Gaia Mazza

Leishmaniasis is a Neglected Tropical Disease caused by protozoa of the Leishmania genus. No human vaccine is available, and current treatments are limited by toxicity and drug resistance. Thus, the identification of new active molecules remains an urgent priority. However, drug discovery is severely hampered by the lack of fast, robust, and high-throughput screening assays, particularly for the clinically relevant intracellular amastigote stage. Here, a transgenic Leishmania infantum line stably co-expressing the bioluminescent marker PpyRE9H fused to the fluorescent reporter tdTomato was generated and used to develop a novel dual-reporter microplate-based platform to identify compounds active against promastigotes or intracellular parasite stages. The engineered strain exhibited comparable growth and infectivity to the wild-type strain, validating its suitability for drug screening assays. The activity of reference drugs was evaluated, and results were compared to those obtained using standard methods, such as MTT assay for promastigotes and Giemsa staining with microscopic quantification for amastigotes. Comparable IC₅₀ values were obtained using fluorescent/bioluminescent assays and conventional methods. These proposed assays provide a sensitive and reproducible alternative to conventional methods. The microplate format enables higher throughput screening, while the dual bioluminescence and fluorescence readouts enable cross-validation of results and reduce the risk of compound-specific signal interference.

Granulomas are organized inflammatory lesions formed in response to persistent stimuli such as infections. Murine infection with Leishmania donovani results in granulomas in the liver, seeded by infected Kupffer cells, and serves as a well-defined model of infection-induced granuloma formation. The resolution of granulomatous inflammation requires dynamic shifts in immune-cell activation states, imposing metabolic demands. As mediators of cell signaling, lipid metabolism plays a key role in regulating inflammation and infection. How lipid changes are spatially linked to altered immune cell transcription remains unresolved. We performed a multimodal imaging analysis combining MALDI mass spectrometry, spatial and single cell transcriptomics, proteomics of flow-sorted macrophages and histopathology of L. donovani induced hepatic granulomas. Using this spatially-integrated approach, we identified LPCAT2-mediated membrane re-modelling of myeloid cells as a novel feature of these granulomas. Our study provides new insights into local immunometabolic changes associated with granuloma formation and macrophage activation.

Leishmania infantum is the etiological agent of visceral leishmaniasis (VL) and is linked to cases of cutaneous leishmaniasis in dogs. Dogs often develop severe systemic disease and serve as the primary reservoir of L. infantum. Although several vaccine candidates are under development, no vaccine for visceral leishmaniasis has been approved for human use to date. Chemotherapeutic treatment is hampered by toxicity, cost, and the emergence of parasite-resistant strains. Immunotherapy, combining chemotherapy with modulation of Th1 responses, is a promising therapeutic approach. Helicobacter pylori neutrophil-activating protein (HP-NAP), an immunomodulatory protein from Helicobacter pylori, is known to promote Th1 immune responses. A Th1 response activates macrophage promoting parasite killing, while a Th2 response favors disease progression. Macrophages are central for infection, either eliminating parasites (Th1 response) or supporting their persistence (Th2 response). IL-12 is a crucial cytokine in driving Th1 immunity and counteracting Th2 responses. We therefore investigated the role of HP-NAP in an in vitro model of L. infantum macrophage infection. Canine monocyte-derived macrophages from seven dogs were incubated with L. infantum promastigotes. More than 85% of macrophages from all donors were infected, with approximately seven amastigotes per cell. HP-NAP treatment significantly reduced all infection parameters and induced IL-12 production. Collectively, these findings suggest that HP-NAP may represent a promising candidate for adjuvant immunotherapies and vaccine development against L. infantum.