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- Title
Highly Sensitive In Vivo Imaging of Trypanosoma brucei Expressing "Red-Shifted" Luciferase.
- Authors
McLatchie, Alex P.; Burrell-Saward, Hollie; Myburgh, Elmarie; Lewis, Michael D.; Ward, Theresa H.; Mottram, Jeremy C.; Croft, Simon L.; Kelly, John M.; Taylor, Martin C.
- Abstract
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. Methodology/Principal findings: Here, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5′-variant surface glycoprotein untranslated region (UTR) and a downstream 3′-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter. Conclusions/Significance: The optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes. Author Summary: Parasites of the Trypanosoma brucei species complex are the causative agents of human African trypanosomiasis. There is an urgent need for new drugs to treat this debilitating and potentially fatal infection, especially in its late stage, when parasites have entered the central nervous system. Factors which hamper drug development include the limitations of the current murine models for stage II disease. In vivo bioluminescence imaging is a non-invasive technique that can be used to monitor infections in real time and is a powerful new approach for studying drug effectiveness. However, application of this imaging technology to trypanosome infections has been restricted because of lack of sensitivity. In this paper, we have taken a major step to resolve this problem. The enhanced sensitivity in infected mice is based on the high level expression in trypanosomes of a "red-shifted" luciferase variant that greatly improves bioluminescence detection in deep tissue. The system which we have developed should be a widely applicable tool for providing new insights into the infection biology of T. brucei.
- Subjects
SUB-Saharan Africa; TRYPANOSOMA brucei; AFRICAN trypanosomiasis; HOST-parasite relationships; GENE expression; DRUG efficacy
- Publication
PLoS Neglected Tropical Diseases, 2013, Vol 7, Issue 11, p1
- ISSN
1935-2727
- Publication type
Article
- DOI
10.1371/journal.pntd.0002571