Infective Trypomastigote Forms Research Articles

Mechanisms by which Factor H protects Trypanosoma cruzi from the alternative pathway of complement.

Chagas disease, a chronic disabling disease caused by the protozoan Trypanosoma cruzi, has no standardized treatment or preventative vaccine. The infective trypomastigote form of T. cruzi is highly resistant to killing by the complement immune system. Factor H (FH), a negative regulator of the alternative pathway (AP) of complement on cell surfaces and in blood, contains 20 short consensus repeat domains. The four N-terminal domains of FH inactivate the AP, while the other domains interact with C3b/d and glycan markers on cell surfaces. Various pathogens bind FH to inactivate the AP. T. cruzi uses its trans-sialidase enzyme to transfer host sialic acids to its own surface, which could be one of the approaches it uses to bind FH. Previous studies have shown that FH binds to complement-opsonized T. cruzi and parasite desialylation increases complement-mediated lysis of trypomastigotes. However, the molecular basis of FH binding to T. cruzi remain unknown. Only trypomastigotes, but not epimastigotes (non-infective, complement susceptible) bound FH directly, independent of C3 deposition, in a dose-dependent manner. Domain mapping experiments using 3-5 FH domain fragments showed that domains 5-8 competitively inhibited FH binding to the trypomastigotes by ~35% but did not decrease survival in complement. FH-Fc or mutant FH-Fc fusion proteins (3-11 contiguous FH domains fused to the IgG Fc) also did not kill trypomastigotes. FH-related protein-5, whose domains bear significant sequence identity to all known polyanion-binding FH domains (6-7, 10-14, 19-20), fully inhibited FH binding to trypomastigotes and reduced trypomastigote survival to < 24% in the presence of serum. In conclusion, we have elucidated the role of FH in complement resistance of trypomastigotes.

Antiparasitic Activity of Oxindolimine–Metal Complexes against Chagas Disease

Some copper(II) and zinc(II) complexes with oxindolimine ligands were tested regarding their trypanocidal properties. These complexes have already shown good biological activity in the inhibition of tumor cell proliferation, having DNA and mitochondria as main targets, through an oxidative mechanism, and inducing apoptosis. Herein, we demonstrate that they also have significant activity against the infective trypomastigote forms and the intracellular amastigote forms of T. cruzi, modulated by the metal ion as well as by the oxindolimine ligand. Selective indexes (LC50/IC50) determined for both zinc(II) and copper(II) complexes, are higher after 24 or 48 h incubation with trypomastigotes, in comparison to traditional drugs used in clinics, such as benznidazole, and other metal-based compounds previously reported in the literature. Additionally, tests against amastigotes indicated infection index &lt;10% (% of infected macrophages/average number of amastigotes per macrophage), after 24 or 48 h in the presence of zinc(II) (60–80 µM) or analogous copper(II) complexes (10–25 µM). The copper complexes exhibit further oxidative properties, being able to damage DNA, proteins and carbohydrates, in the presence of hydrogen peroxide, with the generation of hydroxyl radicals. This redox reactivity could explain its better performance towards the parasites in relation to the zinc analogs. However, both copper and zinc complexes display good selective indexes, indicating that the influence of the ligand is also crucial, and is probably related to the inhibition of some crucial proteins.

Benznidazole and amiodarone combined treatment attenuates cytoskeletal damage in Trypanosoma cruzi-infected cardiac cells.

Chagas disease (CD), a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, is an important public health problem mainly in Latin America, leading to approximately 12,000 annual deaths. Current etiological treatment for CD is limited to two nitro compounds, benznidazole (Bz) and nifurtimox (Nif), both presenting relevant limitations. Different approaches have been employed to establish more effective and safer schemes to treat T. cruzi infection, mostly based on drug repurposing and combination therapies. Amiodarone (AMD), an antiarrhythmic medicament of choice for patients with the chronic cardiac form of CD, is also recognized as a trypanocidal agent. Therefore, our aim is to investigate the combined treatment Bz + AMD on trypomastigote viability, control of T. cruzi intracellular form proliferation, and recovery of the infection-induced cytoskeleton alterations in cardiac cells. The combination of Bz + AMD did not improve the direct trypanocidal effect of AMD on the infective blood trypomastigote and replicative intracellular forms of the parasite. Otherwise, the treatment of T. cruzi-infected cardiac cells with Bz plus AMD attenuated the infection-triggered cytoskeleton damage of host cells and the cytotoxic effects of AMD. Thus, the combined treatment Bz + AMD may favor parasite control and hamper tissue damage.

Experimental infection of Rhodnius robustus Larrousse, 1927 (Hemiptera, Reduviidae, Triatominae) with Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae) IV

Vector competence of triatomines (kissing bugs) for Trypanosoma cruzi transmission depends on the parasite-vector interaction and the genetic constitution of both. This study evaluates the susceptibility and vector competence of Rhodnius robustus experimentally infected with T. cruzi IV (TcIV). Nymphs were fed on infected mice or an artificial feeder with blood containing culture-derived metacyclic trypomastigotes (CMT) or blood trypomastigotes (BT). The intestinal contents (IC) and excreta of the insects were examined by fresh examination and kDNA-PCR. The rate of metacyclogenesis was also determined by differential counts. Fifth instar nymphs fed with CMT ingested a greater blood volume (mean of 74.5 μL) and a greater amount of parasites (mean of 149,000 CMT/μL), and had higher positivity in the fresh examination of the IC. Third instar nymphs fed with CMT had higher positivity (33.3%) in the fresh examination of the excreta. On the 20th day after infection (dai), infective metacyclic trypomastigote (MT) forms were predominant in the excreta of 3/4 experimental groups, and on the 30th dai, the different parasitic forms were observed in the IC of all the groups. Higher percentages of MT were observed in the excreta of the 5th instar nymphs group (84.1%) and in the IC of the 3rd instar nymphs group (80.0%). Rhodnius robustus presented high susceptibility to infection since all nymphs were infected, regardless of the method used for blood meal, in addition these insects demonstrated vector competence for TcIV with high rates of metacyclogenesis being evident.

Exoproteome profiling of Trypanosoma cruzi during amastigogenesis early stages.

Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting around 8 million people worldwide. After host cell invasion, the infective trypomastigote form remains 2–4 hours inside acidic phagolysosomes to differentiate into replicative amastigote form. In vitro acidic-pH-induced axenic amastigogenesis was used here to study this step of the parasite life cycle. After three hours of trypomastigote incubation in amastigogenesis promoting acidic medium (pH 5.0) or control physiological pH (7.4) medium samples were subjected to three rounds of centrifugation followed by ultrafiltration of the supernatants. The resulting exoproteome samples were trypsin digested and analysed by nano flow liquid chromatography coupled to tandem mass spectrometry. Computational protein identification searches yielded 271 and 483 protein groups in the exoproteome at pH 7.4 and pH 5.0, respectively, with 180 common proteins between both conditions. The total amount and diversity of proteins released by parasites almost doubled upon acidic incubation compared to control. Overall, 76.5% of proteins were predicted to be secreted by classical or non-classical pathways and 35.1% of these proteins have predicted transmembrane domains. Classical secretory pathway analysis showed an increased number of mucins and mucin-associated surface proteins after acidic incubation. However, the number of released trans-sialidases and surface GP63 peptidases was higher at pH 7.4. Trans-sialidases and mucins are anchored to the membrane and exhibit an enzyme-substrate relationship. In general, mucins are glycoproteins with immunomodulatory functions in Chagas disease, present mainly in the epimastigote and trypomastigote surfaces and could be enzymatically cleaved and released in the phagolysosome during amastigogenesis. Moreover, evidence for flagella discard during amastigogenesis are addressed. This study provides the first comparative analysis of the exoproteome during amastigogenesis, and the presented data evidence the dynamism of its profile in response to acidic pH-induced differentiation.

Primary evidence of the mechanisms of action of HIV aspartyl peptidase inhibitors on Trypanosoma cruzi trypomastigote forms

The development of HIV aspartyl peptidase inhibitors (HIV-PIs) and their introduction into AIDS therapy preceded a significant decrease in the incidence, morbidity and mortality of relevant protozoan co-infections. However, few data are available about how HIV-PIs act on pathogenic parasites, such as Trypanosoma cruzi, the etiological agent of Chagas disease. Therefore, the aim of the present work was to evaluate different physiological aspects of the treatment of the infective trypomastigote forms of T. cruzi with the HIV-PIs, nelfinavir and lopinavir. At the LD50/4 h doses, both HIV-PIs significantly reduced the trypomastigote size and markedly increased the granularity/complexity. Transmission electron microscopy analysis associated to biochemical assays permitted definition of the main HIV-PIs targets in the parasite. Lopinavir and nelfinavir induced (i) plasma membrane shedding, particularly in the flagellar region, which drastically affected parasite integrity; (ii) strong mitochondrial swelling with rare matrix fragmentation, which were linked to severely reduced hydrolytic activity of dehydrogenases and organelle membrane depolarization; (iii) increased generation of reactive oxygen species (ROS); (iv) dilation of both nuclear envelope (without DNA disruption) and endoplasmic reticulum (with formation of autophagosomes), and (v) accumulation of intracellular lipid droplets, revealing a typical lipid metabolism disorder. Collectively, our study demonstrated that nelfinavir and lopinavir target vital cellular structures of trypomastigotes, culminating in irreversible metabolic injuries that lead to T. cruzi death.

Response to Trypanosoma cruzi by Human Blood Cells Enriched with Dentritic Cells Is Controlled by Cyclooxygenase-2 Pathway.

Chagas disease (Cd) or American human trypanosomiasis is caused by Trypanosoma cruzi and affects ~7 million people, mostly in Latin America. The infective trypomastigote forms of the parasite can invade several human blood cell populations, including monocytes and dendritic cells (DC). Although these cells display a wide functional diversity, their interactions with T. cruzi via cyclooxygenase (COX) and cyclic adenosine monophosphate (cAMP) dependent pathways have not been analyzed. To exploiting this mechanism, DC-enriched peripheral human blood mononuclear cell populations (DC-PBMC) were used as our model. Our results showed that the treatment of these cell populations with celecoxib (CEL), a cyclooxygenase-2 selective inhibitor or SQ 22,536, an adenilate cyclase inhibitor, significantly caused marked inhibition of T. cruzi infection. In contrast, aspirin (ASA, a non-selective COX-1 and COX-2 inhibitor) treatment did not inhibit the infection of the cells by the parasite and was independent of nitric oxide (NO) production. The expression of co-stimulatory molecules CD80 and CD86 were similar on cells treated or not with both COX-inhibitors. The infection stimulated the release of TNF-α, IL-1β, IL-6, IL-8, and IL-10 production by infected cells. Treatment with ASA or CEL did not affect TNF-α, IL-6, IL-8, IL-10, and NO production by infected cells, but increased IL-1β production by them. Our results suggest a key role of COX-2 and cAMP pathways in T. cruzi invasion process of human blood cells and these pathways may represent targets of new therapeutic options for Cd.

Design, Synthesis, and Trypanocidal Activity of Novel 5‐Nitroimidazolyl O‐Benzyloxime Ethers

In this paper, we describe the synthesis and the action against of the trypomastigote form of Trypanosoma cruzi of a new class of nitroimidazole‐2‐carbaldehyde O‐benzyloximes. These derivatives were designed through the application of molecular hybridization concept between two potent antiprotozoal compounds, the 5‐nitrothiophene‐2‐carbaldehyde O‐oxime 6 and the trypanocidal piperidinyl‐4‐carbaldehyde O‐benzyloxime 7 with the intention of reaching two distinct molecular targets of T. cruzi. The activity of these benzyl ether derivatives was tested against the infective trypomastigote forms of T. cruzi, and the derivative (E)‐1‐methyl‐5‐nitro‐1H‐imidazole‐2‐carbaldehyde O‐(4‐nitrobenzyl) oxime (1) presented moderate trypanocidal activity (IC50 = 12.7 μM) when compared with the standard drug benznidazole, which showed to be a good starting point for the design of more effective trypanocide agents.

Virus-like Particle Display of the α-Gal Epitope for the Diagnostic Assessment of Chagas Disease

The α-Gal antigen [Galα(1,3)Galβ(1,4)GlcNAcα] is an immunodominant epitope displayed by infective trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. A virus-like particle displaying a high density of α-Gal was found to be a superior reagent for the ELISA-based serological diagnosis of Chagas disease and the assessment of treatment effectiveness. A panel of sera from patients chronically infected with T. cruzi, both untreated and benznidazole-treated, was compared with sera from patients with leishmaniasis and from healthy donors. The nanoparticle-α-Gal construct allowed for perfect discrimination between Chagas patients and the others, avoiding false negative and false positive results obtained with current state-of-the-art reagents. As previously reported with purified α-Gal-containing glycosylphosphatidylinositol-anchored mucins, the current study also showed concentrations of anti-α-Gal IgG to decrease substantially in patients receiving treatment with benznidazole, suggesting that the semiquantitative assessment of serum levels of this highly abundant type of antibody can report on disease status in individual patients.

HIV aspartic peptidase inhibitors are effective drugs against the trypomastigote form of the human pathogen Trypanosoma cruzi

There is a general lack of effective and non-toxic chemotherapeutic agents against Chagas' disease despite more than a century of research. In this regard, we have verified the impact of human immunodeficiency virus aspartic peptidase inhibitors (HIV-PIs) on the viability and morphology of infective trypomastigote forms of Trypanosoma cruzi as well as on the aspartic peptidase and proteasome activities produced by this parasite. The effects of HIV-PIs on viability were assessed by counting motile parasites in a Neubauer chamber. Morphological alterations were detected by light microscopy of Giemsa-stained smears and scanning electron microscopy. Modulation of aspartic peptidase and proteasome activities by the HIV-PIs was measured by cleavage of fluorogenic peptide substrates. The majority of the HIV-PIs (6/9) were able to drastically decrease the viability of trypomastigotes after 4 h of treatment, with nelfinavir and lopinavir being the most effective compounds presenting LD50 values of 8.6 µM and 10.6 µM, respectively. Additionally, both HIV-PIs were demonstrated to be effective in a time- and cell density-dependent manner. Treatment with nelfinavir and lopinavir caused many morphological/ultrastructural alterations in trypomastigotes; parasites became round in shape, with reduced cell size and flagellar shortening. Nelfinavir and lopinavir were also capable of significantly inhibiting the aspartic peptidase and proteasome activities measured in trypomastigote extracts. These results strengthen the data on the positive effects of HIV-PIs on parasitic infections, possibly by targeting the parasite aspartic peptidase(s) and proteasome(s), opening a new possibility for the use of these clinically approved drugs as an alternative chemotherapy to treat Chagas' disease.

Monitoring of the Parasite Load in the Digestive Tract of Rhodnius prolixus by Combined qPCR Analysis and Imaging Techniques Provides New Insights into the Trypanosome Life Cycle.

BackgroundHere we report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection.Methodology/Principal FindingsqPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3–4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes.Conclusions/SignificanceThese data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence.

Nitric oxide-releasing polymeric nanoparticles against Trypanosoma cruzi

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi (T. cruzi), and the disease remains a major health problem in many Latin American countries. Several papers report that the killing of the parasite is dependent on the production of nitric oxide (NO). The endogenous free radical NO is an important cellular signalling molecule that plays a key role in the defense against pathogens, including T. cruzi. As T. cruzi is able to compromise host macrophages decreasing endogenous NO production, the administration of exogenous NO donors represents an interesting strategy to combat Chagas disease. Thus, the aims of this study were to prepare and evaluate the antimicrobial activity of NO-releasing polymeric nanoparticles against T. cruzi. Biocompatible polymeric nanoparticles composed of chitosan/sodium tripolyphosphate(TPP) were prepared and used to encapsulate mercaptosuccinic acid (MSA), which is a thiol-containing molecule. Nitrosation of free thiols (SH) groups of MSA were performed by the addition of equimolar amount of sodium nitrite (NaNO2), leading to the formation of S-nitroso-MSA-containing nanoparticles. These polymeric nanoparticles act as spontaneous NO donors, with free NO release. The results show the formation of nanoparticles with average hydrodynamic diameter ranging from 270 to 500 nm, average of polydispersity index of 0.35, and encapsulation efficiency in the range of 99%. The NO release kinetics from the S-nitroso-MSA-containing nanoparticles showed sustained and controlled NO release over several hours. The microbicidal activity of S-nitroso-MSA-containing nanoparticles was evaluated by incubating NO-releasing nanoparticles (200 - 600 μg/mL) with replicative and non-infective epimastigote, and non-replicative and infective trypomastigote forms of T. cruzi. In addition, a significant decrease in the percentage of macrophage-infected (with amastigotes) and NO-releasing nanoparticle-treated cells was observed. Taken together, our results reveal a potent toxic effect of NO-releasing polymeric nanoparticles against different life cycle forms of T. cruzi, indicating that the encapsulation of the NO donor S-nitroso-MSA represents an interesting approach to combat and to prevent Chagas disease.

In vitro metacyclogenesis of Trypanosoma cruzi induced by starvation correlates with a transient adenylyl cyclase stimulation as well as with a constitutive upregulation of adenylyl cyclase expression

The Trypanosoma cruzi adenylyl cyclase (AC) multigene family encodes different isoforms (around 15) sharing a variable large N-terminal domain, which is extracellular and receptor-like, followed by a transmembrane helix and a conserved C-terminal catalytic domain. It was proposed that these key enzymes in the cAMP signalling pathway allow the parasite to sense its changing extracellular milieu in order to rapidly adapt to its new environment, which is generally achieved through a differentiation process. One of the critical differentiation events the parasitic protozoan T. cruzi undergoes during its life cycle, known as metacyclogenesis, occurs in the digestive tract of the insect and corresponds to the differentiation from noninfective epimastigotes to infective metacyclic trypomastigote forms. By in vitro monitoring the activity of AC during metacyclogenesis, we showed that both the activity of AC and the intracellular cAMP content follow a similar pattern of transient stimulation in a two-step process, with a first activation peak occurring during the first hours of nutritional stress and a second peak between 6 and 48h, corresponding to the cellular adhesion. During this differentiation process, a general mechanism of upregulation of AC expression of both mRNA and protein is triggered and in particular for a major subclass of these enzymes that are present in various gene copies commonly associated to the THT gene clusters. Although the scattered genome distribution of these gene copies is rather unusual in trypanosomatids and seems to be a recent acquisition in the evolution of the T. cruzi clade, their encoded product redistributed on the flagellum of the parasite upon differentiation could be important to sense the extracellular milieu.

Natural sesquiterpene lactones induce programmed cell death in Trypanosoma cruzi: A new therapeutic target?

BackgroundChagas disease or American Trypanosomiasis is caused by the flagellated protozoan parasite Trypanosoma cruzi (T. cruzi) and is recognized by the WHO as one of the world's 17 neglected tropical diseases. Only two drugs (Benznidazol, Bz and Nifurtimox, Nx) are currently accepted for treatment, however they cause severe adverse effects and their efficacy is still controversial. It is then important to explore for new drugs. PurposeProgrammed cell death (PCD) in parasites offers interesting new therapeutic targets. The aim of this work was to evaluate the induction of PCD in T. cruzi by two natural sesquiterpene lactones (STLs), dehydroleucodine (DhL) and helenalin (Hln) as compared with the two conventional drugs, Bz and Nx. Material and MethodsHln and DhL were isolated from aerial parts of Gaillardia megapotamica and Artemisia douglassiana Besser, respectively. Purity of compounds (greater than 95%) was confirmed by 13C-nuclear magnetic resonance, melting point analysis, and optical rotation. Induction of PCD in T. cruzi epimastigotes and trypomastigotes by DhL, Hln, Bz and Nx was assayed by phosphatidylserine exposure at the parasite surface and by detection of DNA fragmentation using the TUNEL assay. Trypanocidal activity of natural and synthetic compounds was assayed by measuring parasite viability using the MTT method. ResultsThe two natural STLs, DhL and Hln, induce programmed cell death in both, the replicative epimastigote form and the infective trypomastigote form of T. cruzi. Interestingly, the two conventional antichagasic drugs (Bz and Nx) do not induce programmed cell death. A combination of DhL and either Bz or Nx showed an increased effect of natural compounds and synthetic drugs on the decrease of parasite viability. ConclusionDhL and Hln induce programmed cell death in T. cruzi replicative epimastigote and infective trypomastigote forms, which is a different mechanism of action than the conventional drugs to kill the parasite. Therefore DhL and Hln may offer an interesting option for the treatment of Chagas disease, alone or in combination with conventional drugs.

The mRNAs associated to a zinc finger protein from Trypanosoma cruzi shift during stress conditions

Trypanosome gene expression is regulated almost exclusively at the posttranscriptional level, through mRNA stability, storage and degradation. Here, we characterize the ribonucleoprotein complex (mRNPs) corresponding to the zinc finger protein TcZC3H39 from T. cruzi comparing cells growing in normal conditions and under nutritional stress. The nutritional stress is a key step during T. cruzi differentiation from epimastigote form to human infective metacyclic trypomastigote form. The mechanisms by which the stress, altogether with other stimuli, triggers differentiation is not well understood. This work aims to characterize the TcZC3H39 protein during stress response. Using cells cultured in normal and stress conditions, we observed a dynamic change in TcZC3H39 granule distribution, which appeared broader in stressed epimastigotes. The protein core of the TcZC3H39-mRNP is composed of ribosomes, translation factors and RBPs. The TcZC3H39-mRNP could act sequestering highly expressed mRNAs and their associated ribosomes, potentially slowing translation in stress conditions. A shift were observed in the mRNAs associated with TcZC3H39: the number of targets in unstressed epimastigotes was smaller than that in stressed parasites, with no clear functional clustering in normal conditions. By contrast, in stressed parasites, the targets of TcZC3H39 were mRNAs encoding ribosomal proteins and a remarkable enrichment in mRNAs for the cytochrome c complex (COX), highly expressed mRNAs in the replicative form. This identification of a new component of RNA granules in T. cruzi, the TcZC3H39 protein, provides new insight into the mechanisms involved in parasite stress responses and the regulation of gene expression during T. cruzi differentiation.

Characterization of the small RNA content of Trypanosoma cruzi extracellular vesicles

A growing body of evidence in mammalian cells indicates that secreted vesicles can be used to mediate intercellular communication processes by transferring various bioactive molecules, including mRNAs and microRNAs. Based on these findings, we decided to analyze whether Trypanosoma cruzi-derived extracellular vesicles contain RNA molecules and performed a deep sequencing and genome-wide analysis of a size-fractioned cDNA library (16–40nt) from extracellular vesicles secreted by noninfective epimastigote and infective metacyclic trypomastigote forms. Our data show that the small RNAs contained in these extracellular vesicles originate from multiple sources, including tRNAs. In addition, our results reveal that the variety and expression of small RNAs are different between parasite stages, suggesting diverse functions. Taken together, these observations call attention to the potential regulatory functions that these RNAs might play once transferred between parasites and/or to mammalian host cells.

Potential Mechanism of the Anti-trypanosomal Activity of Organoruthenium Complexes with Bioactive Thiosemicarbazones

In the search for new metal-based drugs against diseases produced by trypanosomatid parasites, four organoruthenium(II) compounds [Ru2(p-cymene)2(L)2]X2, where L are bioactive 5-nitrofuryl-containing thiosemicarbazones and X = Cl or PF6, had been previously obtained. These compounds had shown activity on Trypanosoma brucei, the etiological agent of African trypanosomiasis. Because of genomic similarities between trypanosomatides, these ruthenium compounds were evaluated, in the current work, on Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas disease). Two of them showed significant in vitro growth inhibition activity against the infective trypomastigote form of T. cruzi (Dm28c clone, IC50 = 11.69 and 59.42μM for [Ru2(p-cymene)2(L4)2]Cl2 and [Ru2(p-cymene)2(L1)2]Cl2, respectively, where HL4 = 5-nitrofuryl-N-phenylthiosemicarbazone and HL1 = 5-nitrofurylthiosemicarbazone), showing fairly good selectivities toward trypanosomes with respect to mammalian cells (J774 murine macrophages). Moreover, [Ru2(p-cymene)2(L2)2]Cl2, where HL2 = 5-nitrofuryl-N-methylthiosemicarbazone, was synthesized in order to evaluate the effect of improved solubility on biological behavior. This new chloride salt showed higher activity against T. cruzi than that of the previously synthesized hexafluorophosphate one (Dm28c clone, IC50 = 14.30μM for the former and 231.3μM for the latter). In addition, the mode of antitrypanosomal action of the organoruthenium compounds was investigated. The complexes were not only able to generate toxic free radicals through bioreduction but they also interacted with two further potential parasite targets: DNA and cruzipain, a cysteine protease which plays a fundamental role in the biological cycle of these parasites. The results suggest a "multi-target" mechanism of trypanosomicidal action for the obtained complexes.

Article Synthesis and Trypanocidal Activity of Novel 2,4,5-Triaryl-N-Hydroxylimidazole Derivatives

Herein, we report the design, synthesis and trypanocidal activity of some novel trisubstituted imidazole derivatives. These heterocyclic derivatives were structurally planned by exploring the concept of molecular hybridisation between two arylhydrazones derived from megazol, which has potent trypanocidal activity. The trypanocidal activity of these triarylimidazole derivatives was evaluated against infective trypomastigote forms of T. cruzi and the derivative 2'-(4-bromophenyl)-1-methyl-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol showed moderate biological activity (IC50 = 23.9 µM) when compared to benznidazole, a standard trypanocidal drug. These compounds did not present cytotoxic effects at concentrations near the trypanocidal IC50, being considered a good starting point for the development of new anti-Chagas drug candidates.

Regulatory elements in the 3′ untranslated region of the GP82 glycoprotein are responsible for its stage-specific expression in Trypanosoma cruzi metacyclic trypomastigotes

Gene expression in Trypanosoma cruzi is regulated at the post-transcriptional level and cis-acting elements present in the 3′ untranslated region (3′UTR) play an important role by interacting with regulatory proteins. Previous studies demonstrated that the GP82 surface glycoprotein, which is involved in host cell invasion, is up-regulated in the infective metacyclic trypomastigote form, and that GP82 mRNA half-life is longer in this form compared to the non-infective epimastigote form. Here, we demonstrate that the 3′UTR of the GP82 transcript is involved in this developmental regulation, promoting higher expression of the green fluorescent protein (GFP) reporter in metacyclic trypomastigotes than in epimastigotes. A series of stepwise deletions in the 3′UTR was created and results suggest that the mechanism regulating GP82 expression involves multiple elements in the 3′UTR.

Molecular and cellular mechanisms involved in the trypanosoma cruzi/host cell interplay

The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts. In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved. Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes. In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process. Lysosomal-dependent or -independent T. cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells. In this context, we have previously shown that induction of autophagy in host cells before infection favors T. cruzi invasion. Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T. cruzi entry sites and that the newly formed T. cruzi parasitophorous vacuole has characteristics of an autophagolysosome. This review summarizes the current knowledge of the molecular and cellular mechanisms of T. cruzi invasion in nonphagocytic cells. Based on our findings, we propose a new model in which T. cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells. 2012 IUBMB IUBMB Life, 2012