A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor

Background

Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro.

Methodology/Principal Findings

Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace’s Insect Medium mediated their transformation into trypomastigotes. Grace’s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP₃R)—an intracellular Ca²⁺ channel and a key molecule involved in Ca²⁺ signaling in the parasite—is important for the transformation process.

Conclusion/Significance

Our findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.     

Galectin-1 Prevents Infection and Damage Induced by Trypanosoma cruzi on Cardiac Cells

Background

Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection.

Methodology and Principal Findings

Here we investigated the contribution of galectin–1 (Gal–1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL–1 cardiac cells to Gal–1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal–1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL–1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal–1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal–1 to the cell surface. Consistent with these data, Gal–1 deficient (Lgals1 ^-/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain.

Conclusion/Significance

Our results indicate that Gal–1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions.     

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

Background

The unicellular parasite Trypanosoma cruzi is the causative agent of Chagaś disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored.

Methodology/Principal Findings

Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin.

Conclusions/Significance

Herein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.     

Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion

Background

The surface coat of Trypanosoma cruzi is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions.

Methodology/ Principal Findings

Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), which are conserved among different T. cruzi lineages and have orthologs in other Trypanosoma species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all T. cruzi developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the T. cruzi secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca²⁺ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion.

Conclusion/Significance

We hypothesized that the productive interaction of T. cruzi with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by TcSMP may be additive to that triggered by the major surface molecule gp82, further increasing the host cell responses required for infection.     

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.     

Development of a Novel Multiplex Immunoassay Multi-cruzi for the Serological Confirmation of Chagas Disease

Background

Chagas disease is due to the parasite Trypanosoma cruzi, a protist disseminated by a Triatome vector. This disease is endemic to Latin America and considered by WHO as one of the 17 world’s neglected diseases. In Europe and in North America, imported cases are also detected, due to migration of population outside of the endemic region. Diagnosis of T. cruzi infection is usually made indirectly by the detection of specific antibodies to T. cruzi antigens. Following initial diagnostic evaluation or screening test (qualifying or discarding blood donation), a confirmation test is performed for samples initially reactive. The test presented in this study aims at the confirmation/refutation of the infectious status of human blood samples and will permit taking appropriate clinical measures.

Methodology/Principal Findings

We designed a novel array of twelve antigens and printed these antigens onto 96-well plates. We tested 248 positive samples T. cruzi, 94 unscreened blood donors’ samples from non-endemic area, 49 seronegative blood donors, 7 false-positive and 3 doubtful samples. The observed reactivities were analyzed to propose a decision-tree algorithm that correctly classifies all the samples, with the potential to discriminate false-positive results and sticky samples. We observed that antibodies levels (Sum of all antigens) was significantly higher for PCR positive than for PCR negative samples in all studied groups with Multi-cruzi.

Conclusion/Significance

The results described in this study indicate that the Multi-cruzi improves the serological confirmation of Chagas disease. Moreover the “sum of all antigens” detected by Multi-cruzi could reflect parasitemia level in patients–like PCR signals does—and could serve as an indicator of parasite clearance in longitudinal follow-ups. Validation of this assay is still required on an independent large collection of well characterized samples including typical false-reactive samples such as Leishmaniasis.     

Trypanosoma cruzi Needs a Signal Provided by Reactive Oxygen Species to Infect Macrophages

Background

During Trypanosoma cruzi infection, macrophages produce reactive oxygen species (ROS) in a process called respiratory burst. Several works have aimed to elucidate the role of ROS during T. cruzi infection and the results obtained are sometimes contradictory. T. cruzi has a highly efficiently regulated antioxidant machinery to deal with the oxidative burst, but the parasite macromolecules, particularly DNA, may still suffer oxidative damage. Guanine (G) is the most vulnerable base and its oxidation results in formation of 8-oxoG, a cellular marker of oxidative stress.

Methodology/Principal Findings

In order to investigate the contribution of ROS in T. cruzi survival and infection, we utilized mice deficient in the gp91^phox (Phox KO) subunit of NADPH oxidase and parasites that overexpress the enzyme EcMutT (from Escherichia coli) or TcMTH (from T. cruzi), which is responsible for removing 8-oxo-dGTP from the nucleotide pool. The modified parasites presented enhanced replication inside murine inflammatory macrophages from C57BL/6 WT mice when compared with control parasites. Interestingly, when Phox KO macrophages were infected with these parasites, we observed a decreased number of all parasites when compared with macrophages from C57BL/6 WT. Scavengers for ROS also decreased parasite growth in WT macrophages. In addition, treatment of macrophages or parasites with hydrogen peroxide increased parasite replication in Phox KO mice and in vivo.

Conclusions

Our results indicate a paradoxical role for ROS since modified parasites multiply better inside macrophages, but proliferation is significantly reduced when ROS is removed from the host cell. Our findings suggest that ROS can work like a signaling molecule, contributing to T. cruzi growth inside the cells.     

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.     

Use of a Chagas Urine Nanoparticle Test (Chunap) to Correlate with Parasitemia Levels in T. cruzi/HIV Co-infected Patients

Background

Early diagnosis of reactivated Chagas disease in HIV patients could be lifesaving. In Latin America, the diagnosis is made by microscopical detection of the T. cruzi parasite in the blood; a diagnostic test that lacks sensitivity. This study evaluates if levels of T. cruzi antigens in urine, determined by Chunap (Chagas urine nanoparticle test), are correlated with parasitemia levels in T. cruzi/HIV co-infected patients.

Methodology/Principal Findings

T. cruzi antigens in urine of HIV patients (N = 55: 31 T. cruzi infected and 24 T. cruzi serology negative) were concentrated using hydrogel particles and quantified by Western Blot and a calibration curve. Reactivation of Chagas disease was defined by the observation of parasites in blood by microscopy. Parasitemia levels in patients with serology positive for Chagas disease were classified as follows: High parasitemia or reactivation of Chagas disease (detectable parasitemia by microscopy), moderate parasitemia (undetectable by microscopy but detectable by qPCR), and negative parasitemia (undetectable by microscopy and qPCR). The percentage of positive results detected by Chunap was: 100% (7/7) in cases of reactivation, 91.7% (11/12) in cases of moderate parasitemia, and 41.7% (5/12) in cases of negative parasitemia. Chunap specificity was found to be 91.7%. Linear regression analysis demonstrated a direct relationship between parasitemia levels and urine T. cruzi antigen concentrations (p<0.001). A cut-off of > 105 pg was chosen to determine patients with reactivation of Chagas disease (7/7). Antigenuria levels were 36.08 times (95% CI: 7.28 to 64.88) higher in patients with CD4+ lymphocyte counts below 200/mL (p = 0.016). No significant differences were found in HIV loads and CD8+ lymphocyte counts.

Conclusion

Chunap shows potential for early detection of Chagas reactivation. With appropriate adaptation, this diagnostic test can be used to monitor Chagas disease status in T. cruzi/HIV co-infected patients.     

Influence of Parasite Load on Renal Function in Mice Acutely Infected with Trypanosoma cruzi

Background

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi. Despite the vast number of studies evaluating the pathophysiological mechanisms of the disease, the influence of parasite burden on kidney lesions remains unclear. Thus, the main goal of this work was to evaluate the effect of T. cruzi infection on renal function and determine whether there was a correlation between parasite load and renal injury using an acute experimental model of the disease.

Methodology/Principal Findings

Low, medium and high parasite loads were generated by infecting C57BL/6 mice with 300 (low), 3,000 (medium) or 30,000 (high) numbers of “Y” strain trypomastigotes. We found that mice infected with T. cruzi trypomastigotes show increased renal injury. The infection resulted in reduced urinary excretion and creatinine clearance. We also observed a marked elevation in the ratio of urine volume to kidney and body weight, blood urea nitrogen, chloride ion, nitric oxide, pro- and anti-inflammatory cytokines and the number of leukocytes in the blood and/or renal tissues of infected mice. Additionally, we observed the presence of the parasite in the cortical/medullary and peri-renal region, an increase of inflammatory infiltrate and of vascular permeability of the kidney. Overall, most renal changes occurred mainly in animals infected with high parasitic loads.

Conclusions/Significance

These data demonstrate that T. cruzi impairs kidney function, and this impairment is more evident in mice infected with high parasitic loads. Moreover, these data suggest that, in addition to the extensively studied cardiovascular effects, renal injury should be regarded as an important indicator for better understanding the pan-infectivity of the parasite and consequently for understanding the disease in experimental models.     

Trypanosoma cruzi-Infected Pregnant Women without Vector Exposure Have Higher Parasitemia Levels: Implications for Congenital Transmission Risk

Background

Congenital transmission is a major source of new Trypanosoma cruzi infections, and as vector and blood bank control continue to improve, the proportion due to congenital infection will grow. A major unanswered question is why reported transmission rates from T. cruzi-infected mothers vary so widely among study populations. Women with high parasite loads during pregnancy are more likely to transmit to their infants, but the factors that govern maternal parasite load are largely unknown. Better understanding of these factors could enable prioritization of screening programs to target women most at risk of transmission to their infants.

Methodology/Principal Findings

We screened pregnant women presenting for delivery in a large urban hospital in Bolivia and followed infants of infected women for congenital Chagas disease. Of 596 women screened, 128 (21.5%) had confirmed T. cruzi infection; transmission occurred from 15 (11.7%) infected women to their infants. Parasite loads were significantly higher among women who transmitted compared to those who did not. Congenital transmission occurred from 31.3% (9/29), 15.4% (4/26) and 0% (0/62) of women with high, moderate and low parasite load, respectively (χx2 for trend 18.2; p<0.0001). Twin births were associated with higher transmission risk and higher maternal parasite loads. Infected women without reported vector exposure had significantly higher parasite loads than those who had lived in an infested house (median 26.4 vs 0 parasites/mL; p<0.001) with an inverse relationship between years of living in an infested house and parasite load.

Conclusions/Significance

We hypothesize that sustained vector-borne parasite exposure and repeated superinfection by T. cruzi may act as an immune booster, allowing women to maintain effective control of the parasite despite the down-regulation of late pregnancy.     

Overexpression of Cytoplasmic TcSIR2RP1 and Mitochondrial TcSIR2RP3 Impacts on Trypanosoma cruzi Growth and Cell Invasion

Background

Trypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives. Inhibition of sirtuins from Trypanosoma brucei and Leishmania ssp. showed promising results, indicating that these enzymes may be considered as targets for drug discovery in parasite infection. Here, we report the first characterization of the two sirtuins present in T. cruzi.

Methodology

Dm28c epimastigotes that inducibly overexpress TcSIR2RP1 and TcSIR2RP3 were constructed and used to determine their localizations and functions. These transfected lines were tested regarding their acetylation levels, proliferation and metacyclogenesis rate, viability when treated with sirtuin inhibitors and in vitro infectivity.

Conclusion

TcSIR2RP1 and TcSIR2RP3 are cytosolic and mitochondrial proteins respectively. Our data suggest that sirtuin activity is important for the proliferation of T. cruzi replicative forms, for the host cell-parasite interplay, and for differentiation among life-cycle stages; but each one performs different roles in most of these processes. Our results increase the knowledge on the localization and function of these enzymes, and the overexpressing T. cruzi strains we obtained can be useful tools for experimental screening of trypanosomatid sirtuin inhibitors.     

Adipose Tissue-Derived Mesenchymal Stromal Cells Protect Mice Infected with Trypanosoma cruzi from Cardiac Damage through Modulation of Anti-parasite Immunity

Background

Chagas disease, caused by the protozoan Trypanosoma cruzi (T.cruzi), is a complex disease endemic in Central and South America. It has been gathering interest due to increases in non-vectorial forms of transmission, especially in developed countries. The objective of this work was to investigate if adipose tissue-derived mesenchymal stromal cells (ASC) can alter the course of the disease and attenuate pathology in a mouse model of chagasic cardiomyopathy.

Methodology/Principal Findings

ASC were injected intraperitoneally at 3 days post-infection (dpi). Tracking by bioluminescence showed that cells remained in the abdominal cavity for up to 9 days after injection and most of them migrated to the abdominal or subcutaneous fat, an early parasite reservoir. ASC injection resulted in a significant reduction in blood parasitemia, which was followed by a decrease in cardiac tissue inflammation, parasitism and fibrosis at 30 dpi. At the same time point, analyses of cytokine release in cells isolated from the heart and exposed to T. cruzi antigens indicated an anti-inflammatory response in ASC-treated animals. In parallel, splenocytes exposed to the same antigens produced a pro-inflammatory response, which is important for the control of parasite replication, in placebo and ASC-treated groups. However, splenocytes from the ASC group released higher levels of IL-10. At 60 dpi, magnetic resonance imaging revealed that right ventricular (RV) dilation was prevented in ASC-treated mice.

Conclusions/Significance

In conclusion, the injection of ASC early after T. cruzi infection prevents RV remodeling through the modulation of immune responses. Lymphoid organ response to the parasite promoted the control of parasite burden, while the heart, a target organ of Chagas disease, was protected from damage due to an improved control of inflammation in ASC-treated mice.     

Risk Factors and Screening for Trypanosoma cruzi Infection of Dutch Blood Donors

Background

Blood donors unaware of Trypanosoma cruzi infection may donate infectious blood. Risk factors and the presence of T. cruzi antibodies in at-risk Dutch blood donors were studied to assess whether specific blood safety measures are warranted in the Netherlands.

Methodology

Birth in a country endemic for Chagas disease (CEC), having a mother born in a CEC, or having resided for at least six continuous months in a CEC were considered risk factors for T. cruzi infection. From March through September 2013, risk factor questions were asked to all donors who volunteered to donate blood or blood components. Serum samples were collected from donors reporting one or more risk factors, and screened for IgG antibodies to T. cruzi by EIA.

Results

Risk factors for T. cruzi infection were reported by 1,426 of 227,278 donors (0.6%). Testing 1,333 at-risk donors, none (0.0%; 95%, CI 0.0–0.4%) was seroreactive for IgG antibodies to T. cruzi. A total of 472 donors were born in a CEC; 553 donors reported their mother being born in a CEC; and 1,121 donors reported a long-term stay in a CEC. The vast majority of reported risk factors were related to Suriname and Brazil. Overall, the participants resided for 7,694 years in CECs, which equals 2.8 million overnight stays. Of those, 1.9 million nights were spent in Suriname.

Conclusions/Significance

Asymptomatic T. cruzi infection appears to be extremely rare among Dutch blood donors. Blood safety interventions to mitigate the risk of T. cruzi transmission by transfusion would be highly cost-ineffective in the Netherlands, and are thus not required.     

Glycoinositolphospholipids from Trypanosomatids Subvert Nitric Oxide Production in Rhodnius prolixus Salivary Glands

Background

Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands.

Methodology/Principal Findings

Here we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities.

Conclusions/Significance

Taken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.     

Deep Sequencing of the Trypanosoma cruzi GP63 Surface Proteases Reveals Diversity and Diversifying Selection among Chronic and Congenital Chagas Disease Patients

Background

Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology.

Methodology/ Principal Findings

A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target—ND5—was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus.

Conclusions/Significance

Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I gene family suggests a link between genetic diversity within this gene family and survival in the mammalian host.     

Trypanosomiasis-Induced Megacolon Illustrates How Myenteric Neurons Modulate the Risk for Colon Cancer in Rats and Humans

Background

Trypanosomiasis induces a remarkable myenteric neuronal degeneration leading to megacolon. Very little is known about the risk for colon cancer in chagasic megacolon patients. To clarify whether chagasic megacolon impacts on colon carcinogenesis, we investigated the risk for colon cancer in Trypanosoma cruzi (T. cruzi) infected patients and rats.

Methods

Colon samples from T. cruzi-infected and uninfected patients and rats were histopathologically investigated with colon cancer biomarkers. An experimental model for chemical myenteric denervation was also performed to verify the myenteric neuronal effects on colon carcinogenesis. All experiments complied the guidelines and approval of ethical institutional review boards.

Results

No colon tumors were found in chagasic megacolon samples. A significant myenteric neuronal denervation was observed. Epithelial cell proliferation and hyperplasia were found increased in chagasic megacolon. Analyzing the argyrophilic nucleolar organiser regions within the cryptal bottom revealed reduced risk for colon cancer in Chagas’ megacolon patients. T. cruzi-infected rats showed a significant myenteric neuronal denervation and decreased numbers of colon preneoplastic lesions. In chemical myenteric denervated rats preneoplastic lesions were reduced from the 2^nd wk onward, which ensued having the colon myenteric denervation significantly induced.

Conclusion/Significance

Our data suggest that the trypanosomiasis-related myenteric neuronal degeneration protects the colon tissue from carcinogenic events. Current findings highlight potential mechanisms in tropical diseases and cancer research.     

Location and expression kinetics of Tc24 in different life stages of Trypanosoma cruzi

Tc24-C4, a modified recombinant flagellar calcium-binding protein of Trypanosoma cruzi, is under development as a therapeutic subunit vaccine candidate to prevent or delay progression of chronic Chagasic cardiomyopathy. When combined with Toll-like receptor agonists, Tc24-C4 immunization reduces parasitemia, parasites in cardiac tissue, and cardiac fibrosis and inflammation in animal models. To support further research on the vaccine candidate and its mechanism of action, murine monoclonal antibodies (mAbs) against Tc24-C4 were generated. Here, we report new findings made with mAb Tc24-C4/884 that detects Tc24-WT and Tc24-C4, as well as native Tc24 in T. cruzi on ELISA, western blots, and different imaging techniques. Surprisingly, detection of Tc24 by Tc24-C/884 in fixed T. cruzi trypomastigotes required permeabilization of the parasite, revealing that Tc24 is not exposed on the surface of T. cruzi, making a direct role of antibodies in the induced protection after Tc24-C4 immunization less likely. We further observed that after immunostaining T. cruzi–infected cells with mAb Tc24-C4/884, the expression of Tc24 decreases significantly when T. cruzi trypomastigotes enter host cells and transform into amastigotes. However, Tc24 is then upregulated in association with parasite flagellar growth linked to re-transformation into the trypomastigote form, prior to host cellular escape. These observations are discussed in the context of potential mechanisms of vaccine immunity.