Targeting Ergosterol biosynthesis in Leishmania donovani: essentiality of sterol 14 alpha-demethylase.

Laura-Isobel Mccall,Amale El Aroussi,Debora F Vieira,James H Mckerrow,Steven Chen,Geraldine De Muylder,Danielle Kellar,Larissa M Podust,Jair L Siqueira-Neto,Jun Yong Choi,William R Roush,Jonathan B Johnston

doi:10.1371/journal.pntd.0003588

Abstract

Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis.

Highlights

We demonstrate via gene knockout and drug targeting approaches that loss or inhibition of CYP51 inhibits L. donovani growth
Leishmania parasites cause a range of disease manifestations: cutaneous leishmaniasis in which lesions develop at the site of the sandfly bite, mucocutaneous leishmaniasis with destruction of the mucosal tissues in the nose, mouth and throat, and visceral leishmaniasis in which parasites disseminate to the liver, bone marrow and spleen
The infecting species of Leishmania is the major determinant of disease manifestation; parasites from the Leishmania donovani species complex are the main causes of visceral leishmaniasis, while other species, including the Leishmania major species complex, cause cutaneous manifestations [2,3]

Summary

Introduction

They have a digenetic lifecycle; promastigotes are transmitted by the sandfly vector to the mammalian host, where they are taken up by phagocytic cells and differentiate into the amastigote stage within the macrophage phagolysososme. Amastigotes differentiate into promastigotes, thereby completing the parasite lifecycle [1]. Visceral leishmaniasis is the most lethal form of the disease. It is associated with high fever, hepatosplenomegaly and pancytopenia [1]. The infecting species of Leishmania is the major determinant of disease manifestation; parasites from the Leishmania donovani species complex are the main causes of visceral leishmaniasis, while other species, including the Leishmania major species complex, cause cutaneous manifestations [2,3]

Methods

Results

Conclusion