Serine Proteases Profiles of Leishmania (Viannia) Braziliensis Clinical Isolates With Distinct Susceptibilities to Antimony

Abstract

Background: Glucantime® (SbV) is considered the first-line treatment against American Tegumentary Leishmaniasis in South America, though increased parasite resistance towards it have been reported and hampered its effectiveness. In this context, subtilisins serine proteases have been related to parasites’ susceptibility to drugs such as SbV and derivatives, through modulation of the parasite detoxification system. However, little is known about parasites causing ATL and their distinct responses towards this treatment.Methods: The study was conducted using Leishmania (Viannia) braziliensis clinical isolates from patients that presented clinical cure or Responders (R) and relapse/therapeutic failure or Non-responders (NR). Twelve clinical isolates were used to assess their in vitro susceptibility to SbIII and SbV, serine proteases activity, and expression of subtilisins-like and tryparedoxin-peroxidase (TXNPx) transcripts. Results: SbIII was able to better distinguish axenic amastigotes from each clinical group. These isolates were also assessed for serine protease activity, using z-FR-AMC as substrate and detecting distinct enzyme profiles with specific inhibitors. TLCK inhibited almost 100% of activity in both promastigotes and axenic amastigotes while AEBSF inhibited around 70%. PMSF showed low inhibition of specific isolates (35%). Gathering all the quantitative data, we performed principal component analysis and then used the K-means algorithm to cluster the isolates. This analysis yielded one cluster with only one isolate (R isolate), one homogeneous cluster (NR isolates), and three heterogeneous clusters (R and NR isolates). Additionally, gene transcripts of subtilisins and TXNPx were detected in promastigotes and axenic amastigotes from both groups. Conclusions: Cluster analysis showed that there is a phenotypic heterogeneity among the isolates, however, exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization and better understanding of L. (V.) braziliensis clinical isolates.