Proliferation and Differentiation of Trypanosoma cruzi inside Its Vector Have a New Trigger: Redox Status

Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS). We tested the influence of the pro-oxidant molecules H₂O₂ and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. β-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants seem to switch the cycle towards metacyclogenesis. This is a new insight that defines a key role for redox metabolism in governing the parasitic life cycle.     

Cruzipain Promotes Trypanosoma cruzi Adhesion to Rhodnius prolixus Midgut

Background

Trypanosoma cruzi is the etiological agent of Chagas'' disease. Cysteine peptidases are relevant to several aspects of the T. cruzi life cycle and are implicated in parasite-mammalian host relationships. However, little is known about the factors that contribute to the parasite-insect host interaction.

Methodology/Principal Findings

Here, we have investigated whether cruzipain could be involved in the interaction of T. cruzi with the invertebrate host. We analyzed the effect of treatment of T. cruzi epimastigotes with anti-cruzipain antibodies or with a panel of cysteine peptidase inhibitors (cystatin, antipain, E-64, leupeptin, iodocetamide or CA-074-OMe) on parasite adhesion to Rhodnius prolixus posterior midgut ex vivo. All treatments, with the exception of CA074-OMe, significantly decreased parasite adhesion to R. prolixus midgut. Cystatin presented a dose-dependent reduction on the adhesion. Comparison of the adhesion rate among several T. cruzi isolates revealed that the G isolate, which naturally possesses low levels of active cruzipain, adhered to a lesser extent in comparison to Dm28c, Y and CL Brener isolates. Transgenic epimastigotes overexpressing an endogenous cruzipain inhibitor (pCHAG), chagasin, and that have reduced levels of active cruzipain adhered to the insect gut 73% less than the wild-type parasites. The adhesion of pCHAG parasites was partially restored by the addition of exogenous cruzipain. In vivo colonization experiments revealed low levels of pCHAG parasites in comparison to wild-type. Parasites isolated after passage in the insect presented a drastic enhancement in the expression of surface cruzipain.

Conclusions/Significance

These data highlight, for the first time, that cruzipain contributes to the interaction of T. cruzi with the insect host.     

Trypanosoma cruzi TcSMUG L-surface Mucins Promote Development and Infectivity in the Triatomine Vector Rhodnius prolixus

Background

TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector.

Methodology and Principal Findings

Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy.

Conclusion and Significance

Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.     

Involvement of STI1 protein in the differentiation process of Trypanosoma cruzi

The protozoan Trypanosoma cruzi is a parasite exposed to several environmental stressors inside its invertebrate and vertebrate hosts. Although stress conditions are involved in its differentiation processes, little information is available about the stress response proteins engaged in these activities. This work reports the first known association of the stress-inducible protein 1 (STI1) with the cellular differentiation process in a unicellular eukaryote. Albeit STI1 expression is constitutive in epimastigotes and metacyclic trypomastigotes, higher protein levels were observed in late growth phase epimastigotes subjected to nutritional stress. Analysis by indirect immunofluorescence revealed that T. cruzi STI1 (TcSTI1) is located throughout the cell cytoplasm, with some cytoplasmic granules appearing in greater numbers in late growing epimastigotes and late growing epimastigotes subjected to nutritional stress. We observed that part of the fluorescence signal from both TcSTI1 and TcHSP70 colocalized around the nucleus. Gene silencing of sti1 in Trypanosoma brucei did not affect cell growth. Similarly, the growth of T. cruzi mutant parasites with a single allele sti1 gene knockout was not affected. However, the differentiation of epimastigotes in metacyclic trypomastigotes (metacyclogenesis) was compromised. Lower production rates and numbers of metacyclic trypomastigotes were obtained from the mutant parasites compared with the wild-type parasites. These data indicate that reduced levels of TcSTI1 decrease the rate of in vitro metacyclogenesis, suggesting that this protein may participate in the differentiation process of T. cruzi.     

The Calpain Inhibitor MDL28170 Induces the Expression of Apoptotic Markers in Leishmania amazonensis Promastigotes

Background

Human cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals.

Methodology/Principal Findings

In this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC₅₀ (15 µM) and two times the IC₅₀ doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G₀/G₁ phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC₅₀ dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis.

Conclusions/Significance

The data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the potential clinical utility of calpain inhibitors in the chemotherapy of leishmaniases.     

Decoding the Anti-Trypanosoma cruzi Action of HIV Peptidase Inhibitors Using Epimastigotes as a Model

Background

Aspartic peptidase inhibitors have shown antimicrobial action against distinct microorganisms. Due to an increase in the occurrence of Chagas'' disease/AIDS co-infection, we decided to explore the effects of HIV aspartic peptidase inhibitors (HIV-PIs) on Trypanosoma cruzi, the etiologic agent of Chagas'' disease.

Methodology and Principal Findings

HIV-PIs presented an anti-proliferative action on epimastigotes of T. cruzi clone Dm28c, with IC₅₀ values ranging from 0.6 to 14 µM. The most effective inhibitors, ritonavir, lopinavir and nelfinavir, also had an anti-proliferative effect against different phylogenetic T. cruzi strains. The HIV-PIs induced some morphological alterations in clone Dm28c epimastigotes, as reduced cell size and swollen of the cellular body. Transmission electron microscopy revealed that the flagellar membrane, mitochondrion and reservosomes are the main targets of HIV-PIs in T. cruzi epimastigotes. Curiously, an increase in the epimastigote-into-trypomastigote differentiation process of clone Dm28c was observed, with many of these parasites presenting morphological alterations including the detachment of flagellum from the cell body. The pre-treatment with the most effective HIV-PIs drastically reduced the interaction process between epimastigotes and the invertebrate vector Rhodnius prolixus. It was also noted that HIV-PIs induced an increase in the expression of gp63-like and calpain-related molecules, and decreased the cruzipain expression in epimastigotes as judged by flow cytometry and immunoblotting assays. The hydrolysis of a cathepsin D fluorogenic substrate was inhibited by all HIV-PIs in a dose-dependent manner, showing that the aspartic peptidase could be a possible target to these drugs. Additionally, we verified that ritonavir, lopinavir and nelfinavir reduced drastically the viability of clone Dm28c trypomastigotes, causing many morphological damages.

Conclusions and Significance

The results contribute to understand the possible role of aspartic peptidases in T. cruzi physiology, adding new in vitro insights into the possibility of exploiting the use of HIV-PIs in the clinically relevant forms of the parasite.     

Dynasore,a Dynamin Inhibitor,Inhibits Trypanosoma cruzi Entry into Peritoneal Macrophages

Background

Trypanosoma cruzi is an intracellular parasite that, like some other intracellular pathogens, targets specific proteins of the host cell vesicular transport machinery, leading to a modulation of host cell processes that results in the generation of unique phagosomes. In mammalian cells, several molecules have been identified that selectively regulate the formation of endocytic transport vesicles and the fusion of such vesicles with appropriate acceptor membranes. Among these, the GTPase dynamin plays an important role in clathrin-mediated endocytosis, and it was recently found that dynamin can participate in a phagocytic process.

Methodology/Principal Findings

We used a compound called dynasore that has the ability to block the GTPase activity of dynamin. Dynasore acts as a potent inhibitor of endocytic pathways by blocking coated vesicle formation within seconds of its addition. Here, we investigated whether dynamin is involved in the entry process of T. cruzi in phagocytic and non-phagocytic cells by using dynasore. In this aim, peritoneal macrophages and LLC-MK2 cells were treated with increasing concentrations of dynasore before interaction with trypomastigotes, amastigotes or epimastigotes. We observed that, in both cell lines, the parasite internalization was drastically diminished (by greater than 90% in LLC-MK2 cells and 70% in peritoneal macrophages) when we used 100 µM dynasore. The T. cruzi adhesion index, however, was unaffected in either cell line. Analyzing these interactions by scanning electron microscopy and comparing peritoneal macrophages to LLC-MK2 cells revealed differences in the stage at which cell entry was blocked. In LLC-MK2 cells, this blockade is observed earlier than it is in peritoneal macrophages. In LLC-MK2 cells, the parasites were only associated with cellular microvilli, whereas in peritoneal macrophages, trypomastigotes were not completely engulfed by a host cell plasma membrane.

Conclusions/Significance

Taken together our results demonstrate that dynamin is an essential molecule necessary for cell invasion and specifically parasitophorous vacuole formation by host cells during interaction with Trypanosoma cruzi.     

Treatment of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> 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.

     

Cultivation of Trypanosoma cruzi epimastigotes in low glucose axenic media shifts its competence to differentiate at metacyclic trypomastigotes

This study offers an insight into why Trypanosoma cruzi epimastigotes lose their capacity to differentiate into metacyclic forms, if maintained in culture media long-term through serial passages. The biological and metabolic behaviour of two T. cruzi strains isolated from various origins (human, opossum), and maintained under two schedules (alternate triatomine/mouse passages and serial culture media) were compared. To determine the effect of the environment on the parasites, the epimastigotes were grown under extreme conditions (high and low glucose concentrations), and the glucose consumption, ammonia production and changes in pH, either in one compartment (along the growth curve) or two compartments (induced metacyclogenesis) were compared. The glucose effect on the stages involved in metacyclogenesis at antigenic level was also evaluated. The results indicate that T. cruzi adapts to various environmental conditions and also that the ability of epimastigotes to undergo metacyclogenesis are influenced by the maintenance schedule. Antigenic profile analysis supports the idea that epimastigotes adapted to culture media do not complete their molecular differentiation into the trypomastigote metacyclic stage. These transition forms conserve some degree of gene expression of the epimastigote stage.     

Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

Background

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models.

Methodology/Principal Findings

We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites.

Conclusions/Significance

Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.     

A Quinoxaline Derivative as a Potent Chemotherapeutic Agent,Alone or in Combination with Benznidazole,against Trypanosoma cruzi

Background

Chagas’ disease is a condition caused by the protozoan Trypanosoma cruzi that affects millions of people, mainly in Latin America where it is considered endemic. The chemotherapy for Chagas disease remains a problem; the standard treatment currently relies on a single drug, benznidazole, which unfortunately induces several side effects and it is not successful in the cure of most of the chronic patients. In order to improve the drug armamentarium against Chagas’ disease, in the present study we describe the synthesis of the compound 3-chloro-7-methoxy-2-(methylsulfonyl) quinoxaline (quinoxaline 4) and its activity, alone or in combination with benznidazole, against Trypanosoma cruzi in vitro.

Methodology/Principal Findings

Quinoxaline 4 was found to be strongly active against Trypanosoma cruzi Y strain and more effective against the proliferative forms. The cytotoxicity against LLCMK₂ cells provided selective indices above one for all of the parasite forms. The drug induced very low hemolysis, but its anti-protozoan activity was partially inhibited when mouse blood was added in the experiment against trypomastigotes, an effect that was specifically related to blood cells. A synergistic effect between quinoxaline 4 and benznidazole was observed against epimastigotes and trypomastigotes, accompanied by an antagonistic interaction against LLCMK₂ cells. Quinoxaline 4 induced several ultrastructural alterations, including formations of vesicular bodies, profiles of reticulum endoplasmic surrounding organelles and disorganization of Golgi complex. These alterations were also companied by cell volume reduction and maintenance of cell membrane integrity of treated-parasites.

Conclusion/Significance

Our results demonstrated that quinoxaline 4, alone or in combination with benznidazole, has promising effects against all the main forms of T. cruzi. The compound at low concentrations induced several ultrastructural alterations and led the parasite to an autophagic-like cell death. Taken together these results may support the further development of a combination therapy as an alternative more effective in Chagas’ disease treatment.     

Fatty acid oxidation participates in resistance to nutrient-depleted environments in the insect stages of Trypanosoma cruzi

Trypanosoma cruzi, the parasite causing Chagas disease, is a digenetic flagellated protist that infects mammals (including humans) and reduviid insect vectors. Therefore, T. cruzi must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard, it is well known that T. cruzi has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established has been the capability of T. cruzi to use glucose and amino acids to support the differentiation process occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities of using externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-¹⁴C]-palmitate can be taken up from the medium, leading to CO₂ production. Additionally, we show that electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid (TCA) cycle, which can be used to feed anabolic pathways such as the de novo biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization into the mitochondrion diminishes the survival to severe starvation, and impairs metacyclogenesis.     

Differentiation of Trypanosoma cruzi epimastigotes: metacyclogenesis and adhesion to substrate are triggered by nutritional stress

Differentiation of Trypanosoma cruzi epimastigotes to metacyclic trypomastigotes occurs in the insect rectum, after adhesion of the epimastigotes to the intestinal wall. We investigated the effect of the nutritional stress on the metacyclogenesis process in vitro by incubating epimastigotes in the chemically defined TAU3AAG medium supplemented with different nutrients. Addition of fetal bovine serum induced epimastigote growth but inhibited metacyclogenesis. In this medium, few parasites attached to the substrate. Ultrastructural analysis demonstrated reservosomes at the posterior end of the epimastigotes. Incubation of the cells in TAU3AAG medium containing gold-labeled transferrin resulted in high endocytosis of the marker by both adhered and free-swimming epimastigotes. No intracellular gold particles could be detected in trypomastigotes. Addition of transferrin gold complexes to adhered epimastigotes cultivated for 4 days in TAU3AAG medium resulted in decrease of both metacyclogenesis and adhesion to the substrate, as compared with parasites maintained in transferrin-free medium. Adhesion to the substrate is triggered by nutritional stress, and proteins accumulated in reservosomes are used as energy source during the differentiation. A close relationship exists among nutritional stress, endocytosis of nutrients, adhesion to the substrate, and cell differentiation in T. cruzi epimastigotes.     

Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

Background

Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease.

Methodology/Principal Findings

In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene.

Conclusions/Significance

Analyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite.     

Inhibition of poly(ADP-ribose) Polymerase Interferes with Trypanosoma cruzi Infection and Proliferation of the Parasite

Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.     

The MASP Family of Trypanosoma cruzi: Changes in Gene Expression and Antigenic Profile during the Acute Phase of Experimental Infection

Background

Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host–parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms.

Methods

By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice.

Findings and Conclusions

We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.