Authors

L Marriott¹; PW Denny¹;  ¹ Durham University, UK

Discussion

Leishmaniasis and invasive fungal diseases, both caused by eukaryotic pathogens, are diseases for which historical neglect has left us with a limited armoury of therapeutics. Emerging issues with the few drugs we do have for the treatment of these diseases present an increasing risk to global health, especially within vulnerable populations. As such there is a pressing need for novel antimicrobials and a comprehensive understanding of their mode of action. Previous study has implicated the central role of lipids and their synthesis in the modes of action of both amphotericin B and miltefosine, two anti-microbials currently used in the clinical treatment of leishmaniasis and fungal infections. To further the goal of building a comprehensive understanding of the modes of action of these drugs in the context of lipid metabolism, a curated library of CRISPR/Cas9 modified Leishmania mexicana, engineered with genetic knockout of key genes within lipid metabolism pathways were subjected to drug sensitivity and phenotypic screening. This library screening approach was able to uncover genes for which knockout contributes to decreased membrane integrity and resultant sensitivity to drug pressure. Of note however are several genes for which the knockout increases resistance to one or both drugs, implicating the gene product or it’s associated metabolites in the drug mode of action. The most significant changes in drug sensitivity were found after knockout of inositol phosphoryl ceramide synthase (IPCS), a key enzyme in Leishmania spp. sphingolipid synthesis, where a 4-fold increase in resistance to miltefosine accompanied by a 2-fold increase in sensitivity to amphotericin B were observed. Initial screening of drug sensitivity in genetically modified L. mexicana has provided a candidate list of genes that will be further studied in parallel investigations in fungi to further elucidate key areas within the mode of action of these drugs. Additionally, the library screening approach in both organisms will be applied to assess the mode of action of novel compounds with anti-leishmanial activity.