Comparative proteomic profiling reveals distinct molecular signatures of drug resistance in clinical isolates of Leishmania donovani.

Leishmaniasis remains a major public health concern, with treatment options limited to amphotericin B (AmpB), sodium stibogluconate (SSG), miltefosine, and paromomycin. Despite extensive clinical and field evaluations, the efficacy of these agents is increasingly undermined by toxicity, high cost, patient non-compliance, and the emergence of drug-resistance. Given this growing challenge, we conducted a comparative proteomic analysis of five Leishmania donovani clinical isolates: one drug-sensitive (BHU1066) and four drug-unresponsive to SSG (NS2), miltefosine (BHU1064), amphotericin B (BHU919), and paromomycin (BHU1020). Liquid chromatography-mass spectrometry identified proteins across these clinical isolates, with each drug-resistant isolate exhibiting a distinct profile of up-regulated proteins compared to the sensitive. Functional annotation revealed that genes involved in metabolic pathways, translation, RNA modification, chaperones, heat shock proteins, and signaling were prominently up-regulated in all drug-unresponsive clinical isolates. A subset of metabolic enzymes associated with glycolysis unique to Leishmania were consistently elevated, suggesting their potential role in mediating drug resistance. Approximately 20% of up-regulated proteins remain uncharacterized, highlighting novel candidates for future drug target discovery. Quantitative RT-PCR of selected differentially expressed genes confirmed significant drug-resistance-specific transcriptional modulation. These findings demonstrate that drug resistance in L. donovani is accompanied by extensive proteomic remodeling, emphasizing the need for comprehensive molecular studies to inform new therapeutic strategies against both drug-sensitive and drug-resistant leishmaniasis.