Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity.

BACKGROUND: The palmitate analogue 2-bromopalmitate (2-BP) is a non-selective membrane tethered cysteine alkylator of many membrane-associated enzymes that in the last years emerged as a general inhibitor of protein S-palmitoylation. Palmitoylation is a post-translational protein modification that adds palmitic acid to a cysteine residue through a thioester linkage, promoting membrane localization, protein stability, regulation of enzymatic activity, and the epigenetic regulation of gene expression. Little is known on such important process in the pathogenic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. RESULTS: The effect of 2-BP was analyzed on different developmental forms of Trypanosoma cruzi. The IC50/48 h value for culture epimastigotes was estimated as 130 µM. The IC50/24 h value for metacyclic trypomastigotes was 216 nM, while for intracellular amastigotes it was 242 µM and for cell derived trypomasigotes was 262 µM (IC50/24 h). Our data showed that 2-BP altered T. cruzi: 1) morphology, as assessed by bright field, scanning and transmission electron microscopy; 2) mitochondrial membrane potential, as shown by flow cytometry after incubation with rhodamine-123; 3) endocytosis, as seen after incubation with transferrin or albumin and analysis by flow cytometry/fluorescence microscopy; 4) in vitro metacyclogenesis; and 5) infectivity, as shown by host cell infection assays. On the other hand, lipid stress by incubation with palmitate did not alter epimastigote growth, metacyclic trypomastigotes viability or trypomastigote infectivity. CONCLUSION: Our results indicate that 2-BP inhibits key cellular processes of T. cruzi that may be regulated by palmitoylation of vital proteins and suggest a metacyclic trypomastigote unique target dependency during the parasite development.

Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes.

Dynamic S-palmitoylation of proteins is the addition of palmitic acid by zDHHC palmitoyl transferases (PATs) and depalmitoylation by palmitoyl protein thioesterases (PPTs). A putative PAT (TcPAT1) has been previously identified in Trypanosoma cruzi, the etiological agent of Chagas disease. Here we analyse other 14 putative TcPATs and 2 PPTs in the parasite genome. T. cruzi cell lines expressing TcPATs and TcPPTs plus a FLAG tag at the C terminus were produced for most enzymes, with positive detection by indirect immunofluorescence. Overexpressed TcPATs were mostly found as single spots at the parasite anterior end, while the TcPPTs were dispersed throughout the parasite body.

Knockdown of Host Antioxidant Defense Genes Enhances the Effect of Glucantime on Intracellular Leishmania braziliensis in Human Macrophages.

Leishmaniasis is a neglected tropical disease that affects millions of people worldwide and represents a major public health problem. Information on protein expression patterns and functional roles within the context of Leishmania-infected human monocyte-derived macrophages (MDMs) under drug treatment conditions is essential for understanding the role of these cells in leishmaniasis treatment. We analyzed functional changes in the expression of human MDM genes and proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime (SbV), using quantitative PCR (qPCR) arrays, Western blotting, confocal microscopy, and small interfering RNA (siRNA) human gene inhibition assays. Comparison of the results from gene transcription and protein expression analyses revealed that glutathione S-transferase π1 (GSTP1), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione synthetase (GSS), thioredoxin (TRX), and ATP-binding cassette, subfamily B, member 5 (ABCB5), were strongly upregulated at both the mRNA and protein levels in human MDMs that were infected and treated, compared to the control group. Subcellular localization studies showed a primarily phagolysosomal location for the ABCB5 transporter, indicating that this protein may be involved in the transport of SbV By inducing a decrease in L. braziliensis intracellular survival in THP-1 macrophages, siRNA silencing of GSTP1, GSS, and ABCB5 resulted in an increased leishmanicidal effect of SbV exposure in vitro Our results suggest that human MDMs infected with L. braziliensis and treated with SbV express increased levels of genes participating in antioxidant defense, whereas our functional analyses provide evidence for the involvement of human MDMs in drug detoxification. Therefore, we conclude that GSS, GSTP1, and ABCB5 proteins represent potential targets for enhancing the leishmanicidal activity of Glucantime.

Nerolidol, the main constituent of Piper aduncum essential oil, has anti-Leishmania braziliensis activity.

Leishmania (Viannia) braziliensis is a protozoan that causes mucocutaneous leishmaniasis, which is an infectious disease that affects more than 12 million people worldwide. The available treatment is limited, has side-effects or is inefficient. In a search for alternative compounds of natural origin, we tested the microbicidal activity of Piper aduncum essential oil (PaEO) on this parasite. Our data showed that PaEO had an inhibitory effect on the growth of L. braziliensis promastigotes with an IC50/24 h=77·9 µg mL-1. The main constituent (nerolidol: 25·22%) presented a similar inhibitory effect (IC50/24 h = 74·3 µg mL-1). Ultrastructural observation of nerolidol-treated parasites by scanning and transmission electron microscopies revealed cell shrinkage and morphological alterations in the mitochondrion, nuclear chromatin and flagellar pocket. Flow cytometry analysis showed a reduction in the cell size, loss of mitochondrial membrane potential, phosphatidylserine exposure and DNA degradation, which when associated with the morphological changes indicated that nerolidol induced incidental cell death in the L. braziliensis promastigotes. The results presented here indicate that nerolidol derivatives are promising compounds for further evaluation against Leishmania parasites.

Linalool, a Piper aduncum essential oil component, has selective activity against Trypanosoma cruzi trypomastigote forms at 4°C.

BACKGROUND: Recent studies showed that essential oils from different pepper species (Piper spp.) have promising leishmanicidal and trypanocidal activities. OBJECTIVES: In search for natural compounds against Trypanosoma cruzi, different forms of the parasite were incubated for 24 h at 28ºC or 4ºC with Piper aduncum essential oil (PaEO) or its main constituents linalool and nerolidol. METHODS: PaEO chemical composition was obtained by GC-MS. Drug activity assays were based on cell counting, MTT data or infection index values. The effect of PaEO on the T. cruzi cell cycle and mitochondrial membrane potential was evaluated by flow cytometry. FINDINGS: PaEO was effective against cell-derived (IC50/24 h: 2.8 µg/mL) and metacyclic (IC50/24 h: 12.1 µg/mL) trypomastigotes, as well as intracellular amastigotes (IC50/24 h: 9 µg/mL). At 4ºC - the temperature of red blood cells (RBCs) storage in blood banks - cell-derived trypomastigotes were more sensitive to PaEO (IC50/24 h = 3.8 µg/mL) than to gentian violet (IC50/24 h = 24.7 mg/mL). Cytotoxicity assays using Vero cells (37ºC) and RBCs (4ºC) showed that PaEO has increased selectivity for cell-derived trypomastigotes. Flow cytometry analysis showed that PaEO does not affect the cell cycle of T. cruzi epimastigotes, but decreases their mitochondrial membrane potential. GC-MS data identified nerolidol and linalool as major components of PaEO, and linalool had trypanocidal effect (IC50/24 h: 306 ng/mL) at 4ºC. MAIN CONCLUSION: The trypanocidal effect of PaEO is likely due to the presence of linalool, which may represent an interesting candidate for use in the treatment of potentially contaminated RBCs bags at low temperature.

Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi.

BACKGROUND: Nucleotide sugar transporters (NSTs) play an essential role in translocating nucleotide sugars into the lumen of the endoplasmic reticulum and Golgi apparatus to be used as substrates in glycosylation reactions. This intracellular transport is an essential step in the biosynthesis of glycoconjugates. RESULTS: We have identified a family of 11 putative NSTs in Trypanosoma cruzi, the etiological agent of Chagas' disease. A UDP-N-acetylglucosamine transporter, TcNST1, was identified by a yeast complementation approach. Based on a phylogenetic analysis four candidate genes were selected and used for complementation assays in a Kluyveromyces lactis mutant strain. The transporter is likely expressed in all stages of the parasite life cycle and during differentiation of epimastigotes to infective metacyclics. Immunofluorescence analyses of a GFP-TcNST1 fusion protein indicate that the transporter is localized to the Golgi apparatus. As many NSTs are multisubstrate transporters, we also tested the capacity of TcNST1 to transport GDP-Man. CONCLUSIONS: We have identified a UDP-N-acetylglucosamine transporter in T. cruzi, which is specifically localized to the Golgi apparatus and seems to be expressed, at the mRNA level, throughout the parasite life cycle. Functional studies of TcNST1 will be important to unravel the role of NSTs and specific glycoconjugates in T. cruzi survival and infectivity.

Clathrin expression in Trypanosoma cruzi.

BACKGROUND: Clathrin-mediated vesicular trafficking, the mechanism by which proteins and lipids are transported between membrane-bound organelles, accounts for a large proportion of import from the plasma membrane (endocytosis) and transport from the trans-Golgi network towards the endosomal system. Clathrin-mediated events are still poorly understood in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In this study, clathrin heavy (TcCHC) and light (TcCLC) chain gene expression and protein localization were investigated in different developmental forms of T. cruzi (epimastigotes, trypomastigotes and amastigotes), using both polyclonal and monoclonal antibodies raised against T. cruzi recombinant proteins. RESULTS: Analysis by confocal microscopy revealed an accumulation of TcCHC and TcCLC at the cell anterior, where the flagellar pocket and Golgi complex are located. TcCLC partially colocalized with the Golgi marker TcRAB7-GFP and with ingested albumin, but did not colocalize with transferrin, a protein mostly ingested via uncoated vesicles at the cytostome/cytopharynx complex. CONCLUSION: Clathrin heavy and light chains are expressed in T. cruzi. Both proteins typically localize anterior to the kinetoplast, at the flagellar pocket and Golgi complex region. Our data also indicate that in T. cruzi epimastigotes clathrin-mediated endocytosis of albumin occurs at the flagellar pocket, while clathrin-independent endocytosis of transferrin occurs at the cytostome/cytopharynx complex.

In vitro biological evaluation of eight different essential oils against Trypanosoma cruzi, with emphasis on Cinnamomum verum essential oil.

BACKGROUND: Essential oils (EOs) are complex mixtures of secondary metabolites from various plants. It has been shown that several EOs, or their constituents, have inhibitory activity against trypanosomatid protozoa. Thus, we analyzed the biological activity of different EOs on Trypanosoma cruzi, as well as their cytotoxicity on Vero cells. METHODS: The following EOs were evaluated on T. cruzi epimastigote forms: Cinnamomum verum, Citrus limon, Cymbopogon nardus, Corymbia citriodora, Eucalyptus globulus, Eugenia uniflora, Myrocarpus frondosus, and Rosmarinus officinalis. Inhibitory activity against T. cruzi (IC50/24 h) and cytotoxicity against Vero cells (CC50/24 h) were evaluated by the MTT assay. The EO of C. verum was selected for further evaluation against trypomastigotes and intracellular amastigotes, as well as on parasite metacyclogenesis. Constituents of C. verum EO were identified by GC-MS. One-way ANOVA statistical analysis was performed with GraphPad version 5.01. RESULTS: Cinnamomum verum EO was the most effective against T. cruzi epimastigotes (IC50/24 h = 24.13 µg/ml), followed by Myrocarpus frondosus (IC50/24 h = 60.87 µg/ml) and Eugenia uniflora (IC50/24 h = 70 µg/ml). The EOs of C. citriodora, E. globulus, and R. officinalis showed no activity at concentrations up to 300 µg/ml. Incubation of T. cruzi metacyclic trypomastigotes and intracellular amastigotes with C. verum EO resulted in IC50/24 h values of 5.05 µg/ml and 20 µg/ml, respectively. Therefore, trypomastigotes are more susceptible than epimastigotes, with selectivity index (SI) about 4.7-fold higher (9.78 and 2.05, respectively). Analysis of C. verum EO by GC-MS showed mainly (E)-cinnamaldehyde (81.52%) and eugenol (16.68%). CONCLUSIONS: C. verum essential oil is effective against T. cruzi (epimastigotes, trypomastigotes and amastigotes) and interferes with the parasite differentiation process in vitro. Thus, it represents a strong candidate for further studies to improve its activity on pathogenic trypanosomatids.

Identification and subcellular localization of TcHIP, a putative Golgi zDHHC palmitoyl transferase of Trypanosoma cruzi.

Protein palmitoylation is a post-translational modification that contributes to determining protein localization and function. Palmitoylation has been described in trypanosomatid protozoa, but no zDHHC palmitoyl transferase has been identified in Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. In this study we identify and show the subcellular localization of TcHIP (Tc00.1047053508199.50), a putative T. cruzi zDHHC palmitoyl transferase. Analysis of the deduced protein sequence indicates that it contains ankyrin repeats (Ank and Ank2) and the zDHHC conserved domain, typical of zDHHC palmitoyl transferases. A TcHIP polyclonal antiserum obtained from mice immunized with the purified recombinant protein was used to study the presence and subcellular localization of the native enzyme. In western blots this antiserum recognized a protein of about 95 kDa, consistent with the predicted molecular mass of TcHIP (95.4 kDa), in whole extracts of T. cruzi epimastigotes, metacyclic trypomastigotes and intracellular amastigotes. Immunolocalization by confocal microscopy showed TcHIP labeling at the Golgi complex, co-localizing with the T. cruzi Golgi marker TcRab7-GFP. Transfectant T. cruzi epimastigotes containing a construct encoding TcHIP fused to proteins A and C (TcHIP/AC) were obtained. In western blotting experiments, the TcHIP polyclonal antiserum recognized both native and TcHIP/AC proteins in extracts of the transfectants. Confocal microscopy showed co-localization of native TcHIP with TcHIP/AC. These findings demonstrate the presence of a putative zDHHC palmitoyl transferase (TcHIP) containing ankyrin and zDHHC domains in different developmental forms of T. cruzi, and its association with the Golgi complex.

Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea).

Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids.