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An immunostimulatory glycolipid molecule from the intestinal protozoan parasite <i>Entamoeba histolytica</i> (<i>Eh</i>) and its synthetic analogs derived from its phosphatidylinositol-b-anchor (<i>Eh</i>PIb) previously showed considerable immunotherapeutic effects against <i>Leishmania major</i> infection in vitro and in vivo. Here, we describe a high content screening assay, based on primary murine macrophages. Parasites detection is based on a 90 kDA heat shock protein-specific staining, enabling the detection of several <i>Leishmania</i> species. We validated the assay using <i>L. major</i>, <i>L. braziliensis</i>, <i>L. donovani,</i> and <i>L. infantum</i> as well as investigated the anti-leishmanial activity of six immunostimulatory <i>Eh</i>PIb-compounds (Eh-1 to Eh-6). Macrophages infected with dermotropic species were more sensitive towards treatment with the compounds as their viability showed a stronger reduction compared to macrophages infected with viscerotropic species. Most compounds caused a significant reduction of the infection rates and the parasite burdens depending on the infecting species. Only compound Eh-6 was found to have activity against all <i>Leishmania</i> species. Considering the challenges in anti-leishmanial drug discovery, we developed a multi-species screening assay capable of utilizing non-recombinant parasite strains, and demonstrated its usefulness by screening macrophage-targeting <i>Eh</i>PIb-compounds showing their potential for the treatment of cutaneous and visceral leishmaniasis.