Co-infection with Leptomonas seymouri enhances macrophage survival and promotes intracellular parasite persistence during Leishmania donovani infection

BackgroundLeishmania donovani (LD) is an obligate intracellular parasite that survives and replicates within macrophages. Leptomonas seymouri (LS), a traditionally monoxenous trypanosomatid, has been repeatedly co-isolated with LD from visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients in India, often together with Leptomonas seymouri narna-like virus 1 (Lepsey NLV1). Whether LS can survive and replicate within mammalian macrophages, and how co-infection influences parasite and viral dynamics, remains unresolved. Methods and FindingsUsing murine (RAW 264.7) and human (THP-1) macrophages, we systematically evaluated intracellular survival, replication, and revival of LS alone and during co-infection with LD. Quantitative ITS1 PCR demonstrated significant increases in intracellular parasite DNA over 48-168 h post-infection, with mean intracellular loads rising up to [~]7.6-fold, indicating active replication rather than persistence. Reduced extracellular parasite load suggested restricted cell lysis and enhanced macrophage survival in co-infection compared to mono-infections. In co-infection scenario, generally LS displayed higher persistence compared to LD. Giemsa staining confirmed intracellular localization of LS. Parasites recovered from infected macrophages remained viable and revived as motile promastigotes, whereas extracellular parasites failed to survive beyond 48 h, confirming macrophages as the exclusive niche for prolonged viability. Interestingly, co-infection dampened macrophage IL-12 production, suggesting altered host immune activation. Lepsey NLV1 RNA accumulated predominantly within macrophages and persisted up to 168 h post-infection. Virus load within LS in co-infection state was [~]2.5-4 and [~]7.5-23 fold higher (for RAW 264.7 and THP-1 respectively) compared to LS mono-infection. Purified virus alone failed to enter macrophages, indicating LS-dependent viral delivery. ConclusionsOur findings question the prevailing view of LS as a strictly non-infective parasite, demonstrating its capacity to replicate within mammalian macrophages and persist during mono-or LD co-infection. The identification of a stable LD-LS-virus interaction highlights a previously underappreciated "triple-pathogen" biology with potential implications for VL and PKDL pathogenesis.