Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model

Abstract

Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT), also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51) is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF) cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51 ^RNAi ) in vitro. Following reduction of Tb CYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51 ^RNAi induction caused morphological defects with multiflagellated cells ( p<0.05), suggesting cytokinesis dysfunction. The survival of CYP51 ^RNAi Doxycycline-treated mice ( p = 0.053) and of CYP51 ^RNAi 5-day pre-induced Doxycycline-treated mice ( p = 0.008) were improved compared to WT showing a CYP51 RNAi effect on trypanosomal virulence in mice. The posaconazole concentrations that inhibited parasite growth by 50% (IC ₅₀) were 8.5, 2.7, 1.6 and 0.12 μM for T. b. brucei 427 90–13, T. b. brucei Antat 1.1, T. b. gambiense Feo (Feo/ITMAP/1893) and T. b. gambiense Biyamina (MHOM/SD/82), respectively. During infection with these last three virulent strains, posaconazole-eflornithine and nifurtimox-eflornithine combinations showed similar improvement in mice survival ( p≤0.001). Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic alternative for trypanosomiasis.

Author Summary

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic disease exclusively located in sub-Saharan and southern Africa where it is transmitted by infected tsetse flies and is caused by a blood parasite named Trypanosoma brucei. Because of limited alternatives, toxicity and high expense new treatment options are urgently needed for patients with this deadly disease and related diseases. Sterols are important for building cell membranes and are precursors for the synthesis of powerful bioactive molecules, such as steroid hormones in mammals. Among the enzymes required for sterol production, one class (named CYP51), share similar functions and importantly these enzymes are found in all biological kingdoms, including in the sleeping sickness parasite. We have studied the role of trypanosome CYP51 and found that when native levels are diminished experimentally, parasite survival and division are radically diminished resulting in cell death but this can be rescued by the addition of ergosterol. We observed that parasites with diminished level of CYP51 enzyme were less virulent in mice. We also show that posaconazole, a clinically used antifungal and potential CYP51 inhibitor, augments parasite killing when used in combination with currently used trypanocidal drugs. Thus our results suggest that a specific targeting of the enzyme could be a potential strategy to treat HAT.