High-throughput Cos-Seq screen with intracellular Leishmania infantum for the discovery of novel drug-resistance mechanisms

Abstract

Increasing drug resistance towards first line antimony-derived compounds has forced the introduction of novel therapies in leishmaniasis endemic areas including amphotericin B and miltefosine. However, their use is threatened by the emergence and spread of drug-resistant strains. In order to discover stage-dependent resistance genes, we have adapted the Cos-Seq approach through the introduction of macrophage infections in the pipeline. A L. infantum intracellular amastigote population complemented with a L. infantum cosmid library was submitted to increasing concentrations of miltefosine, amphotericin B and pentavalent antimonials in experimental infections of THP-1 cells. For each step of selection, amastigotes were extracted and cosmids were isolated and submitted to next-generation sequencing, followed by subsequent gene-enrichment analyses. Cos-Seq screen in amastigotes revealed four highly enriched loci for antimony, five for miltefosine and one for amphotericin B. Of these, a total of seven cosmids were recovered and tested for resistance in both promastigotes and amastigotes. Candidate genes within the pinpointed genomic regions were validated using single gene overexpression in wild-type parasites and/or gene disruption by means of a CRISPR-Cas9-based approach. This led to the identification and validation of a stage-independent antimony-resistance gene (LinJ.06.1010) coding for a putative leucine rich repeat protein and a novel amastigote-specific miltefosine-resistance gene (LinJ.32.0050) coding for a member of the SEC13 family of WD-repeat proteins. This study further reinforces the power of Cos-Seq approach to discover novel drug-resistance genes, some of which are life-stages specific.

Unravelling the proteomic signature of extracellular vesicles released by drug-resistant Leishmania infantum parasites

“…Different parasite molecular modifications, such as ABC transporters, membrane composition changes or oxidative stress, among others, can contribute both directly and indirectly to the phenomena of drug resistance [35,46,58]. Moreover, it has been shown that Leishmania is able to actively alter macrophage and neutrophil environments to resist current antileishmanial agents [59].…”

RNA Polymrase II gene expression in clinical Leishmania major isolates with no-response-to-drug pattern

“…LRR protein is an important gene in antimonial resistance in Leishmania spp. [26]. This gene is located in chromosome 6.…”

Experimental Strategies to Explore Drug Action and Resistance in Kinetoplastid Parasites

“…All these studies have used the extracellular promastigote stage as it is known to be easier and faster. Only two studies opted to use intracellular amastigotes for the selection of cosmid bearing parasites [ 113 , 116 ], resulting in the identification of predominantly different genes compared to those identified in promastigotes [ 116 ]. These differences could be caused by a number of factors, not necessarily directly related to the drug, such as the host cell environment, parasite infectivity and intracellular multiplication [ 116 ].…”