Structural Characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei Bound to the Antifungal Drugs Posaconazole and Fluconazole

Background

Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14α-demethylase (CYP51), which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding.

Methodology/Principal Findings

We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 Å and 2.27 Å), and from the related pathogen T. brucei (resolutions of 2.7 Å and 2.6 Å), co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180° depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target.

Conclusions/Significance

The structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and offer a starting point for rationally designed anti-Chagasic drugs with improved efficacy and reduced toxicity.     

Trypanosoma cruzi CYP51 Inhibitor Derived from a Mycobacterium tuberculosis Screen Hit

Background

The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas'' disease chemotherapy is sterol 14α-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols.

Methodology/Principal Finding

In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51_Mt), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51_Mt. Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51_Tc, demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site.

Conclusions/Significance

CYP51_Mt-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51_Tc.     

Testing the efficacy of a multi-component DNA-prime/DNA-boost vaccine against Trypanosoma cruzi infection in dogs

Background

Trypanosoma cruzi, the etiologic agent of Chagas Disease, is a major vector borne health problem in Latin America and an emerging infectious disease in the United States.

Methods

We tested the efficacy of a multi-component DNA-prime/DNA-boost vaccine (TcVac1) against experimental T. cruzi infection in a canine model. Dogs were immunized with antigen-encoding plasmids and cytokine adjuvants, and two weeks after the last immunization, challenged with T. cruzi trypomastigotes. We measured antibody responses by ELISA and haemagglutination assay, parasitemia and infectivity to triatomines by xenodiagnosis, and performed electrocardiography and histology to assess myocardial damage and tissue pathology.

Results

Vaccination with TcVac1 elicited parasite-and antigen-specific IgM and IgG (IgG2>IgG1) responses. Upon challenge infection, TcVac1-vaccinated dogs, as compared to non-vaccinated controls dogs, responded to T. cruzi with a rapid expansion of antibody response, moderately enhanced CD8⁺ T cell proliferation and IFN-γ production, and suppression of phagocytes’ activity evidenced by decreased myeloperoxidase and nitrite levels. Subsequently, vaccinated dogs controlled the acute parasitemia by day 37 pi (44 dpi in non-vaccinated dogs), and exhibited a moderate decline in infectivity to triatomines. TcVac1-immunized dogs did not control the myocardial parasite burden and electrocardiographic and histopatholgic cardiac alterations that are the hallmarks of acute Chagas disease. During the chronic stage, TcVac1-vaccinated dogs exhibited a moderate decline in cardiac alterations determined by EKG and anatomo-/histo-pathological analysis while chronically-infected/non-vaccinated dogs continued to exhibit severe EKG alterations.

Conclusions

Overall, these results demonstrated that TcVac1 provided a partial resistance to T. cruzi infection and Chagas disease, and provide an impetus to improve the vaccination strategy against Chagas disease.     

Real-time PCR in HIV/Trypanosoma cruzi coinfection with and without Chagas disease reactivation: association with HIV viral load and CD4 level

Background

Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed.

Methodology

Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not.

Principal Findings

qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman''s correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4⁺ cells or the CD4⁺/CD8⁺ ratio.

Conclusions

qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4⁺ cells/mm³ and the CD4⁺/CD8⁺ ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.     

HLA Class I-T cell epitopes from trans-sialidase proteins reveal functionally distinct subsets of CD8+ T cells in chronic Chagas disease

Background

Previously, we identified a set of HLA-A020.1-restricted trans-sialidase peptides as targets of CD8⁺ T cell responses in HLA-A0201⁺ individuals chronically infected by T. cruzi.

Methods and Findings

Herein, we report the identification of peptides encoded by the same trans-sialidase gene family that bind alleles representative of the 6 most common class I HLA-supertypes. Based on a combination of bioinformatic predictions and HLA-supertype considerations, a total of 1001 epitopes predicted to bind to HLA A01, A02, A03, A24, B7 and B44 supertypes was selected. Ninety-six supertype-binder epitopes encoded by multiple trans-sialidase genes were tested for the ability to stimulate a recall CD8⁺ T cell response in the peripheral blood from subjects with chronic T. cruzi infection regardless the HLA haplotype. An overall hierarchy of antigenicity was apparent, with the A02 supertype peptides being the most frequently recognized in the Chagas disease population followed by the A03 and the A24 supertype epitopes. CD8⁺ T cell responses to promiscuous epitopes revealed that the CD8⁺ T cell compartment specific for T. cruzi displays a functional profile with T cells secreting interferon-γ alone as the predominant pattern and very low prevalence of single IL-2-secreting or dual IFN-γ/IL-2 secreting T cells denoting a lack of polyfunctional cytokine responses in chronic T. cruzi infection.

Conclusions

This study identifies a set of T. cruzi peptides that should prove useful for monitoring immune competence and changes in infection and disease status in individuals with chronic Chagas disease.     

High throughput selection of effective serodiagnostics for Trypanosoma cruzi infection

Background

Diagnosis of Trypanosoma cruzi infection by direct pathogen detection is complicated by the low parasite burden in subjects persistently infected with this agent of human Chagas disease. Determination of infection status by serological analysis has also been faulty, largely due to the lack of well-characterized parasite reagents for the detection of anti-parasite antibodies.

Methods

In this study, we screened more than 400 recombinant proteins of T. cruzi, including randomly selected and those known to be highly expressed in the parasite stages present in mammalian hosts, for the ability to detect anti-parasite antibodies in the sera of subjects with confirmed or suspected T. cruzi infection.

Findings

A set of 16 protein groups were identified and incorporated into a multiplex bead array format which detected 100% of >100 confirmed positive sera and also documented consistent, strong and broad responses in samples undetected or discordant using conventional serologic tests. Each serum had a distinct but highly stable reaction pattern. This diagnostic panel was also useful for monitoring drug treatment efficacy in chronic Chagas disease.

Conclusions

These results substantially extend the variety and quality of diagnostic targets for Chagas disease and offer a useful tool for determining treatment success or failure.     

Profile of Central and Effector Memory T Cells in the Progression of Chronic Human Chagas Disease

Background

Chronic Chagas disease presents several different clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive clinical forms. Several studies have demonstrated that immunoregulatory mechanisms are important processes for the control of the intense immune activity observed in the chronic phase. T cells play a critical role in parasite specific and non-specific immune response elicited by the host against Trypanosoma cruzi. Specifically, memory T cells, which are basically classified as central and effector memory cells, might have a distinct migratory activity, role and function during the human Chagas disease.

Methodology/Principal Findings

Based on the hypothesis that the disease severity in humans is correlated to the quality of immune responses against T. cruzi, we evaluated the memory profile of peripheral CD4⁺ and CD8⁺ T lymphocytes as well as its cytokine secretion before and after in vitro antigenic stimulation. We evaluated cellular response from non-infected individuals (NI), patients with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas disease. The expression of CD45RA, CD45RO and CCR7 surface molecules was determined on CD4⁺ and CD8⁺ T lymphocytes; the pattern of intracellular cytokines (IFN-γ, IL-10) synthesized by naive and memory cells was determined by flow cytometry. Our results revealed that IND and CARD patients have relatively lower percentages of naive (CD45RA^high) CD4⁺ and CD8⁺ T cells. However, statistical analysis of ex-vivo profiles of CD4⁺ T cells showed that IND have lower percentage of CD45RA^high in relation to non-infected individuals, but not in relation to CARD. Elevated percentages of memory (CD45RO^high) CD4⁺ T cells were also demonstrated in infected individuals, although statistically significant differences were only observed between IND and NI groups. Furthermore, when we analyzed the profile of secreted cytokines, we observed that CARD patients presented a significantly higher percentage of CD8⁺CD45RA^high IFN-γ-producing cells in control cultures and after antigen pulsing with soluble epimastigote antigens.

Conclusions

Based on a correlation between the frequency of IFN-γ producing CD8+ T cells in the T cell memory compartment and the chronic chagasic myocarditis, we propose that memory T cells can be involved in the induction of the development of the severe clinical forms of the Chagas disease by mechanisms modulated by IFN-γ. Furthermore, we showed that individuals from IND group presented more T_CM CD4⁺ T cells, which may induce a regulatory mechanism to protect the host against the exacerbated inflammatory response elicited by the infection.     

Trypanosoma cruzi in the chicken model: Chagas-like heart disease in the absence of parasitism

Background

The administration of anti-trypanosome nitroderivatives curtails Trypanosoma cruzi infection in Chagas disease patients, but does not prevent destructive lesions in the heart. This observation suggests that an effective treatment for the disease requires understanding its pathogenesis.

Methodology/Principal Findings

To understand the origin of clinical manifestations of the heart disease we used a chicken model system in which infection can be initiated in the egg, but parasite persistence is precluded. T. cruzi inoculation into the air chamber of embryonated chicken eggs generated chicks that retained only the parasite mitochondrial kinetoplast DNA minicircle in their genome after eight days of gestation. Crossbreeding showed that minicircles were transferred vertically via the germ line to chicken progeny. Minicircle integration in coding regions was shown by targeted-primer thermal asymmetric interlaced PCR, and detected by direct genomic analysis. The kDNA-mutated chickens died with arrhythmias, shortness of breath, cyanosis and heart failure. These chickens with cardiomyopathy had rupture of the dystrophin and other genes that regulate cell growth and differentiation. Tissue pathology revealed inflammatory dilated cardiomegaly whereby immune system mononuclear cells lyse parasite-free target heart fibers. The heart cell destruction implicated a thymus-dependent, autoimmune; self-tissue rejection carried out by CD45⁺, CD8γδ⁺, and CD8α lymphocytes.

Conclusions/Significance

These results suggest that genetic alterations resulting from kDNA integration in the host genome lead to autoimmune-mediated destruction of heart tissue in the absence of T. cruzi parasites.     

Complexes of Trypanosoma cruzi Sterol 14α-Demethylase (CYP51) with Two Pyridine-based Drug Candidates for Chagas Disease: STRUCTURAL BASIS FOR PATHOGEN SELECTIVITY*

Chagas disease, caused by the eukaryotic (protozoan) parasite Trypanosoma cruzi, is an alarming emerging global health problem with no clinical drugs available to treat the chronic stage. Azole inhibitors of sterol 14α-demethylase (CYP51) were proven effective against Chagas, and antifungal drugs posaconazole and ravuconazole have entered clinical trials in Spain, Bolivia, and Argentina. Here we present the x-ray structures of T. cruzi CYP51 in complexes with two alternative drug candidates, pyridine derivatives (S)-(4-chlorophenyl)-1-(4-(4-(trifluoromethyl)phenyl)-piperazin-1-yl)-2-(pyridin-3-yl)ethanone (UDO; Protein Data Bank code 3ZG2) and N-[4-(trifluoromethyl)phenyl]-N-[1-[5-(trifluoromethyl)-2-pyridyl]-4-piperi-dyl]pyridin-3-amine (UDD; Protein Data Bank code 3ZG3). These compounds have been developed by the Drugs for Neglected Diseases initiative (DNDi) and are highly promising antichagasic agents in both cellular and in vivo experiments. The binding parameters and inhibitory effects on sterol 14α-demethylase activity in reconstituted enzyme reactions confirmed UDO and UDD as potent and selective T. cruzi CYP51 inhibitors. Comparative analysis of the pyridine- and azole-bound CYP51 structures uncovered the features that make UDO and UDD T. cruzi CYP51-specific. The structures suggest that although a precise fit between the shape of the inhibitor molecules and T. cruzi CYP51 active site topology underlies their high inhibitory potency, a longer coordination bond between the catalytic heme iron and the pyridine nitrogen implies a weaker influence of pyridines on the iron reduction potential, which may be the basis for the observed selectivity of these compounds toward the target enzyme versus other cytochrome P450s, including human drug-metabolizing P450s. These findings may pave the way for the development of novel CYP51-targeted drugs with optimized metabolic properties that are very much needed for the treatment of human infections caused by eukaryotic microbial pathogens.     

Benznidazole Therapy Modulates Interferon-�� and M2 Muscarinic Receptor Autoantibody Responses in Trypanosoma cruzi-Infected Children

Objective

The presence of autoantibodies with adrenergic and cholinergic activity, capable of triggering neurotransmitter receptor-mediated effects, has been associated with pathogenesis in T. cruzi-infected hosts. The goal of this study was to investigate the production of anti-M2 muscarinic receptor autoantibodies (Anti-M2R AAbs) as well as the IFN-γ profile in children at the early stage of Chagas disease, and to examine whether trypanocidal chemotherapy with benznidazole (BZ) could modify both response patterns.

Methods

This study comprised 30 T. cruzi-infected children (mean age: 13.8 years) and 19 uninfected controls (mean age: 12.7 years). Infected patients were treated with BZ and followed-up. Blood samples collected at diagnosis-T0, end of treatment-T1, and six months later-T2 were analysed by ELISA for detection of Anti-M2R AAbs and circulating levels of IFN-γ.

Results

At T0, anti-M2R AAbs were demonstrated in 56.7% of T. cruzi-infected patients, whereas uninfected controls were 100% negative. The average age of Anti-M2R AAbs⁺ patients was higher than that from negative population. Infected children also displayed significantly stronger serum IFN-γ responses than controls. Upon BZ treatment, a significant linear decreasing trend in Anti-M2R AAb reactivity was recorded throughout the follow-up, with 29.7–88.1% decrease at T2. IFN-γ circulating levels also declined by T2.

Conclusion

Anti-M2R AAbs and IFN-γ raise early during chagasic infection in children and are downmodulated by BZ therapy. These findings reinforce the usefulness of early BZ treatment not only to eliminate the parasite but also to reduce potentially pathogenic immune responses.     

Trypanosoma cruzi Coaxes Cardiac Fibroblasts into Preventing Cardiomyocyte Death by Activating Nerve Growth Factor Receptor TrkA

Rationale

Cardiomyocytes express neurotrophin receptor TrkA that promotes survival following nerve growth factor (NGF) ligation. Whether TrkA also resides in cardiac fibroblasts (CFs) and underlies cardioprotection is unknown.

Objective

To test whether CFs express TrkA that conveys paracrine signals to neighbor cardiomyocytes using, as probe, the Chagas disease parasite Trypanosoma cruzi, which expresses a TrkA-binding neurotrophin mimetic, named PDNF. T cruzi targets the heart, causing chronic debilitating cardiomyopathy in ∼30% patients.

Methods and Results

Basal levels of TrkA and TrkC in primary CFs are comparable to those in cardiomyocytes. However, in the myocardium, TrkA expression is significantly lower in fibroblasts than myocytes, and vice versa for TrkC. Yet T cruzi recognition of TrkA on fibroblasts, preferentially over cardiomyocytes, triggers a sharp and sustained increase in NGF, including in the heart of infected mice or of mice administered PDNF intravenously, as early as 3-h post-administration. Further, NGF-containing T cruzi- or PDNF-induced fibroblast-conditioned medium averts cardiomyocyte damage by H₂O₂, in agreement with the previously recognized cardioprotective role of NGF.

Conclusions

TrkA residing in CFs induces an exuberant NGF production in response to T cruzi infection, enabling, in a paracrine fashion, myocytes to resist oxidative stress, a leading Chagas cardiomyopathy trigger. Thus, PDNF-TrkA interaction on CFs may be a mechanism orchestrated by T cruzi to protect its heart habitat, in concert with the long-term (decades) asymptomatic heart parasitism that characterizes Chagas disease. Moreover, as a potent booster of cardioprotective NGF in vivo, PDNF may offer a novel therapeutic opportunity against cardiomyopathies.     

Chagas disease among the Latin American adult population attending in a primary care center in Barcelona, Spain

Background/Aims

The epidemiology of Chagas disease, until recently confined to areas of continental Latin America, has undergone considerable changes in recent decades due to migration to other parts of the world, including Spain. We studied the prevalence of Chagas disease in Latin American patients treated at a health center in Barcelona and evaluated its clinical phase. We make some recommendations for screening for the disease.

Methodology/Principal Findings

We performed an observational, cross-sectional prevalence study by means of an immunochromatographic test screening of all continental Latin American patients over the age of 14 years visiting the health centre from October 2007 to October 2009. The diagnosis was confirmed by serological methods: conventional in-house ELISA (cELISA), a commercial kit (rELISA) and ELISA using T cruzi lysate (Ortho-Clinical Diagnostics) (oELISA). Of 766 patients studied, 22 were diagnosed with T. cruzi infection, showing a prevalence of 2.87% (95% CI, 1.6–4.12%). Of the infected patients, 45.45% men and 54.55% women, 21 were from Bolivia, showing a prevalence in the Bolivian subgroup (n = 127) of 16.53% (95% CI, 9.6–23.39%).All the infected patients were in a chronic phase of Chagas disease: 81% with the indeterminate form, 9.5% with the cardiac form and 9.5% with the cardiodigestive form. All patients infected with T. cruzi had heard of Chagas disease in their country of origin, 82% knew someone affected, and 77% had a significant history of living in adobe houses in rural areas.

Conclusions

We found a high prevalence of T. cruzi infection in immigrants from Bolivia. Detection of T. cruzi–infected persons by screening programs in non-endemic countries would control non-vectorial transmission and would benefit the persons affected, public health and national health systems.     

A history of chagas disease transmission, control, and re-emergence in peri-rural La Joya, Peru

Background

The history of Chagas disease control in Peru and many other nations is marked by scattered and poorly documented vector control campaigns. The complexities of human migration and sporadic control campaigns complicate evaluation of the burden of Chagas disease and dynamics of Trypanosoma cruzi transmission.

Methodology/Principal Findings

We conducted a cross-sectional serological and entomological study to evaluate temporal and spatial patterns of T. cruzi transmission in a peri-rural region of La Joya, Peru. We use a multivariate catalytic model and Bayesian methods to estimate incidence of infection over time and thereby elucidate the complex history of transmission in the area. Of 1,333 study participants, 101 (7.6%; 95% CI: 6.2–9.0%) were confirmed T. cruzi seropositive. Spatial clustering of parasitic infection was found in vector insects, but not in human cases. Expanded catalytic models suggest that transmission was interrupted in the study area in 1996 (95% credible interval: 1991–2000), with a resultant decline in the average annual incidence of infection from 0.9% (95% credible interval: 0.6–1.3%) to 0.1% (95% credible interval: 0.005–0.3%). Through a search of archival newspaper reports, we uncovered documentation of a 1995 vector control campaign, and thereby independently validated the model estimates.

Conclusions/Significance

High levels of T. cruzi transmission had been ongoing in peri-rural La Joya prior to interruption of parasite transmission through a little-documented vector control campaign in 1995. Despite the efficacy of the 1995 control campaign, T. cruzi was rapidly reemerging in vector populations in La Joya, emphasizing the need for continuing surveillance and control at the rural-urban interface.     

Targeted screening strategies to detect Trypanosoma cruzi infection in children

Background

Millions of people are infected with Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Anti-trypanosomal drug therapy can cure infected individuals, but treatment efficacy is highest early in infection. Vector control campaigns disrupt transmission of T. cruzi, but without timely diagnosis, children infected prior to vector control often miss the window of opportunity for effective chemotherapy.

Methods and Findings

We performed a serological survey in children 2–18 years old living in a peri-urban community of Arequipa, Peru, and linked the results to entomologic, spatial and census data gathered during a vector control campaign. 23 of 433 (5.3% [95% CI 3.4–7.9]) children were confirmed seropositive for T. cruzi infection by two methods. Spatial analysis revealed that households with infected children were very tightly clustered within looser clusters of households with parasite-infected vectors. Bayesian hierarchical mixed models, which controlled for clustering of infection, showed that a child''s risk of being seropositive increased by 20% per year of age and 4% per vector captured within the child''s house. Receiver operator characteristic (ROC) plots of best-fit models suggest that more than 83% of infected children could be identified while testing only 22% of eligible children.

Conclusions

We found evidence of spatially-focal vector-borne T. cruzi transmission in peri-urban Arequipa. Ongoing vector control campaigns, in addition to preventing further parasite transmission, facilitate the collection of data essential to identifying children at high risk of T. cruzi infection. Targeted screening strategies could make integration of diagnosis and treatment of children into Chagas disease control programs feasible in lower-resource settings.     

Differential Infectivity by the Oral Route of Trypanosoma cruzi Lineages Derived from Y Strain

Background

Diversity of T. cruzi strains is a central problem in Chagas disease research because of its correlation with the wide range of clinical manifestations and the biogeographical parasite distribution. The role played by parasite microdiversity in Chagas disease epidemiology is still debatable. Also awaits clarification whether such diversity is associated with the outcome of oral T. cruzi infection, responsible for frequent outbreaks of acute Chagas disease.

Methods and Findings

We addressed the impact of microdiversity in oral T. cruzi infection, by comparative analysis of two strains, Y30 and Y82, both derived from Y strain, a widely used experimental model. Network genealogies of four nuclear genes (SSU rDNA, actin, DHFR-TS, EF1α) revealed that Y30 is closely related to Discrete Typing Unit TcII while Y82 is more closely related to TcVI, a group containing hybrid strains. Nevertheless, excepting one A-G transition at position 1463, Y30 and Y82 SSU rDNAs were identical. Y82 strain, expressing the surface molecule gp82, infected mice orally more efficiently than Y30, which expresses a related gp30 molecule. Both molecules are involved in lysosome exocytosis-dependent host cell invasion, but exhibit differential gastric mucin-binding capacity, a property critical for parasite migration toward the gastric mucosal epithelium. Upon oral infection of mice, the number of Y30 and Y82 parasites in gastric epithelial cells differed widely.

Conclusions

We conclude that metacyclic forms of gp82-expressing Y82 strain, closely related to TcVI, are better adapted than Y30 strain (TcII) to traverse the stomach mucous layer and establish oral route infection. The efficiency to infect target cell is the same because gp82 and gp30 strains have similar invasion-promoting properties. Unknown is whether differences in Y30 and Y82 are natural parasite adaptations or a product of lab-induced evolution by differential selection along the 60 years elapsed since the Y strain isolation.     

Low Frequency of Circulating CD8+ T Stem Cell Memory Cells in Chronic Chagasic Patients with Severe Forms of the Disease

Background

CD8+ T cells have been shown to play a crucial role in Trypanosoma cruzi infection. Memory CD8+ T cells can be categorised based on their distinct differentiation stages and functional activities as follows: stem cell memory (TSCM), central memory (TCM), transitional memory (TTM), effector memory (TEM) and terminal effector (TTE) cells. Currently, the immune mechanisms that control T. cruzi in the chronic phase of the infection are unknown.

Methodology/Principal Findings

To characterise the CD8+ T cell subsets that could be participating in the control of T. cruzi infection, in this study, we compared total and T. cruzi-specific circulating CD8+ T cells with distinctive phenotypic and functional features in chronic chagasic patients (CCPs) with different degrees of cardiac dysfunction. We observed a decreased frequency of total TSCM along with an increased frequency of TTE in CCPs with severe disease. Antigen-specific TSCM cells were not detectable in CCPs with severe forms of the disease. A functional profile of CD8+ T cell subsets among CCPs revealed a high frequency of monofunctional CD8+ T cells in the most severe patients with IFN-γ+- or TNF-α+-producing cells.

Conclusions/Significance

These findings suggest that CD8+ TSCM cells may be associated with the immune response to T. cruzi and outcome of Chagas disease, given that these cells may be involved in repopulating the T cell pool that controls infection.     

Impact of aetiological treatment on conventional and multiplex serology in chronic Chagas disease

Background

The main criterion for treatment effectiveness in Chagas Disease has been the seronegative conversion, achieved many years post-treatment. One of the main limitations in evaluating treatment for chronic Chagas disease is the lack of reliable tests to ensure parasite clearance and to examine the effects of treatment. However, declines in conventional serological titers and a new multiplex assay can be useful tools to monitor early the treatment impact.

Methodology/Principal Findings

Changes in antibody levels, including seronegative conversion as well as declines in titers, were serially measured in 53 benznidazole-treated and 89 untreated chronic patients in Buenos Aires, Argentina with a median follow-up of 36 months. Decrease of titers (34/53 [64%] treated vs. 19/89 [21%] untreated, p<0.001) and seronegative conversion (21/53, [40%] treated vs. 6/89, [7%] untreated, p<0.001) in at least one conventional serological test were significantly higher in the benznidazole-treated group compare with the untreated group. When not only complete seronegative conversion but also seronegative conversion on 2 tests and the decreases of titers on 2 or 3 tests were considered, the impact of treatment on conventional serology increased from 21% (11/53 subjects) to 45% (24/53 subjects). A strong concordance was found between the combination of conventional serologic tests and multiplex assay (kappa index 0.60) to detect a decrease in antibody levels pos-treatment.

Conclusions/Significance

Treatment with benznidazole in subjects with chronic Chagas disease has a major impact on the serology specific for T. cruzi infection in a shorter follow-up period than previously considered, reflected either by a complete or partial seronegative conversion or by a significant decrease in the levels of T. cruzi antibodies, consistent with a possible elimination or reduction of parasite load.     

Analyses of 32 loci clarify phylogenetic relationships among Trypanosoma cruzi lineages and support a single hybridization prior to human contact

Background

The genetic diversity of Trypanosoma cruzi, the etiological agent of Chagas disease, has been traditionally divided in two major groups, T. cruzi I and II, corresponding to discrete typing units TcI and TcII-VI under a recently proposed nomenclature. The two major groups of T. cruzi seem to differ in important biological characteristics, and are thus thought to represent a natural division relevant for epidemiological studies and development of prophylaxis. To understand the potential connection between the different manifestations of Chagas disease and variability of T. cruzi strains, it is essential to have a correct reconstruction of the evolutionary history of T. cruzi.

Methodology/Principal Findings

Nucleotide sequences from 32 unlinked loci (>26 Kilobases of aligned sequence) were used to reconstruct the evolutionary history of strains representing the known genetic variability of T. cruzi. Thorough phylogenetic analyses show that the original classification of T. cruzi in two major lineages does not reflect its evolutionary history and that there is only strong evidence for one major and recent hybridization event in the history of this species. Furthermore, estimates of divergence times using Bayesian methods show that current extant lineages of T. cruzi diverged very recently, within the last 3 million years, and that the major hybridization event leading to hybrid lineages TcV and TcVI occurred less than 1 million years ago, well before the contact of T. cruzi with humans in South America.

Conclusions/Significance

The described phylogenetic relationships among the six major genetic subdivisions of T. cruzi should serve as guidelines for targeted epidemiological and prophylaxis studies. We suggest that it is important to reconsider conclusions from previous studies that have attempted to uncover important biological differences between the two originally defined major lineages of T. cruzi especially if those conclusions were obtained from single or few strains.     

Immunization with Hexon Modified Adenoviral Vectors Integrated with gp83 Epitope Provides Protection against Trypanosoma cruzi Infection

Background

Trypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary.

Methodology/Principal Findings

The “antigen capsid-incorporation” strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus'' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His₆ were incorporated into the Ad serotype 5 (Ad5) vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83). This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies.

Conclusions/Significance

This data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses, as evidenced by the production of several Ig isotypes. Taken together, these novel results show that the recombinant Ad5 presenting T. cruzi gp83 antigen is a useful candidate for the development of a vaccine against Chagas disease.