Research Summary: Drug unresponsive Leishmania donovani accumulates excess low-density lipoprotein (LDL) to fuel aggressive growth and form lipid droplets, which hinder antileishmanial Amphotericin B activity. Disrupting these droplets enhances drug efficacy against the parasite.
Author interview: Supratim Pradhan just completed his Ph.D. synopsis at the School of Medical Science and Technology (SMST), IIT Kharagpur and is currently awaiting the award of his degree.
Lab: Dr. Budhaditya Mukherjee, University: School of Medical Science and Technology, Indian Institute of Technology Kharagpur, India
What was the core problem you aimed to solve with this research?
Visceral leishmaniasis (VL), caused by the protozoan parasite Leishmania, remains a serious public health challenge, particularly in endemic regions. Although initially responsive to antimonial derivatives, an alarming rise in drug resistance first to antimonials in the late 1990s and now increasingly to newer treatments like Amphotericin B and Miltefosine has complicated disease management.
Our research aimed to address the fundamental question: What drives the survival and rapid intracellular growth of drug-unresponsive Leishmania parasites, particularly within liver-resident macrophages (Kupffer cells)?
Using a combination of in vitro and in vivo experimental approaches, we discovered that drug-unresponsive Leishmania donovani strains exhibit markedly accelerated proliferation within liver-resident macrophages, known as Kupffer cells, compared to their drug-sensitive counterparts. Interestingly, these parasites inhabit in membrane-bound compartments called parasitophorous vacuoles (PVs) during their intracellular lifecycle, structures that demand substantial lipid resources for maintenance and expansion.
What makes this particularly intriguing is the fact that Leishmania parasites are incapable of synthesizing cholesterol de-novo. This led us to hypothesize that their rapid growth may be driven by hijacking host-derived lipids. Supporting this hypothesis, we found that infecting macrophages under lipid-depleted conditions resulted in severely compromised intracellular replication of the drug-unresponsive strains. Conversely, enriching the macrophage environment with lipid sources significantly boosted their growth. These findings revealed a critical dependency of the drug-unresponsive parasites on host lipid acquisition to sustain their aggressive intracellular proliferation.
Further investigations pinpointed low-density lipoprotein (LDL) as the primary lipid source fuelling the rapid growth of drug-unresponsive Leishmania donovani parasites. Remarkably, we found that LDL uptake in infected macrophages occurred via cofilin-mediated fluid-phase endocytosis, effectively bypassing the classical LDL receptor (LDLr)-dependent pathway. This receptor-independent mechanism underscores the parasite’s ability to rewire host cellular processes to its advantage.
Adding clinical relevance to our findings, we observed that patients with relapsed visceral leishmaniasis consistently exhibited reduced LDL levels in their blood, strongly suggesting active LDL internalization by Leishmania-infected cells. Once internalized, LDL not only fulfilled the parasite’s metabolic requirements but also contributed to the formation of dense lipid droplet (LD) networks surrounding the intracellular amastigotes.
These lipid droplets played a dual role: not only acting as a metabolic reservoir to fuel the parasite’s rapid growth, but also physically trapping lipophilic antileishmanial drugs like Amphotericin B within the host cell. This sequestration reduces the drug’s bioavailability at the site of infection, effectively shielding the parasite from its action. This mechanism offers a compelling explanation for the clinical observation of cross-resistance in patients those unresponsive to traditional antimonial therapy were also increasingly failing to respond to Amphotericin B.
In essence, our study revealed a novel mechanism by which drug-resistant Leishmania parasites exploit host lipid metabolism for both survival and drug evasion highlighting host lipid pathways as potential therapeutic targets in the fight against drug-resistant leishmaniasis.
Drug-resistant Leishmania donovani (LD-R) reside within parasitophorous vacuoles (PVs) in host macrophages and trigger active remodeling of the host cell’s actin cytoskeleton via cofilin activation. This promotes fluid-phase endocytosis, allowing high uptake of low-density lipoprotein (LDL). The LDL-loaded vesicles then fuse with the parasite-containing PVs, supplying lipids that fuel rapid intracellular growth.
Within these fused compartments, the cholesterol export protein NPC-1 is downregulated, leading to cholesterol accumulation inside the PVs. Some of this cholesterol, possibly after processing in the endoplasmic reticulum (ER), contributes to the formation of lipid droplets. These lipid droplets accumulate around the parasite and form a barrier, which impairs the action of Amphotericin-B.
Additionally, LD-R parasites suppress inflammatory responses by downregulating MSR-1, a receptor responsible for recognizing oxidized LDL. They also block cholesterol biosynthesis by inhibiting the nuclear translocation of SREBP2, a key regulator of this pathway. Together, these mechanisms help LD-R survive, proliferate, and evade drug action within the host.
How did you go about solving this problem?
To address the challenge of Amphotericin B unresponsiveness in Leishmania infections, we began by hypothesizing that the accumulation of lipid droplets around intracellular parasites acts as a physical barrier trapping the drug and preventing it from reaching and binding to the parasite’s membrane.
To test this, we first cultured infected macrophages in a low-lipid medium. Under these conditions, we observed a significant reduction in lipid droplet formation, and critically Amphotericin B regained its ability to effectively kill the intracellular parasites. However, replicating such a lipid-deprived environment in human VL patients is not feasible due to the metabolic complexities associated with the disease.
Seeking a more practical solution, we turned to pharmacological intervention. We identified Aspirin a well-known cyclooxygenase (COX) inhibitor as a potential candidate to suppress lipid droplet formation. Remarkably, when aspirin was administered alongside Amphotericin B, the drug’s efficacy was restored.
Through this approach, we not only uncovered the mechanistic basis of Amphotericin B unresponsiveness, lipid droplet-mediated drug sequestration but also proposed a clinically translatable strategy to overcome it. By co-targeting the host lipid environment, we offer a promising therapeutic avenue to restore treatment efficacy in patients with drug-unresponsive visceral leishmaniasis.
Lipid buildup fuels Leishmania’s rapid growth and forms a barrier, shielding it from lipophilic drugs like Amphotericin B.
How would you explain your research outcomes (Key findings) to the non-scientific community?
Drug unresponsiveness in parasites like Leishmania, the cause of visceral leishmaniasis, is a growing concern, especially as more patients stop responding to life-saving medications like Amphotericin B. In our study, we investigated why some strains of Leishmania are no longer affected by these treatments.
We observed that these drug-resistant parasites grow much faster than drug-sensitive ones. But to fuel this rapid growth, they need large amounts of lipid, especially cholesterol. Interestingly, these parasites can’t make cholesterol on their own. Instead, they hijack the host’s resources by triggering the host cell to absorb lots of lipid molecule called LDL (low-density lipoprotein). The host cell then forms large lipid droplets around the parasite.
These lipid droplets serve a dual purpose: not only do they act as metabolic reservoirs, supplying the energy required for the parasite’s rapid intracellular growth, but more critically, they form a protective lipid shield that physically sequesters lipophilic drugs such as Amphotericin B, thereby hindering the drug’s access to the parasite.
We hypothesized that the prolonged use of lipophilic antileishmanial drugs like Amphotericin B may have inadvertently exerted selective pressure, favouring the emergence of parasite strains capable of excessive lipid accumulation within infected host cells. This adaptation, while enhancing survival, may also represent an evolutionary trade-off where the ability to harness and store host lipids confers both a metabolic advantage and a mechanism for drug evasion.
Here’s the breakthrough: we found that using aspirin, a common and inexpensive drug, significantly reduces the formation of these lipid droplets. When aspirin was combined with Amphotericin B, the drug was able to reach and kill the parasite effectively in resistant strains.
Our findings offer a promising new strategy: by using aspirin to disrupt the parasite’s protective barrier, we can restore the effectiveness of existing drugs and potentially improve treatment outcomes for patients with drug-resistant leishmaniasis.
What are the potential implications of your findings for the field and society?
Drug unresponsiveness to Amphotericin B is increasingly being reported across regions where visceral leishmaniasis is endemic including the Indian subcontinent. Until now, the underlying reasons for this treatment failure remained poorly understood.
Our research provides a critical breakthrough by uncovering the mechanism behind this unresponsiveness. We demonstrate how drug-resistant Leishmania parasites manipulate host cells to form protective lipid droplets that block Amphotericin B from reaching its target. This insight not only explains why current treatments may fail but also reveals a clear strategy to overcome this barrier.
We also propose a practical solution: combining Amphotericin B with aspirin a widely available and affordable drug can disrupt this protective lipid shield, restoring the drug’s effectiveness against resistant parasites.
Beyond Leishmania, our findings may have broader implications. The mechanism of lipid-based drug shielding could be relevant in other parasitic infections like Plasmodium (malaria) and Toxoplasma, as well as certain fungal and bacterial diseases. This opens new avenues for research and treatment strategies across a wide range of infectious diseases, offering hope for more effective therapies in the face of rising drug resistance.
What was the exciting moment during your research?
The entire journey of this project has been incredibly exciting, especially as a newly established lab working under tight timelines. But if I had to highlight one defining moment it was the realization that aspirin, a simple and widely used drug, could restore the efficacy of Amphotericin-B against drug-unresponsive Leishmania strains. Seeing the once-resistant parasites being cleared in aspirin-treated cells was truly exhilarating and felt like unlocking a critical piece of the puzzle.
Equally thrilling was the hands-on experience of using advanced microscopy techniques. Visualizing the dynamic cellular changes post-infection especially the formation of lipid droplets and their impact on drug accessibility brought the data to life in a way that was both scientifically meaningful and personally rewarding. Each image captured told a compelling story that pushed the research forward.
Paper reference: Supratim Pradhan, Dhruba Dhar, Debolina Manna, Shubhangi Chakraborty, Arkapriya Bhattacharyya, Khushi Chauhan, Rimi Mukherjee, Abhik Sen, Krishna Pandey, Soumen Das, Budhaditya Mukherjee (2025) Scrutinized lipid utilization disrupts Amphotericin-B responsiveness in clinical isolates of Leishmania donovani eLife 14:RP102857 https://doi.org/10.7554/eLife.102857.3
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