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

Abstract

Glucantime® (SbV) is the first-line treatment against leishmaniasis in South America. Its effectiveness has been associated with modulation of the parasite detoxification system that, in turn, is related to serine proteases such as subtilisins. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders - R) and relapse or therapeutic failure (Non-responders - NR) were used. The parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity – measured with z-FR-AMC as substrate and specific inhibitors – and expression of subtilisins and tryparedoxin-peroxidase (TXNPx). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. TLCK inhibited almost 100 % of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70 %, and PMSF showed lower inhibition of specific isolates. Principal component and clustering analysis yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, transcripts of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) were detected in promastigotes and axenic amastigotes from both groups. The data presented here show a phenotypic heterogeneity among the parasites, suggesting that exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization of L. (V.) braziliensis clinical isolates.

Impact of collaborative actions of Leishmania (Viannia) braziliensis subpopulations on the infection profile

“…The heterogeneous composition of parasite populations in Leishmania spp. can be illustrated by phenotypic variations in antimony susceptibility, as observed for L. (V.) braziliensis, Leishmania (V.) guyanensis and Leishmania (L.) amazonensis isolates (Moreira et al ., 1998; Zabala-Peñafiel et al ., 2021), and for Leishmania (L.) donovani isolates which were described as a mixture of subpopulations susceptible and resistant to sodium stibogluconate (Pal et al ., 2001; Bhattacharyya et al ., 2002). Another example for the role of population structure in the environmental adaptive success of the parasites is the study performed with L. (L.) major submitted to in vitro treatment with miltefosine, which indicated that resistance is related to population heterogeneity, with subpopulations that differ in susceptibility phenotypes (Coelho et al ., 2012).…”

First report of Leishmania RNA virus 1 in Leishmania (Viannia) braziliensis clinical isolates from Rio de Janeiro State - Brazil

“…In fact, the results presented here contribute to reinforce the heterogeneity previously seen for these clinical isolates. (31,32)…”