Functional Cloning as a Means to Identify Leishmania Genes Involved in Drug Resistance

Abstract

Resistance to anti-leishmanial drugs is a mounting problem in high-endemicity regions of South Asia and, potentially, in the context of HIV-Leishmania coinfections in Southern Europe. The molecular basis for clinical drug resistance is still largely unknown. It is important, however, to identify all relevant drug resistance markers for further drug development and for epidemiological surveys. An elegant and powerful method to identify such drug resistance markers without bias is functional cloning, using cosmid-based genomic DNA libraries. This review discusses the merits and caveats of this approach.

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

“…1A), which might favor the propagation of parasites bearing cosmids providing a more modest selective advantage. Parasites were likewise passaged in the absence of drug to control for the selection of cosmids conferring selectable growth fitness traits unrelated to drug pressure (12).…”

“…In Leishmania spp., copy number variation and single-nucleotide polymorphism were detected in drug-resistant parasites using NGS (9, 10; reviewed in ref 11). Gain-of-function screening using genomic cosmid libraries is also a proven approach to studying drug resistance in Leishmania (12). Cosmid-based functional cloning was first implemented for studying lipophosphoglycan biosynthesis (13) and later successfully applied to study nucleoside transport (14,15) and drug resistance (16)(17)(18)(19)(20)(21).…”

Reduced Antimony Accumulation in ARM58 -Overexpressing Leishmania infantum

“…Only those parasites carrying cosmids with dominant drug resistance markers will survive the challenge and grow under selection. Recovering and analyzing the cosmids within the surviving parasites will then make it possible to identify resistance marker genes (20). This approach has already been used to identify novel markers of resistance against antimonials and other antileishmanial compounds (21,22).…”

No stress – Hsp90 and signal transduction inLeishmania

“…The lack of RNA interference, however, paves the way for functional cloning strategies and stable, episomal transfection using circular DNA constructs such as plasmids and cosmids. Since the leishmaniae show a high variability of gene copy numbers, both chromosomal and extrachromosomal, functional cloning and episomal transgenes are valid models for copy number-dependent gene functionality (Clos and Choudhury, 2006).…”