Serine proteases profiles of Leishmania (Viannia) braziliensis clinical isolates with distinct susceptibilities to antimony

Abstract

Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders—R) and relapse or therapeutic failure (Non-responders—NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, differential expression of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) was evaluated in promastigotes and axenic amastigotes from both groups. The results showed a higher expression of LbrM.13.0860 and LbrM.15.1080 genes in axenic amastigotes, while LbrM.28.2570 gene had the lowest expression in all isolates, regardless of the parasite form. 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.

Novel Insights Into Leishmania (Viannia) braziliensis In Vitro Fitness Guided by Temperature Changes Along With Its Subtilisins and Oligopeptidase B

“…In this context, it has been described that L. (V.) braziliensis clinical isolates have different gene expression profiles throughout their life cycle ( Adaui et al., 2011a ). This was also previously reported in the context of L. (V.) braziliensis clinical isolates heterogeneity based on higher expression of S13 gene in axenic amastigotes, and lowest expression of S28 gene in both promastigote and axenic amastigote forms ( Zabala-Peñafiel et al., 2021 ). Moreover, advancing on the exploratory search and understanding of these heterogeneous phenotypes, based on serine proteases profiles, we describe an increased S13 and OPB expression in intracellular amastigotes of L. ( V. ) braziliensis clinical isolates, while S28 was not significantly expressed here and in our previous publication ( Zabala-Peñafiel et al., 2021 ).…”

“…Altogether, the weakness behind understanding this diverse scenario is the lack of factors that better characterize the phenotype variations within the same species. In the search for these factors, our group separated different phenotypes based on in vitro Sb III susceptibility and serine proteases activity ( Zabala-Peñafiel et al., 2021 ). In the present study, we continue in the search for possible discriminatory elements focusing on serine proteases, subtilisins and oligopeptidase B from L. (V.) braziliensis , and propose a relationship between them and the parasites’ in vitro fitness.…”

“…Gene expression was expressed as fold change (2 -ΔΔCt ), in relation to the sample with the lowest expression for each evaluated gene ( Ennes-Vidal et al, 2019 ). For subtilisins, the reference sample was previously determined ( Zabala-Peñafiel et al., 2021 ) and used for this study; meanwhile, for OPB, all samples (promastigotes and intracellular amastigotes cDNA) were run in order to set the reference sample and analyze differential expression.…”

“…The set of primers used in this study were designed based on the L. (V.) braziliensis Subtilisin 13 (S13 - LbrM.13.0860), Subtilisin 28 (S28 - LbrM.28.2570), Oligopeptidase B (OPB - LbrM.9.0850), Actin housekeeping (LbrM.04.1250) and 40S Ribosomal protein S8 housekeeping gene (LbrM.24.2160) sequences recorded in the GeneDB database ( ). Subtilisins as well as Actin and 40S Ribosomal protein S8 housekeeping genes were previously designed and analyzed for L. (V.) braziliensis ( Zabala-Peñafiel et al., 2021 ). In this study, the primers for OPB were designed using the online software Primer3 v.0.4.0 ( ), with all parameters set to default except the product size range, which was adjusted to 80–200 base pairs (bp), and the sequence of this amplicon resulted in sequences compatible with the expected target sequence ( Supplementary Table 2 ).…”

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 …”