Gabriel Quintanilha dos Santos¹, Rebeca Santana Souza¹, Lara Auymi Dias Nakamura¹, Fátima Ribeiro Dias², Marina Tiemi Shio¹

Introduction: Cutaneous leishmaniasis is the most common clinical manifestation of leishmaniasis in Brazil. This disease is caused by species of the genus Leishmania, including L. (Leishmania) amazonensis, L. (Viannia) braziliensis, L. (Viannia) guyanensis, and, less frequently, L. (Viannia) naiffi. Transmission occurs through the bite of infected phlebotomine sandflies, in which the parasite differentiates into the metacyclic promastigote form—the infective stage for the mammalian host. Once inside the host, these promastigotes are phagocytosed and subsequently transform into amastigotes. The promastigote form possesses several virulence factors on its surface membrane, including ectophosphatases. These enzymes, by dephosphorylating a range of substrates, facilitate parasite internalization and intracellular survival. Although L. naiffi was first described in 1989, relatively little is known about its biology and pathogenic significance compared to other Leishmania species.

Objective: The present study aims to characterize the ectophosphatase activity of Leishmania naiffi isolates and its impact on metacyclogenesis and macrophage infection.

Materials and Methods: Two isolates (JM and RM) of L. naiffi and L. braziliensis (used as a control) were employed. Parasites were cultured at 28 °C in medium 199 supplemented with 10% FBS, antibiotics, 2% male urine, and 10 µg/mL hemin. RAW 264.7 macrophages were maintained in complete RPMI with 10% or 5% FBS at 37 °C. Ectophosphatase activity was assessed using the p-nitrophenyl phosphate (pNPP) assay, and parasite viability was evaluated by resazurin reduction. Metacyclogenesis was determined by morphological counting.

Results: Both isolates displayed similar ectophosphatase activity, which was significantly higher at pH 4.5 and 6.5 during the log phase (day 4) compared to the stationary phase, indicating prominent acid ectophosphatase activity. To assess its role, parasites on day 0 of culture were treated with 1–100 µM ammonium molybdate or 0.001–0.1 µM BpV(phen). At 100 µM, molybdate decreased L. naiffi JM viability after 7 days, whereas BpV(phen) showed greater parasite tolerance. Both inhibitors reduced ectophosphatase activity in a dose-dependent manner, with molybdate being more effective. Based on these findings, 10 µM molybdate and 0.1 µM BpV(phen) were selected for infection assays. Following 7 days of treatment, parasites were used to infect RAW 264.7 macrophages. Molybdate did not alter L. braziliensis infection or amastigote numbers, but L. naiffi JM showed a trend toward reduced infection at 48 h. BpV(phen) treatment of L. braziliensis decreased infection rates and amastigote numbers at both 2 h and 48 h. For L. naiffi, BpV(phen) reduced both the percentage of infected cells and amastigote counts at 48 h. Neither inhibitor affected metacyclogenesis.

Conclusion: Acid ectophosphatase activity is enhanced during the log phase but does not appear to influence metacyclogenesis under the tested conditions. However, these enzymes may positively modulate macrophage infection, particularly in L. naiffi, through mechanisms independent of metacyclic form generation.

Agradecimentos: FAPESP aid nº 2022/04395-9