Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates

Area of Science:

• Parasitology
• Biochemistry
• Molecular Biology

Background:

• Leishmania amazonensis is a protozoan parasite responsible for leishmaniasis.
• Understanding parasite peptidases is crucial for drug development.
• Leishmania amastigotes possess uncharacterized peptidase activities essential for survival.

Purpose of the Study:

• To investigate and characterize peptidase activity in live Leishmania amazonensis amastigotes.
• To determine the types of peptidases present and their substrate specificities.
• To explore the influence of environmental conditions on parasite peptidase function.

Main Methods:

• Confocal microscopy was used to visualize peptidase activity in live amastigotes using peptidyl-MCA substrates.
• Peptidase inhibitors (E64, TLCK) and specific substrates (Z-FR-MCA, Z-RR-MCA) were employed to identify enzyme classes.
• Peptidase activity in amastigote and promastigote lysates was analyzed across a pH range (4.5-9.0), with varying temperatures and salt concentrations.
• FRET peptide hydrolysis was used to identify oligopeptidase B activity.

Main Results:

• Cysteine and serine peptidases were detected in Leishmania amazonensis amastigotes.
• Parasites selectively incorporated substrates and inhibitors with positive charges.
• Optimal activity for cysteine peptidases was observed under various environmental conditions.
• Serine peptidase activity was confirmed, and oligopeptidase B was identified in the parasite.

Conclusions:

• Leishmania amazonensis amastigotes possess distinct cysteine and serine peptidase activities.
• The parasite exhibits substrate selectivity based on charge, suggesting specific transport mechanisms.
• Identified peptidases and their characteristics provide potential targets for anti-Leishmania drug development.