Nifurtimox and benznidazole are the front-line drugs used to treat Chagas disease, the most important parasitic infection in the Americas. These agents function as prodrugs and must be activated within the parasite to have trypanocidal effects. Despite >40 years of research, the mechanism(s) of action and resistance have remained elusive. Here, we report that in trypanosomes, both drugs are activated by a NADH-dependent, mitochondrially localized, bacterial-like, type I nitroreductase (NTR), and that down-regulation of this explains how resistance may emerge. Loss of a single copy of this gene in Trypanosoma cruzi, either through in vitro drug selection or by targeted gene deletion, is sufficient to cause significant cross-resistance to a wide range of nitroheterocyclic drugs. In Trypanosoma brucei, loss of a single NTR allele confers similar cross-resistance without affecting growth rate or the ability to establish an infection. This potential for drug resistance by a simple mechanism has important implications, because nifurtimox is currently undergoing phase III clinical trials against African trypanosomiasis.activation ͉ nitroheterocyclic drugs ͉ Trypanosoma brucei ͉ Trypanosoma cruzi

SummaryChronic Trypanosoma cruzi infections lead to cardiomyopathy in 20–30% of cases. A causal link between cardiac infection and pathology has been difficult to establish because of a lack of robust methods to detect scarce, focally distributed parasites within tissues. We developed a highly sensitive bioluminescence imaging system based on T. cruzi expressing a novel luciferase that emits tissue-penetrating orange-red light. This enabled long-term serial evaluation of parasite burdens in individual mice with an in vivo limit of detection of significantly less than 1000 parasites. Parasite distributions during chronic infections were highly focal and spatiotemporally dynamic, but did not localize to the heart. End-point ex vivo bioluminescence imaging allowed tissue-specific quantification of parasite loads with minimal sampling bias. During chronic infections, the gastro-intestinal tract, specifically the colon and stomach, was the only site where T. cruzi infection was consistently observed. Quantitative PCR-inferred parasite loads correlated with ex vivo bioluminescence and confirmed the gut as the parasite reservoir. Chronically infected mice developed myocarditis and cardiac fibrosis, despite the absence of locally persistent parasites. These data identify the gut as a permissive niche for long-term T. cruzi infection and show that canonical features of Chagas disease can occur without continual myocardium-specific infection.

The kinetoplastid Protozoa are responsible for devastating diseases. In the Americas, Trypanosoma cruzi is the agent of Chagas' disease--a widespread disease transmissible from animals to humans (zoonosis)--which is transmitted by exposure to infected faeces of blood-sucking triatomine bugs. The presence of genetic exchange in T. cruzi and in Leishmania is much debated. Here, by producing hybrid clones, we show that T. cruzi has an extant capacity for genetic exchange. The mechanism is unusual and distinct from that proposed for the African trypanosome, Trypanosoma brucei. Two biological clones of T. cruzi were transfected to carry different drug-resistance markers, and were passaged together through the entire life cycle. Six double-drug-resistant progeny clones, recovered from the mammalian stage of the life cycle, show fusion of parental genotypes, loss of alleles, homologous recombination, and uniparental inheritance of kinetoplast maxicircle DNA. There are strong genetic parallels between these experimental hybrids and the genotypes among natural isolates of T. cruzi. In this instance, aneuploidy through nuclear hybridization results in recombination across far greater genetic distances than mendelian genetic exchange. This mechanism also parallels genome duplication.

SummaryHost and parasite diversity are suspected to be key factors in Chagas disease pathogenesis. Experimental investigation of underlying mechanisms is hampered by a lack of tools to detect scarce, pleiotropic infection foci. We developed sensitive imaging models to track Trypanosoma cruzi infection dynamics and quantify tissue‐specific parasite loads, with minimal sampling bias. We used this technology to investigate cardiomyopathy caused by highly divergent parasite strains in BALB/c, C3H/HeN and C57BL/6 mice. The gastrointestinal tract was unexpectedly found to be the primary site of chronic infection in all models. Immunosuppression induced expansion of parasite loads in the gut and was followed by widespread dissemination. These data indicate that differential immune control of T. cruzi occurs between tissues and shows that the large intestine and stomach provide permissive niches for active infection. The end‐point frequency of heart‐specific infections ranged from 0% in TcVI‐CLBR‐infected C57BL/6 to 88% in TcI‐JR‐infected C3H/HeN mice. Nevertheless, infection led to fibrotic cardiac pathology in all models. Heart disease severity was associated with the model‐dependent frequency of dissemination outside the gut and inferred cumulative heart‐specific parasite loads. We propose a model of cardiac pathogenesis driven by periodic trafficking of parasites into the heart, occurring at a frequency determined by host and parasite genetics.

The antifungal drug posaconazole has shown significant activity against Trypanosoma cruzi in vitro and in experimental murine models. Despite this, in a recent clinical trial it displayed limited curative potential. Drug testing is problematic in experimental Chagas disease because of difficulties in demonstrating sterile cure, particularly during the chronic stage of infection when parasite burden is extremely low and tissue distribution is ill defined. To better assess posaconazole efficacy against acute and chronic Chagas disease, we have exploited a highly sensitive bioluminescence imaging system which generates data with greater accuracy than other methods, including PCR-based approaches. Mice inoculated with bioluminescent T. cruzi were assessed by in vivo and ex vivo imaging, with cyclophosphamide-induced immunosuppression used to enhance the detection of relapse. Posaconazole was found to be significantly inferior to benznidazole as a treatment for both acute and chronic T. cruzi infections. Whereas 20 days treatment with benznidazole was 100% successful in achieving sterile cure, posaconazole failed in almost all cases. Treatment of chronic infections with posaconazole did however significantly reduce infection-induced splenomegaly, even in the absence of parasitological cure. The imaging-based screening system also revealed that adipose tissue is a major site of recrudescence in mice treated with posaconazole in the acute, but not the chronic stage of infection. This in vivo screening model for Chagas disease is predictive, reproducible and adaptable to diverse treatment schedules. It should provide greater assurance that drugs are not advanced prematurely into clinical trial.

A BSTR ACTThe unusual DNA base ␤-D-glucosylhydroxymethyluracil, called ''J,'' replaces Ϸ0.5-1% of Thy in DNA of African trypanosomes but has not been found in other organisms thus far. In Trypanosoma brucei, J is located predominantly in repetitive DNA, and its presence correlates with the silencing of telomeric genes. Using antibodies specific for J, we have developed sensitive assays to screen for J in a range of organisms and have found that J is not limited to trypanosomes that undergo antigenic variation but is conserved among Kinetoplastida. In all kinetoplastids tested, including the human pathogens Leishmania donovani and Trypanosoma cruzi, J was found to be abundantly present in the (GGGTTA) n telomere repeats. Outside Kinetoplastida, J was found only in Diplonema, a small phagotrophic marine f lagellate, in which we also identified 5-MeCyt. Fractionation of Diplonema DNA showed that the two modifications are present in a common genome compartment, which suggests that they may have a similar function. Dinof lagellates appear to contain small amounts of modified bases that may be analogs of J. The evolutionary conservation of J in kinetoplastid protozoans suggests that it has a general function, repression of transcription or recombination, or a combination of both. T. brucei may have recruited J for the control of genes involved in antigenic variation.In the nuclear DNA of Trypanosoma brucei, Ϸ0.5-1% of Thy is replaced by the modified base ␤-D-glucosyl-hydroxymethyluracil (␤-gluc-HOMeUra) (1). This base that we call ''J'' was detected initially by 32 P-nucleotide postlabeling combined with twodimensional TLC (2D-TLC) (2), and we used this technique to show that approximately one-half of the cellular J is present in both strands of the telomeric (GGGTTA) n repeats (3). To map the location of J more precisely, we have generated antisera that immunoprecipitate J-containing duplex DNA and that detect this DNA with high sensitivity and specificity on dot blots (4). We have used these antisera to demonstrate that J is present in other repetitive DNA sequences but not in housekeeping genes or transcribed repeats (4). Moreover, we have shown that J is responsible for the blocked restriction sites that are present in silent telomeric variant surface glycoprotein (VSG) genes but not in actively transcribed VSG genes (4-6). This result has linked J to the transcriptional control of VSG genes.Thus far, J has been detected only in African trypanosome species that undergo antigenic variation (2). The availability of antibodies acting against J has prompted us to reinvestigate whether J is also present in other organisms. With anti-J-DNA immunoblots, approximately one J per 10 7 bases can be detected, which is Ϸ1,000-fold more sensitive than MATERIALS AND METHODSCells and DNA Analysis. DNA was derived from: T. brucei brucei (427); Trypanosoma congolense (WG81 and TSW13 bloodstream forms and WG81 procyclics); Trypanosoma vivax (Y58); Crithidia fasciculata (ϭ C. luciliae); Leishmania donovani (HU3); Leishmania tarentol...

Trypanosoma cruzi undergo PCD (programmed cell death) under appropriate stimuli, the mechanisms of which remain to be established. In the present study, we show that stimulation of PCD in T. cruzi epimastigotes by FHS (fresh human serum) results in rapid (<1 h) externalization of phosphatidylserine and depletion of the low molecular mass thiols dihydrotrypanothione and glutathione. Concomitantly, enhanced generation of oxidants was established by EPR and immuno-spin trapping of radicals using DMPO (5,5-dimethylpyrroline-N-oxide) and augmentation of the glucose flux through the pentose phosphate pathway. In the early period (<20 min), changes in mitochondrial membrane potential and inhibition of respiration, probably due to the impairment of ADP/ATP exchange with the cytosol, were observed, conditions that favour the generation of O2*-. Accelerated rates of mitochondrial O2*- production were detected by the inactivation of the redox-sensitive mitochondrial aconitase and by oxidation of a mitochondrial-targeted probe (MitoSOX). Importantly, parasites overexpressing mitochondrial FeSOD (iron superoxide dismutase) were more resistant to the PCD stimulus, unambiguously indicating the participation of mitochondrial O2*- in the signalling process. In summary, FHS-induced PCD in T. cruzi involves mitochondrial dysfunction that causes enhanced O(2)(*-) formation, which leads to cellular oxidative stress conditions that trigger the initiation of PCD cascades; moreover, overexpression of mitochondrial FeSOD, which is also observed during metacyclogenesis, resulted in cytoprotective effects.

Benznidazole is the frontline drug used against Trypanosoma cruzi, the causative agent of Chagas disease. However, treatment failures are often reported. Here, we demonstrate that independently acquired mutations in the gene encoding a mitochondrial nitroreductase (TcNTR) can give rise to distinct drug-resistant clones within a single population. Following selection of benznidazole-resistant parasites, all clones examined had lost one of the chromosomes containing the TcNTR gene. Sequence analysis of the remaining TcNTR allele revealed 3 distinct mutant genes in different resistant clones. Expression studies showed that these mutant proteins were unable to activate benznidazole. This correlated with loss of flavin mononucleotide binding. The drug-resistant phenotype could be reversed by transfection with wild-type TcNTR. These results identify TcNTR as a central player in acquired resistance to benznidazole. They also demonstrate that T. cruzi has a propensity to undergo genetic changes that can lead to drug resistance, a finding that has implications for future therapeutic strategies.