Abstract
The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of L. donovani HSP78 (LdHSP78+/-) and Leishmania mexicana HSP78 (LmxHSP78+/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The LdHSP78+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that LdHSP78+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with LdHSP78+/- parasites, compared with WT parasites. Virulence of the LdHSP78+/- strain was restored by episomal addition of the LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P1,P5-di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents.
Highlights
The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis
Involvement of HSP78 in visceral leishmaniasis (VL) was initially perceived from semiquantitative PCR of Leishmania donovani heat shock protein 78 (LdHSP78) transcripts from L. donovani-infected bone marrow-derived macrophages (BMDMs) (Fig. 1A)
Almost 40–55-fold higher expression of LdHSP78 mRNA was obtained at pH 5.5 and 37 °C, compared with that obtained at pH 7.0 and 22 °C (Fig. 1C)
Summary
The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. We identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Using high-throughput virtual screening, we identified P1,P5-di(adenosine-59)-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. Present address for Anindyajit Banerjee: Tata Translation Cancer Research. For some pathogens like Leishmania donovani, which have a digenetic life cycle, inducible heat stress is a vital part of their biology They are transmitted from the poikilothermic sand fly gut to the homeothermic mammalian macrophage, which involves a shift in temperature of almost 10 °C. LdHSP1002/2 null mutants of L. donovani showed impaired amastigote development, and LdHSP100 is found to be involved in the transport of exosome-mediated virulence factors during infection [6]
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