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

Abstract

Glucantime (Sb^V) 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 Sb^III and Sb^V, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to Sb^III 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 Sb^III and serine proteases profiles can aid in the characterization of L. (V.) braziliensis clinical isolates.

Figure 1

Promastigotes and axenic amastigotes in vitro susceptibility profile to antimony. Both parasite forms of each isolate (n = 12), 4 × 10⁶ promastigotes/well and 5 × 10⁵ axenic amastigotes/well were exposed to serial dilutions of trivalent (Sb^III) and pentavalent (Sb^V) antimonial for 48 h and 24 h in 96-well plates, respectively. The half-maximal inhibitory concentration (IC₅₀ [mg/mL]) of parasites viability was measured using a fluorescence method with AlamarBlue reagent. Parasites isolated before treatment of patients with ATL cured after antimonial therapy (R: Responder, filled circle) or with poor clinical response to therapy, either therapeutic failure or relapse (NR: Non-responder, open circle). The data is presented by boxplot diagrams as the mean of three biological replicates for each isolate. Asterisks indicate statistically significant differences: *p = 0.04. The figure was generated using GraphPad Prism version 9.0.1.

Figure 2

Enzymatic residual activity of whole soluble proteins from the clinical isolates. In these assays, protein extract [5 µg] of promastigotes and axenic amastigotes were measured using a specific fluorogenic peptide substrate of serine proteinases, z-FR-AMC (1 mM). The enzymatic activities (mmol min⁻¹ mg of protein⁻¹) were assessed without inhibitor (w/i) and in the presence of inhibitors: E-64 [10 µM], PMSF [1 mM], AEBSF [1 mM] and TLCK [100 µM]. R: Responder (black bars); NR: Non-responder (white bars). The results are shown as mean and the standard deviation (±) of three independent experiments. Asterisks indicate statistically significant differences of enzymatic activities in the absence (w/i) and presence (E-64, PMSF, AEBSF, TLCK) of inhibitors for each isolate: *p = 0.01; **p = 0.005; ^#p ≤ 0.0005. The figure was generated using GraphPad Prism version 9.0.1.

Figure 3

Cluster analysis of L. (V.) braziliensis clinical isolates. Principal components analysis (PCA) was performed to group clinical isolates based on the normalized quantitative variables. Each point represents the first three principal components of a clinical isolate. The points are colored according to the cluster they belong to, C1 (blue filled circle): NR6 and R3; C2 (red filled circle): R1, R3, NR5 and NR7; C3 (green filled circle): NR3; C4 (violet filled circle): NR2, R2 and R4; C5 (yellow filled circle): NR1 and NR4. The figure was generated using R version 1.4.1106.

Figure 4

Differential gene expression of subtilisins and TXNPx of promastigotes and axenic amastigotes of L. (V.) braziliensis clinical isolates. Transcript levels were evaluated by qRT-PCR and the resulting relative quantification (fold-change) values of subtilisins (LbrM.13.0860, silver bars), (LbrM.28.2570, nickel bars) and TXNPx (LbrM.15.1080, iron bars) are presented. Actin and protein S8 were used as endogenous controls. The ∆∆Ct value of each gene was calculated pair-to-pair between promastigotes and axenic amastigotes, and the promastigote sample with lowest expression for each gene was set as reference sample. The dashed lines indicate RQ level of the reference samples: NR1 promastigote for subtilisins; NR2 promastigote for TXNPx. The graph presents the mean of two independent experiments performed in triplicate. *p < 0.05. The figure was generated using GraphPad Prism version 9.0.1.