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

Author summary Visceral leishmaniasis is a life-threatening disease caused byLeishmania infantumparasites, which are transmitted by sand flies. In the absence of vaccines, current control of this disease is based on chemotherapy, which is comprised of a very limited arsenal threatened by the emergence and spread of drug-resistant strains. In the shadow of growing concern and treatment failure due to resistance, the characterization of extracellular vesicles (EVs) released by drug-resistantL.infantumparasites could shed some light on the complex nature of drug resistance inLeishmaniaand increase our understanding of the biology of the parasite. EVs are vesicles secreted by all eukaryotic cells whose contents (proteins, DNA/RNAs, lipids) vary as a function of their cellular origin. Our results demonstrate for the first time that EVs released by drug-resistant parasites are enriched in unique protein markers that reflect the drug-resistance mechanisms involved in the survival of parental cells. These unique proteins included several virulence and transcription factors, as well as drug-resistance genes; this offers a potential benefit for drug-resistant parasites in terms of parasite-to-parasite communication and host-parasite interactions. Collectively, our initial results could serve as a jumping-off point for the future development of novel EV-based diagnostic tools for the detection and appraisal of antimicrobial-resistantLeishmaniapopulations. Leishmaniasis constitutes the 9(th)largest disease burden among all infectious diseases. Control of this disease is based on a short list of chemotherapeutic agents headed by pentavalent antimonials, followed by miltefosine and amphotericin B; drugs that are far from ideal due to host toxicity, elevated cost, limited access, and high rates of drug resistance. Knowing that the composition of extracellular vesicles (EVs) can vary according to the state of their parental cell, we hypothesized that EVs released by drug-resistantLeishmania infantumparasites could contain unique and differently enriched proteins depending on the drug-resistance mechanisms involved in the survival of their parental cell line. To assess this possibility, we studied EV production, size, morphology, and protein content of three well-characterized drug-resistantL.infantumcell lines and a wild-type strain. Our results are the first to demonstrate that drug-resistance mechanisms can induce changes in the morphology, size, and distribution ofL.infantumEVs. In addition, we identifiedL.infantum's core EV proteome. This proteome is highly conserved among strains, with the exception of a handful of proteins that are enriched differently depending on the drug responsible for induction of antimicrobial resistance. Furthermore, we obtained the first snapshot of proteins enriched in EVs released by antimony-, miltefosine- and amphotericin-resistant parasites. These include several virulence factors, transcription factors, as well as proteins encoded by drug-resistance genes. This detailed study ofL.infantumEVs sheds new light on the potential roles of EVs inLeishmaniabiology, particularly with respect to the parasite's survival in stressful conditions. This work outlines a crucial first step towards the discovery of EV-based profiles capable of predicting response to antileishmanial agents.