In Vivo Bioluminescence Imaging to Assess Compound Efficacy Against Trypanosoma brucei (original) (raw)
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Highly Sensitive In Vivo Imaging of Trypanosoma brucei Expressing “Red-Shifted” Luciferase
PLoS Neglected Tropical Diseases, 2013
Background: Human African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids.
PLoS Neglected Tropical Diseases, 2009
Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs.
Drug Development Research, 2020
African trypanosomiasis is a major problem for human and animal health in endemic countries, where it threatens millions of people and affects economic development. New drugs are needed to overcome the toxicity, administration, low efficacy, and resistance issues of the current chemotherapy. Robust, simple, and economical highthroughput, whole-cell-based assays are required to accelerate the identification of novel chemical entities. With this aim, we generated a bioluminescent cell line of the bloodstream stage of Trypanosoma brucei brucei and established a screening assay. Trypanosomes were stably transfected to constitutively express a thermostable redshifted luciferase. The growth phenotype and drug sensitivity of the reporter cell line were essentially identical to that of the parental cell line. The endogenous luciferase activity, measured by a simple bioluminescence assay, proved to be proportional to parasite number and metabolic status. The assay, optimized to detect highly potent compounds in a 96-well-plate format, was validated by screening a small compound library (inter-assay values for Z' factor and coefficient variation were 0.77 and 5.8%, respectively). With a hit-confirmation ratio of~97%, the assay was potent enough to identify several hits with EC 50 ≤ 10 μM. Preliminary tests indicated that the assay can be scaled up to a 384-well-plate format without compromising its robustness. In summary, we have generated reporter trypanosomes and a simple, robust, and affordable bioluminescence screening assay with great potential to speed up the early-phase drug discovery against African trypanosomes.
J Antimicrob Chemother, 2014
To optimize the Trypanosoma brucei brucei GVR35 VSL-2 bioluminescent strain as an innovative drug evaluation model for late-stage human African trypanosomiasis. Methods: An IVIS w Lumina II imaging system was used to detect bioluminescent T. b. brucei GVR35 parasites in mice to evaluate parasite localization and disease progression. Drug treatment was assessed using qualitative bioluminescence imaging and real-time quantitative PCR (qPCR). Results: We have shown that drug dose-response can be evaluated using bioluminescence imaging and confirmed quantification of tissue parasite load using qPCR. The model was also able to detect drug relapse earlier than the traditional blood film detection and even in the absence of any detectable peripheral parasites. Conclusions: We have developed and optimized a new, efficient method to evaluate novel anti-trypanosomal drugs in vivo and reduce the current 180 day drug relapse experiment to a 90 day model. The non-invasive in vivo imaging model reduces the time required to assess preclinical efficacy of new anti-trypanosomal drugs.
2013
Human African trypanosomiasis (HAT) manifests in two stages of disease: firstly, haemolymphatic, and secondly, an encephalitic phase involving the central nervous system (CNS). New drugs to treat the second-stage disease are urgently needed, yet testing of novel drug candidates is a slow process because the established animal model relies on detecting parasitemia in the blood as late as 180 days after treatment. To expedite compound screening, we have modified the GVR35 strain of Trypanosoma brucei brucei to express luciferase, and have monitored parasite distribution in infected mice following treatment with trypanocidal compounds using serial, non-invasive, bioluminescence imaging. Parasites were detected in the brains of infected mice following treatment with diminazene, a drug which cures stage 1 but not stage 2 disease. Intravital multi-photon microscopy revealed that trypanosomes enter the brain meninges as early as day 5 postinfection but can be killed by diminazene, whereas those that cross the blood-brain barrier and enter the parenchyma by day 21 survived treatment and later caused bloodstream recrudescence. In contrast, all bioluminescent parasites were permanently eliminated by treatment with melarsoprol and DB829, compounds known to cure stage 2 disease. We show that this use of imaging reduces by two thirds the time taken to assess drug efficacy and provides a dual-modal imaging platform for monitoring trypanosome infection in different areas of the brain.
Journal of biomolecular screening, 2015
The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, one of the world's major neglected infections. Although development of improved antiparasitic drugs is considered a priority, there have been no significant treatment advances in the past 40 years. Factors that have limited progress include an incomplete understanding of pathogenesis, tissue tropism, and disease progression. In addition, in vivo models, which allow parasite burdens to be tracked throughout the chronic stage of infection, have been lacking. To address these issues, we have developed a highly sensitive in vivo imaging system based on bioluminescent T. cruzi, which express a red-shifted luciferase that emits light in the tissue-penetrating orange-red region of the spectrum. The exquisite sensitivity of this noninvasive murine model has been exploited to monitor parasite burden in real time throughout the chronic stage, has allowed the identification of the gastrointestinal tract as the major ...
Parasitology International, 2014
The advances in microscopy combined to the invaluable progress carried by the utilization of molecular, immunological or immunochemical markers and the implementation of more powerful imaging technologies have yielded great improvements to the knowledge of the interaction between microorganisms and their hosts, notably a better understanding of the establishment of infectious processes. Still today, the intricacies of the dialog between parasites, cells and tissues remain limited. Some improvements have been attained with the stable integration and expression of the green fluorescence protein or firefly luciferase and other reporter genes, which have allowed to better approach the monitoring of gene expression and protein localization in vivo, in situ and in real time. Aiming at better exploring the well-established models of murine infections with the characterized strains of Trypanosoma cruzi and Trypanosoma vivax, we revisited in the present report the state of the art about the tools for the imaging of Trypanosomatids in vitro and in vivo and show the latest transgenic parasites that we have engineered in our laboratory using conventional transfection methods. The targeting of trypanosomes presented in this study is a promising tool for approaching the biology of parasite interactions with host cells, the progression of the diseases they trigger and the screening of new drugs in vivo or in vitro.
Bioluminescent imaging of Trypanosoma cruzi infection
International Journal for Parasitology, 2008
Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.
Discovery of Trypanocidal Compounds by Whole Cell HTS of Trypanosoma brucei
Chemical Biology <html_ent glyph="@amp;" ascii="&"/> Drug Design, 2006
Chemotherapy against human African trypanosomiasis relies on four drugs that cause frequent and occasionally severe side-effects. Because human African trypanosomiasis is a disease of poor people in Africa, the traditional market-driven pathways to drug development are not available. One potentially rapid and cost-effective approach to identifying and developing new trypanocidal drugs would be high throughput-screening of existing drugs already approved for other uses, as well as clinical candidates in late development. We have developed an ATP-bioluminescence assay that could be used to rapidly and efficiently screen compound libraries against trypanosomes in a high throughput-screening format to validate this notion. We screened a collection of 2160 FDAapproved drugs, bioactive compounds and natural products to identify hits that were cytotoxic to cultured Trypanosoma brucei at a concentration of 1 lM or less. This meant that any hit identified would be effective at a concentration readily achievable by standard drug dosing in humans. From the screen, 35 hits from seven different drug categories were identified. These included the two approved trypanocidal drugs, suramin and pentamidine, several other drugs suspected but never validated as trypanocidal, and 17 novel trypanocidal drugs.