The protozoan parasite Leishmania causes leishmaniasis, a neglected tropical disease, that disproportionately affects underdeveloped countries. This disease has major health, economic, and social implications, particularly because of the limited treatment options, high cost, the severe side effects associated with available therapeutics, and the high rate of treatment failure caused by the parasites’ growing resistance to current medications. In this review, we describe first the common strategies used by pathogens to develop drug resistance and then focus on the arsenal of available drugs to treat leishmaniasis, their modes of action, and the molecular mechanisms contributing to drug resistance in Leishmania spp., including the role of genomic, transcriptional, and translational control. We focus more specifically on our recent discovery of translational reprogramming as a major driver of drug resistance leading to coordinated changes in the translation of transcripts and orchestrating changes in metabolome and lipidome to support drug resistance. A thorough understanding of these mechanisms is essential to identify the key elements needed to combat resistance and improve leishmaniasis treatment methods.

Persistence - characterized by the transient ability of subpopulations of drug-susceptible parasites to survive exposure to drug - is a major driver of treatment failure and clinical relapses in leishmaniasis. Persisters are characterized by non-dividing or slow-growing state and increased drug tolerance. However, the molecular mechanisms governing formation of persisters remain poorly understood in Leishmania parasites. Here, we developed a model to explore persistence in Leishmania mexicana . Viable promastigote persister-like subpopulations were enriched using Ficoll density gradient centrifugation following lethal exposure to antimonial drugs that killed 80% of parasites. The surviving parasites exhibited delayed growth in drug-free medium that is characteristic for persisters, and significantly higher tolerance to drug upon rechallenge. Transcriptomic profiling across acute stress, drug-free recovery, and rechallenge phases revealed a global remodeling in persisters under all tested conditions. Induction phase in Leishmania persisters was characterized by downregulation of several biological processes and a robust upregulation of nucleolar pathways, supporting epitranscriptomic changes during formation of persisters. Upon drug removal, this profile rapidly reverted, initiating ribosomal biogenesis to exit latency and resume cellular proliferation. Resuscitation phase exhibited activated protein synthesis and upregulation in many biological processes associated with metabolic and mitochondrial functions. Furthermore, comparative analysis of drug responses in rechallenged drug-tolerant persisters and parental parasites exposed to the drug for the first time, revealed that persisters exhibit distinct drug response profiles compared to parental parasites by rapidly implementing a highly conserved, coordinated survival reprogramming, where 316 genes were uniquely downregulated, and 241 genes were upregulated. The distinct features of the drug response in rechallenged persister cells were characterized by the downregulation of mitochondrial function and protein synthesis machinery to induce a dormant, idling state, and the upregulation of drug-response and stress-tolerance genes to survive immediate toxicity. In contrast, parental parasites displayed a broad and disorganized drug response. Additionally, rechallenged persisters exhibited a distinct transcriptomic memory that transiently phenocopies stable genetic resistance. This pre-adapted state is characterized by the targeted upregulation of epigenetic modulators, heavy metal transporters, and catabolic enzymes to maintain viability. These findings demonstrate that drug persistence in Leishmania is not merely a metabolic collapse, but rather a sophisticated survival strategy involving active transcriptome remodeling, downregulation of translation and epigenetic adaptations. This transient state constitutes an initial evolutionary step toward permanent drug resistance and highlights new molecular vulnerabilities for therapeutic interventions aimed at preventing clinical relapse. Author summary: The efficacy of leishmaniasis chemotherapy is often fails because Leishmania parasites survive the drugs by entering a dormant state, causing relapses in patients. In this study, however, we discovered that these parasites are not simply ‘quiescent’. On the contrary, they orchestrate a highly active survival response: they shut down energy-consuming functions while keeping their cellular alert mechanisms active. Remarkably, upon re-exposure to antileishmanial drugs, the parasites recall previous stress events and transiently activate defensive pathways characteristic of stable drug resistance. Understanding how Leishmania employs this temporary adaptive strategy provides critical targets for developing therapies designed to eradicate the infection and avert relapses.

Persistence - characterized by the transient ability of subpopulations of Leishmania parasites to survive exposure to drugs - is a major driver of treatment failure and clinical relapses in leishmaniasis. Persisters are characterized by non-dividing or slow-growing state and increased drug tolerance. However, the molecular mechanisms governing formation of persisters remain poorly understood in Leishmania parasites. Here, we developed a model to explore persistence in Leishmania mexicana . Viable promastigote persister-like subpopulations were enriched using Ficoll density gradient centrifugation following lethal exposure to potassium antimonyl tartrate (PAT) that killed 80% of parasites. The surviving parasites exhibited delayed growth in drug-free medium that is characteristic for persisters, and higher drug tolerance upon rechallenge. Transcriptomic profiling across acute stress, drug-free recovery, and rechallenge phases revealed a global remodeling in persister-like cells under all tested conditions. Induction was characterized by downregulation of several biological processes and a robust upregulation of nucleolar pathways, supporting epitranscriptomic changes during the formation of persister-like cells. Upon drug removal, this profile rapidly reverted, initiating ribosome biogenesis to exit latency and resume proliferation. Resuscitation phase exhibited active protein synthesis and upregulation in biological processes associated with metabolic and mitochondrial functions. Furthermore, persister-like parasites displayed distinct drug response profiles compared to parental parasites by rapidly implementing a highly conserved, coordinated survival reprogramming, where 316 genes were uniquely downregulated, and 241 genes were upregulated. The distinct features of the drug response in rechallenged persister-like cells were characterized by the downregulation of mitochondrial function and protein synthesis to induce dormant state, and the upregulation of drug-response and stress-tolerance genes to survive immediate toxicity. In contrast, parental parasites displayed a broad and disorganized drug response. Additionally, rechallenged persisters exhibited distinct transcriptomic features that transiently phenocopies stable genetic resistance. This pre-adapted state is characterized by the targeted upregulation of epigenetic and epitranscriptomic modulators, heavy metal transporters, and catabolic enzymes to maintain viability. These findings demonstrate that drug persistence in Leishmania is not merely a metabolic collapse, but rather a sophisticated survival strategy involving active transcriptome remodeling, downregulation of translation and epigenetic/epitranscriptomic changes. This transient state constitutes an initial evolutionary step toward permanent drug resistance and highlights new molecular vulnerabilities for therapeutic interventions aimed at preventing clinical relapse.

Leishmania braziliensis cutaneous leishmaniasis (CL) remains a major public health challenge due to the toxicity, variable efficacy, and emerging resistance associated with systemic therapies. However, few topical candidates have been successfully developed despite growing guideline support for local treatment in many patients. This study evaluated the antileishmanial, immunomodulatory, and proregenerative effects of a topical formulation combining triterpene saponins from Sapindus saponaria (SS) and a synthetic chroman hydrazone (TC2) using a standardized hamster model of L. braziliensis CL. The antiparasitic and immunomodulatory effects of TC2-SS were examined in human monocytederived macrophages infected with L. braziliensis, assessing intracellular parasite burden, macrophage morphology, nitric oxide (NO) and reactive oxygen species (ROS) production, and cytokine expression by ELISA and RT-qPCR. In vivo efficacy and early wound-healing responses were evaluated in a dorsalskin hamster model treated topically with 4% TC2SS and compared with intralesional meglumine antimoniate, including epidermal growth factor (EGF) and transforming growth factor β1 (TGFβ1) expression. TC2-SS reduced intracellular infection (EC₅₀ 12.83 ± 0.97 µg/mL), decreased parasite burden, and induced macrophage activation-like morphological changes. The formulation preserved infectiondriven NO and ROS and selectively enhanced late NO in infected cells. Cytokine analyses revealed coordinated suppression of IL-1β, TNF-α, IL-4, IL-10, and TGF-β1, preserving MIP-1α. In vivo, TC2-SS achieved cure rates comparable to those of antimonials, with uniform lesion regression, no treatment failures, no systemic toxicity, and an increased ratio of EGF/TGF-β1. Thus, TC2-SS provided dual antileishmanial and host-directed benefits, supporting its advancement as a topical therapeutic candidate for L. braziliensis CL.