Immunocompetent individuals are generally asymptomatic, while severe disease may occur in immunocompromised subjects and in congenital toxoplasmosis, which is caused by transplacental acquisition of T. gondii. Genetic diversity of T. gondii has often been studied using a PCR-RFLP scheme based on nine molecular markers. These studies led to the description of a clonal population structure with three main lineages (I, II and III) in North America and Europe and higher genetic diversity in South America. The aim of this study was to develop molecular markers that could allow the discrimination of genetic variants within each clonal lineage. We analyzed the genome of T. gondii to identify genes containing variable number tandem repeats (VNTRs). The coding sequences of T. gondii ME49 genome were processed with Tandem Repeat Finder software. A panel of candidate markers was selected based on the following parameters: the repeat unit size (>9 bp) and composition (to avoid single and dinucleotide runs), the number of copies (<20), and the absence of introns within the repeat region. The selected panel of eight molecular markers was analyzed in PRU and RH strains. As a first step, the variability of the sequence size allowed us to differentiate PRU from ME49 (two type II strains) and RH from GT1 (two type I strains). Additionally, amplification products from PRU and RH strains were sequenced to study intra-lineage variability. Aside from size polymorphisms in the amplification products we were able to identify sequence variability in polymorphic markers.

Characterisation of susceptibility of chicken macrophages to infection with Toxoplasma gondii of type II and III strains.

Toxoplasma gondii is known to be able to infect any nucleated cell including immune cells like macrophages. In addition, it is assumed that macrophages serve as trojan horse during distribution in hosts. The underlying causes of parasite host interaction remain yet not fully understood. The aim of the present study was to investigate susceptibility of chicken macrophages to infection with T. gondii and the process of infection in avian cells in comparison to cells of mammalian origin. Primary avian blood monocyte-derived macrophages were infected with tachyzoites of type II (ME49) and III (NED) strains. Long term observations of parasite replication in primary macrophages were compared to data obtained in an avian macrophage cell line (HD11) and a standard cultivation mammalian cell line (VERO). Furthermore, we assessed the immune response of the primary macrophages by long-term investigation of gene expression of IL-1 beta, IL-12p40, Lipopolysaccharide induced TNF-alpha factor (LITAF) and inducible nitric oxide synthase (iNOS) comparing viable and heat-inactivated tachyzoites of the ME49 strain. Albeit, we found no differences between both strains, replication of tachyzoites in avian primary macrophages was significantly different from immortalized cell lines HD11 and VERO. The crucial period of parasite replication was between 8 and 24 h post-infection coinciding with the upregulation of gene expression of cytokines and iNOS revealing an active macrophage response at this period. Gene expression in macrophages was higher after infection with viable tachyzoites than by exposure of cells to heat-inactivated tachyzoites. Hence, we conclude that the process of penetration is pivotal for host cell response to the parasite both in avian as in mammalian cells.

Pyrimidine-chloroquinoline hybrids: Synthesis and antiplasmodial activity.

Triazole tethered 7-chloroquinoline-pyrimidine-5-carboxylate hybrids were synthesized and evaluated for antiplasmodial activity against chloroquine sensitive (CQS) NF54 strain of Plasmodium falciparum. The most active hybrids of the series were further screened against the chloroquine resistant (CQR) Dd2 strain of the parasite and for in vitro cytotoxicity against mammalian Vero cell lines. Further, their physico-chemical properties, binding studies with hemin (monomeric &µ-oxo dimeric) and DNA [pUC-18, calf thymus (CT)] led us to plausible proposed binding mode of the most active member of the present series.

Cloning, localization and differential expression of the Trypanosoma cruzi TcOGNT-2 glycosyl transferase.

The surface of Trypanosoma cruzi is covered by a dense glycocalix which is characteristic of each stage of the life cycle. Its composition and complexity depend mainly on mucin-like proteins. A remarkable feature of O-glycan biosynthesis in trypanosomes is that it initiates with the addition of a GlcNAc instead of the GalNAc residue that is commonly used in vertebrate mucins. The fact that the interplay between trans-sialidase and mucin is crucial for pathogenesis, and both families have stage-specific members is also remarkable. Recently the enzyme that transfers the first GlcNAc from UDP-GlcNAc to a serine or threonine residue was kinetically characterized. The relevance of this enzyme is evidenced by its role as catalyzer of the first step in O-glycosylation. In this paper we describe how this gene is expressed differentially along the life cycle with a pattern that is very similar to that of trans-sialidases. Its localization was determined, showing that the protein predicted to be in the Golgi apparatus is also present in reservosomes. Finally our results indicate that this enzyme, when overexpressed, enhances T. cruzi infectivity.

[Efficiency of in vitro culture of Toxoplasma gondii in THP1 and Vero cell lines]. / Eficiencia de cultivo in vitro de Toxoplasma gondii en las líneas celulares THP1 y Vero.

INTRODUCTION: Cell culture is an important method for isolating Toxoplasma gondii to make clinical diagnosis or for biotechnological purposes. OBJECTIVE: The percentage of invasion and production levels of T. gondii was determined for THP1 and Vero cell lines. MATERIALS AND METHODS: The growth conditions for T. gondii in Vero and THP1 cell lines were determined by counting in hemocytometer chamber. The percentage of invasion of T. gondii in THP1 and Vero cells was determined by flow cytometry in different cell/tachyzoite ratios: 1/5, 1/20, 1/50. The growth performance index of the T. gondii RH strain and the CIBM1 isolate was calculated for THP1 cells. RESULTS: Vero cells multiplied faster than the THP1 cells, showing an exponential and a sigmoidal growth curve respectively, within a period of 7 days. The CIBM1 isolate infected the THP1 cells in three different parasite concentrations: 1/5, 1/20 and 1/50, with invasion percentages in THP1 cells of 57.1%, 15.5% and 12.2% and for the Vero cells 25.3%, 17.8% and 8.8% respectively. The RH strain of T. gondii had the lowest invasion percentage with 32.6%, 14.8% and 8.1% in THP1 cells and 22.3%, 14.1% and 3.4% in Vero cells. CONCLUSIONS: The CIBM1 isolate had a higher yield than the RH strain of T. gondii in THP1 cells. THP1 cells were indicated to be a good model for the study of invasion and for the assays of new pharmacological candidates against T. gondii.

[Production of Toxoplasma gondii in Vero cell culture]. / Vero Hücre Kültüründe Toxoplasma gondii Üretimi.

OBJECTIVE: To investigate parasite-host dynamics in cultures of Toxoplasma gondii (T. gondii). METHODS: T. gondii tachyzoites were incubated in Vero-E6 cell cultures at 37°C, 5％CO2. Tachyzoites and host cells were characterized by light microscopy. Growth kinetics of the parasite were determined. RESULTS: Doubling time of tachyzoites and viability of host cells were determined by comparing tachyzoite cultures and control Vero cell cultures that were free of parasites. Tachyzoites harvested from cell cultures were established as a line for future studies. Purified tachyzoit line was named as GPK-001, a catalog name for the line. CONCLUSION: In this study, we showed that an intracellular parasite, T. gondii can be produced in cell cultures under sterile conditions. We believe that the tachyzoite line established in this study would be useful in many other studies and provide answers to questions regarding biology and treatment of T. gondii infections.

Trypanocidal properties, structure-activity relationship and computational studies of quinoxaline 1,4-di-N-oxide derivatives.

Pyrazole and propenone quinoxaline derivatives were tested against intracellular forms of Leishmania peruviana and Trypanosoma cruzi. Both series were tested for toxicity against proliferative and non-proliferative cells. The pyrazole quinoxaline series was quite inactive against T. cruzi; however, the compound 2,6-dimethyl-3-f-quinoxaline 1,4-dioxide was found to inhibit 50% of Leishmania growth at 8.9 µM, with no impact against proliferative kidney cells and with low toxicity against THP-1 cells and murine macrophages. The compounds belonging to the propenone quinoxaline series were moderately active against T. cruzi. Among these compounds, two were particularly interesting, (2E)-1-(7-fluoro-3-methyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-phenyl)-propenone and (2E)-3-(3,4,5-trimethoxy-phenyl)-1-(3,6,7-trimethyl-quinoxalin-2-yl)-propenone. The former possessed selective activity against proliferative cells (cancer and parasites) and was inactive against murine peritoneal macrophages; the latter was active against Leishmania and inactive against the other tested cells. Furthermore, insilico studies showed that both series respected Lipinski's rules and that they confirmed a linear correlation between trypanocidal activities and LogP. Docking studies revealed that compounds of the second series could interact with the poly (ADP-ribose) polymerase protein of Trypanosoma cruzi.

A new thrombospondin-related anonymous protein homologue in Neospora caninum (NcMIC2-like1).

Neospora caninum is an Apicomplexan protozoan that has the dog as a definitive host and cattle (among other animals) as intermediate hosts. It causes encephalopathy in dogs and abortion in cows, with significant loss in worldwide livestock. As any Apicomplexan, the parasite invades the cells using proteins contained in the phylum-specific organelles, like the micronemes, rhoptries and dense granules. The aim of this study was the characterization of a homologue (denominated NcMIC2-like1) of N. caninum thrombospondin-related anonymous protein (NcMIC2), a micronemal protein previously shown to be involved in the attachment and connection with the intracellular motor responsible for the active process of invasion. A polyclonal antiserum raised against the recombinant NcMIC2-like1 functional core (thrombospondin and integrin domains) recognized the native form of NcMIC2-like1, inhibited the in vitro invasion process and localized NcMIC2-like1 at the apical complex of the parasite by confocal immunofluorescence, indicating its micronemal localization. The new molecule, NcMIC2-like1, has features that differentiates it from NcMIC2 in a substantial way to be considered a homologue.

Roles of CD122+ cells in resistance against Neospora caninum infection in a murine model.

Innate cells, such as natural killer (NK) cells and NKT cells, play essential roles as primary effector cells at the interface between the host and parasite until establishment of adaptive immunity. However, the roles of NK and NKT cells in defense against Neospora caninum have not been well clarified. NK and NKT cells were depleted by the treatment with an anti-CD122 (interleukin-2 receptor beta chain) monoclonal antibody (mAb, TM-ß1) in vivo. The parasite burden in the brain of mice was promoted by the treatment with anti-CD122 mAb. However, there was no significant difference in the infection rates between controls and the mice treated with anti-asialoGM1 antibody to deplete NK cells. Activation of CD4+ T cells was suppressed in the mice treated with anti-CD122 mAb compared with controls and the mice treated with anti-asialoGM1 antibody. On the other hand, depletion of CD122+ cells or NK cells did not affect the number of activated CD8+ T cells, dendritic cells and B cells following N. caninum infection. These results indicate that CD122+ cells (probably NKT cells) play a crucial role in host defense by activating CD4+ T cells against N. caninum infection.

Anti-parasitic action and elimination of intracellular Toxoplasma gondii in the presence of novel thiosemicarbazone and its 4-thiazolidinone derivatives

Toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. In the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of Toxoplasma. The compounds were tested in infected Vero cell culture using concentration screens (0.1 to 20 mM). The final concentration of 1 mM was chosen for biological assay. The following results were obtained: 1) These new derivatives decreased T. gondii infection with an in vitro parasite IC50 percent of 0.2-0.7 mM, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) The main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) Ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) Vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) The death and elimination of T. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. Our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mM concentration for 24 h of treatment.

Anti-parasitic action and elimination of intracellular Toxoplasma gondii in the presence of novel thiosemicarbazone and its 4-thiazolidinone derivatives.

Toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. In the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of Toxoplasma. The compounds were tested in infected Vero cell culture using concentration screens (0.1 to 20 mM). The final concentration of 1 mM was chosen for biological assay. The following results were obtained: 1) These new derivatives decreased T. gondii infection with an in vitro parasite IC50% of 0.2-0.7 mM, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) The main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) Ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) Vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) The death and elimination of T. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. Our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mM concentration for 24 h of treatment.

Novel strategy in Trypanosoma cruzi cell invasion: implication of cholesterol and host cell microdomains.

Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligatory intracellular parasite in the mammalian host. In order to invade a wide variety of mammalian cells, T. cruzi engages parasite components that are differentially expressed among strains and infective forms. Because the identification of putative protein receptors has been particularly challenging, we investigated whether cholesterol and membrane rafts, sterol- and sphingolipid-enriched membrane domains, could be general host surface components involved in invasion of metacyclic trypomastigotes and extracellular amastigotes of two parasite strains with distinct infectivities. HeLa or Vero cells treated with methyl-beta-cyclodextrin (MbetaCD) are less susceptible to invasion by both infective forms, and the effect was dose-dependent for trypomastigote but not amastigote invasion. Moreover, treatment of parasites with MbetaCD only inhibited trypomastigote invasion. Filipin labeling confirmed that host cell cholesterol concentrated at the invasion sites. Binding of a cholera toxin B subunit (CTX-B) to ganglioside GM1, a marker of membrane rafts, inhibited parasite infection. Cell labeling with CTX-B conjugated to fluorescein isothiocyanate revealed that not only cholesterol but also GM1 is implicated in parasite entry. These findings thus indicate that microdomains present in mammalian cell membranes, that are enriched in cholesterol and GM1, are involved in invasion by T. cruzi infective forms.

EnP1, a microsporidian spore wall protein that enables spores to adhere to and infect host cells in vitro.

Microsporidia are spore-forming fungal pathogens that require the intracellular environment of host cells for propagation. We have shown that spores of the genus Encephalitozoon adhere to host cell surface glycosaminoglycans (GAGs) in vitro and that this adherence serves to modulate the infection process. In this study, a spore wall protein (EnP1; Encephalitozoon cuniculi ECU01_0820) from E. cuniculi and Encephalitozoon intestinalis is found to interact with the host cell surface. Analysis of the amino acid sequence reveals multiple heparin-binding motifs, which are known to interact with extracellular matrices. Both recombinant EnP1 protein and purified EnP1 antibody inhibit spore adherence, resulting in decreased host cell infection. Furthermore, when the N-terminal heparin-binding motif is deleted by site-directed mutagenesis, inhibition of adherence is ablated. Our transmission immunoelectron microscopy reveals that EnP1 is embedded in the microsporidial endospore and exospore and is found in high abundance in the polar sac/anchoring disk region, an area from which the everting polar tube is released. Finally, by using a host cell binding assay, EnP1 is shown to bind host cell surfaces but not to those that lack surface GAGs. Collectively, these data show that given its expression in both the endospore and the exospore, EnP1 is a microsporidian cell wall protein that may function both in a structural capacity and in modulating in vitro host cell adherence and infection.

Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal.

Little is known of Toxoplasma gondii infections in animals in Portugal. In the present paper, we report the first isolation of viable T. gondii from pigs in Portugal. Antibodies to T. gondii were found in 52 (15.6%) of 333 pigs prior to slaughter using the modified agglutination test (MAT) at a serum dilution of 1:20. Attempts were made to isolate T. gondii from 37 seropositive pigs. Samples of brain and/or heart from each pig were digested in acid pepsin, and bioassayed into mice. Viable T. gondii was isolated from 15 pigs. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR and microsatellite analysis revealed that 11 isolates were Type II and four were Type III. The results indicate that phenotypically and genetically T. gondii are similar to isolates from pigs from the U.S.

Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms.

Trypanosoma cruzi, the etiological agent of Chagas disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

Interaction of Leishmania (L.) chagasi with the Vero cell line.

The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L.) chagasi. This strain (MHOM/BR/501/MS00) was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20% of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L.) chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan.

In vitro induction of Neospora caninum bradyzoites in vero cells reveals differential antigen expression, localization, and host-cell recognition of tachyzoites and bradyzoites.

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 microM 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.

Cis and trans factors involved in apicoplast targeting in Toxoplasma gondii.

Ribosomal subunit protein 9 (rps9) is a nuclearly encoded protein that resides in the apicoplast organelle of Toxoplasma gondii. Two cis-acting regions within the rps9 transit domain (amino acids 38-49 and 79-86), when combined with the rps9 signal sequence, were necessary and sufficient for apicoplast targeting. To investigate proteins interacting with the rps9 leader sequence, parasites expressing rps9 leader constructs fused to a glutathione S-transferase (GST) reporter were prepared, and proteins associated with the leader constructs were purified from extracts by affinity chromatography. In addition to GST-containing peptides, proteins with apparent masses of 92, 90, 86, and 160 kDa were purified. Mass spectrometry data suggested that the 92- and 90-kDa polypeptides appear to be subtilisin-like proteins, whereas the 86-kDa polypeptide was identified as the molecular chaperone BiP of T. gondii.

Acidification modulates the traffic of Trypanosoma cruzi trypomastigotes in Vero cells harbouring Coxiella burnetii vacuoles.

We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella burnetii. When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin A1 and related drugs, increased invasion of both metacyclic and tissue-culture trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. The higher tissue-culture trypomastigote hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2 cells facilitate the escape of both forms. Endosomal-lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of C. burnetii vacuolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells.