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

1. Problem Statement

Leishmania donovani (LD) is an obligate intracellular parasite causing visceral leishmaniasis (VL). Leptomonas seymouri (LS) is traditionally classified as a monoxenous (insect-only) trypanosomatid that does not infect mammalian hosts. However, LS has been frequently co-isolated with LD from VL and post-kala-azar dermal leishmaniasis (PKDL) patients in India, often carrying a specific RNA virus, Leptomonas seymouri narna-like virus 1 (Lepsey NLV1).
The Knowledge Gap: It remains unresolved whether LS can survive and replicate within mammalian macrophages, how co-infection with LD affects the dynamics of both parasites and the associated virus, and whether this "triple-pathogen" interaction (LD-LS-Virus) influences disease pathogenesis. Previous studies suggested LS could not infect mammalian macrophages, but recent clinical correlations suggest otherwise.

2. Methodology

The study utilized in vitro infection models using both murine (RAW 264.7) and human (THP-1, PMA-differentiated) macrophages.

• Infection Models: Macrophages were infected with LD, LS, or co-cultures (LD:LS ratios of 2:1 and 10:1) at a multiplicity of infection (MOI) of 10:1.
• Parasite Quantification: Intracellular (IC) and extracellular (EC) parasite loads were quantified over 48–168 hours post-infection (h.p.i.) using quantitative ITS1 PCR and densitometric analysis.
• Viability and Revival Assays: To confirm active replication rather than persistence, infected macrophages were lysed, and the contents were transferred to Schneider's medium to observe the transformation of intracellular amastigotes back into motile promastigotes. Controls were run to ensure extracellular parasites could not survive in culture media at 37°C.
• Viral Dynamics: Lepsey NLV1 RNA loads were quantified via qRT-PCR. Purified Lepsey NLV1 (extracted via sonication/filtration) was tested for its ability to infect macrophages independently.
• Immunological Profiling: IL-12 (p40) levels in supernatants were measured via ELISA to assess host immune activation.
• Microscopy: Giemsa staining was used to visualize intracellular amastigote forms.

3. Key Contributions

• Reclassification of LS Infectivity: The study challenges the prevailing view that LS is strictly non-infective to mammals, demonstrating its capacity to infect, differentiate, and replicate within mammalian macrophages.
• Discovery of "Triple-Pathogen" Biology: The paper establishes a stable interaction between LD, LS, and Lepsey NLV1, suggesting a complex biological niche where the virus is maintained and amplified within the host cell.
• Mechanism of Viral Delivery: It proves that Lepsey NLV1 cannot infect macrophages independently; it requires LS as a carrier vector for cellular entry.
• Immunomodulation: The study identifies that co-infection dampens the host's pro-inflammatory response (specifically IL-12 production), potentially facilitating parasite persistence.

4. Key Results

• Intracellular Replication of LS:
  • LS successfully infected both RAW 264.7 and THP-1 macrophages.
  • Intracellular DNA levels of LS increased significantly (~7.6-fold in RAW 264.7 and ~4.3-fold in THP-1) over 168 hours, confirming active replication rather than mere persistence.
  • Revival assays confirmed that parasites harvested from macrophages transformed into motile promastigotes in Schneider's medium, whereas extracellular parasites died within 48 hours in culture media.
• Co-infection Dynamics:
  • In co-infection scenarios, LS replicated more efficiently than LD. Despite a higher initial inoculum of LD, the intracellular LD:LS ratio shifted dramatically over time (e.g., from 1:150 to 1:66 in RAW 264.7).
  • Co-infection led to higher extracellular LS loads, suggesting enhanced host cell lysis and release of LS compared to LD mono-infection.
• Viral Persistence and Amplification:
  • Lepsey NLV1 RNA accumulated predominantly intracellularly (10–100 fold higher than extracellular levels).
  • In THP-1 cells, viral loads increased significantly (~4-fold) over time in co-infection, whereas they remained static in RAW 264.7.
  • Crucially, the virus load per LS parasite was 2.5–23 fold higher in co-infection compared to LS mono-infection, indicating that LD presence enhances viral replication within LS.
  • Purified virus alone failed to enter macrophages, confirming LS is the essential delivery vehicle.
• Immune Modulation:
  • Co-infected macrophages produced significantly lower levels of IL-12 (p40) compared to LD mono-infected cells, suggesting a dampened Th1 immune response that may aid in parasite survival.
• Morphological Evidence: Giemsa staining confirmed the presence of amastigote-like forms of LS inside macrophages, though they were less distinct than LD amastigotes.

5. Significance

• Pathogenesis Implications: The findings suggest that LS is not a passive bystander but an active participant in VL/PKDL pathogenesis. Its ability to replicate in mammalian cells and carry a virus that may further suppress immunity (via IL-12/IL-18 downregulation) could explain treatment failures or chronicity in co-infected patients.
• Paradigm Shift: This study redefines the ecological niche of Leptomonas seymouri, moving it from a strictly insect-pathogen to a potential opportunistic mammalian pathogen, particularly in immunocompromised or co-infected states.
• Clinical Relevance: The "triple-pathogen" phenomenon (LD + LS + Lepsey NLV1) warrants investigation in clinical samples to determine if this co-infection correlates with severe disease outcomes, drug resistance, or relapse in VL and PKDL.
• Therapeutic Targets: Understanding the mechanism by which LD facilitates LS replication and viral amplification could reveal new targets for interrupting the transmission or persistence of these pathogens.

In conclusion, the paper provides robust molecular and morphological evidence that Leptomonas seymouri can infect and replicate in mammalian macrophages, especially when co-infected with Leishmania donovani, creating a unique environment that supports the amplification of its associated virus and dampens host immunity.