Deborah Caldeira Brandt Almeida¹, Ismael Pretto Sauter², Lina Borda-Samper¹, Patricio R. Orrego2³, Mauro Cortez Javier Veliz¹

INTRODUCTION Leishmania amazonensis manipulates the host immune response to survive and cause leishmaniasis. Amastigotes release extracellular vesicles (EVs), which play a role in inducing CD200 from infected macrophages—this ligand inhibits cellular activation and impacts the inducible nitric oxide synthase/nitric oxide (iNOS/NO) pathway. OBJECTIVE This study aimed to explore the contents of these structures and
to determine whether the release of amastigote-EVs is influenced by different pH conditions or calcium (Ca 2+ ) inhibitors. MATERIALS AND METHODS Leishmania amastigotes were incubated in axenic amastigote media (AA) for one hour to promote vesiculation. The supernatants were then filtered through a 0.45-µm sterile cell strainer, concentrated, and used for various assays, including proteomics analysis (nano LC-MS/MS). Amastigotes were also incubated in phosphate-buffered saline (PBS), RPMI, or AA, prepared at different pH levels. Bone marrow macrophages (BMM) stimulation assays were conducted to assess CD200 induction via immunoprecipitation/western blot (IP/WB), and subsequent analysis was performed using SDS-PAGE with silver staining and WB assays employing a polyclonal antibody against Leishmania EVs. Nanoparticle tracking analysis (NTA) was also utilized to verify the purity and quantity of EVs. RESULTS Proteomic analysis identified 25 proteins shared across replicates,
INTRODUCTION Leishmania amazonensis manipulates the host immune response to survive and cause leishmaniasis. Amastigotes release extracellular vesicles (EVs), which play a role in inducing CD200 from infected macrophages—this ligand inhibits cellular activation and impacts the inducible nitric oxide synthase/nitric oxide (iNOS/NO) pathway. OBJECTIVE This study aimed to explore the contents of these structures and to determine whether the release of amastigote-EVs is influenced by different pH conditions or calcium (Ca2+) inhibitors. MATERIALS AND METHODS Leishmania amastigotes were incubated in axenic amastigote media (AA) for one hour to promote vesiculation. The supernatants were then filtered through a 0.45-µm sterile cell strainer, concentrated, and used for various assays, including proteomics analysis (nano LC-MS/MS). Amastigotes were also incubated in phosphate-buffered saline (PBS), RPMI, or AA, prepared at different pH levels. Bone marrow macrophages (BMM) stimulation assays were conducted to assess CD200 induction via immunoprecipitation/western blot (IP/WB), and subsequent analysis was performed using SDS-PAGE with silver staining and WB assays employing a polyclonal antibody against Leishmania EVs. Nanoparticle tracking analysis (NTA) was also utilized to verify the purity and quantity of EVs. RESULTS Proteomic analysis identified 25 proteins shared across replicates, totaling 56 proteins in replicate 1 and 63 in replicate 2. The majority of these shared proteins were found to have catalytic activity (as determined by molecular function) and were localized in the nucleus (as assessed through cellular component analysis). The induction of CD200 was notably affected when amastigotes were incubated in culture media with a pH higher than 5.2. When incubated at different pH levels (4.5, 5.2, 7.0, and 8.0), a significant impairment of EV release was observed at lower pH (4.5), with more pronounced effects noted at higher pH levels (7.0 and 8.0). Regarding calcium\'s role, the quantity and size of EVs were influenced by the presence of calcium inhibitors, including EDTA, EGTA, and BAPTA-AM. Under these conditions, NTA analysis showed lower quantities of EVs, with the most significant reduction noted with EDTA. Furthermore, the EVs profile in the control group, which displayed a more concentrated range of EVs measuring between 100 to 200 nm, altered under different treatments, suggesting that Ca2+ plays a crucial role in the release of amastigote EVs. CONCLUSION Both pH and Ca2+ are essential for the release of amastigote EVs. This underscores the unique factors that must be taken into account when studying parasite EVs.

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