Transcriptomic profiling of paired clinical Leishmania (Mundinia) martiniquensis isolates reveals candidate pathways associated with reduced amphotericin B susceptibility and identifies a novel leishbuvirus variant

Abstract Background Leishmania ( Mundinia ) martiniquensis is an emerging cause of visceral leishmaniasis in Thailand, particularly among patients with advanced HIV infection. Reports of clinical relapse and treatment failure following amphotericin B (AmB) therapy have raised concerns regarding reduced drug susceptibility. However, the molecular basis of reduced AmB susceptibility in this species remains poorly understood. Methods Paired L. martiniquensis isolates obtained from a single patient before treatment (CULE7.2) and after clinical relapse (CULE8) were compared using in vitro AmB susceptibility assays, transmission electron microscopy (TEM), and transcriptomic profiling of promastigotes and axenic amastigotes. For transcriptomics, CULE8 was cultured under continuous exposure to 0.3 µM sublethal AmB to characterize its transcriptional profile during ongoing drug exposure, whereas CULE7.2 was cultured in parallel without AmB. Differential expression, hierarchical clustering, and functional enrichment analyses were performed independently within each developmental stage. Viral sequences were further characterized by de novo assembly of unmapped RNA-seq reads, abundance analysis, negative-stain TEM, phylogenetic analysis, and comparative structural modeling. Results CULE8 exhibited reduced in vitro AmB susceptibility relative to CULE7.2. Ultrastructural examination of CULE7.2 promastigotes following acute AmB exposure revealed prominent cytoplasmic vacuolization, lipid-like inclusions, mitochondrial swelling, and focal nuclear membrane disruption. Transcriptomic analysis revealed distinct transcriptional differences between the isolates within each developmental stage. In promastigotes, CULE8 showed predominant downregulation of genes involved in sterol and lipid metabolism, membrane transport, protein synthesis, motility, and metabolic and regulatory processes, including sterol C24 reductase and upstream mevalonate-pathway enzymes, together with increased expression of genes associated with thiol-based redox homeostasis. In contrast, axenic amastigotes showed increased expression of genes associated with lipid metabolism, redox homeostasis, protein synthesis, and proteostasis, alongside broader downregulation of transport and genome-maintenance genes. Despite predominantly stage-specific transcriptional patterns, a limited subset of genes showed concordant regulation across both stages, including genes involved in lipid metabolism and transport, while genes associated with thiol-based redox homeostasis showed increased expression in both stages. Because the isolates were examined under different drug-exposure conditions, these patterns likely reflect a combination of isolate-associated characteristics and responses to ongoing AmB exposure. De novo assembly recovered complete L and S genomic segments of a genetically distinct leishbuvirus from CULE8, representing, to our knowledge, the first molecular characterization of leishbuvirus in a Southeast Asian L. martiniquensis isolate. Comparative structural modeling supported conservation of the predicted RNA-dependent RNA polymerase core and Motif C, while negative-stain TEM revealed structures consistent with virus-like particles. Viral RNA abundance was higher in CULE8 across both stages. Conclusions The paired-isolate comparison revealed transcriptional differences extending beyond sterol metabolism to multiple cellular processes, with distinct patterns in promastigotes and axenic amastigotes. These findings broaden the molecular landscape associated with reduced AmB susceptibility in L. martiniquensis and highlight candidate pathways for functional investigation. Higher leishbuvirus RNA abundance was observed in the post-relapse isolate, although its biological significance and relationship to reduced AmB susceptibility or clinical relapse remain unresolved. Overall, this hypothesis-generating study provides transcriptomic insights and virological findings that warrant validation in independent isolates under matched experimental conditions.