Mannosyltransferase (GPI-14) overexpression protects promastigote and amastigote forms of Leishmania braziliensis against trivalent antimony

Abstract

Background: Glycosylphosphatidylinositol is a surface molecule important for host-parasite interactions. Mannosyltransferase (GPI-14) is an essential enzyme for adding mannose on the glycosylphosphatidyl group. This study attempted to overexpress the GPI-14 gene in Leishmania braziliensis to investigate its role in the antimony-resistance phenotype of this parasite.

Results: GPI-14 mRNA levels determined by quantitative real-time PCR (qRT-PCR) showed an increased expression in clones transfected with GPI-14 compared to its respective wild-type line. In order to investigate the expression profile of the surface carbohydrates of these clones, the intensity of the fluorescence emitted by the parasites after concanavalin-A (a lectin that binds to the terminal regions of α-D-mannosyl and α-D-glucosyl residues) treatment was analyzed. The results showed that the clones transfected with GPI-14 express 2.8-fold more mannose and glucose residues than those of the wild-type parental line, indicating effective GPI-14 overexpression. Antimony susceptibility tests using promastigotes showed that clones overexpressing the GPI-14 enzyme are 2.4- and 10.5-fold more resistant to potassium antimonyl tartrate (Sb^III) than the parental non-transfected line. Infection analysis using THP-1 macrophages showed that amastigotes from both GPI-14 overexpressing clones were 3-fold more resistant to Sb^III than the wild-type line.

Conclusions: Our results suggest the involvement of the GPI-14 enzyme in the Sb^III-resistance phenotype of L. braziliensis.

Keywords: Antimony resistance; Concanavalin-A; Glycosylphosphatidylinositol; Leishmania braziliensis; Mannosyltransferase.

Fig. 1

Levels of transcription of the GPI-14 gene (a) and concanavalin-A (Con-A) lectin binding profile (b-d) in the wild type and GPI-14 overexpressor L. braziliensis lines. a Levels of GPI-14 mRNA as determined quantitatively (relative to the DNA polymerase Leishmania gene) by real-time RT-PCR. Transcript levels ratio (GPI-14/DNA polymerase gene) ± standard deviations are indicated from three independent experiments. b Flow cytometric analysis of the differential expression of surface carbohydrate in wild-type (LbWT) and GPI14C10 clone (LbGPI14C10) of L. braziliensis using concanavalin-A (Con-A) lectin conjugated to fluorescein isothiocyanate (FITC). c The GPI-14-overexpressor clones were compared to the wild-type L. braziliensis line by the amount of α-D-mannosyl and α-D-glucosyl on its surface using concanavalin-A FITC agglutination profile and a significant difference is seen in the mean intensity of fluorescence (gMIF) in both clones. d The percentage (%) of Con-A FITC labeled parasites was used as a control parameter of efficiency. Data obtained in duplicates from at least three independent experiments were analyzed by Student’s t-test using GraphPad Prism 5.0 software. Statistically significant differences (P < 0.001) between wild type and GPI-14 overexpressor parasites are showed by asterisks (***). Pairwise comparisons: a LbWT vs LbGPI14C4 (t₍₃₎ = 21.16, P = 0.0002); LbWT vs LbGPI14C10 (t₍₄₎ = 9.130, P = 0.0008); c LbWT vs LbGPI14C4 (t₍₃₎ = 25.03, P = 0.0001); LbWT vs LbGPI14C10 (t₍₃₎ = 23.92, P = 0.0002)

Fig. 2

Sb^III susceptibility assays on the promastigotes and intracellular amastigotes of wild type and GPI-14-overexpressor L. braziliensis clones. a Promastigotes were cultured in the absence or presence of increasing Sb^III concentrations (1.2 to 74.9 μM) for 48 h and the percentage of relative growth was determined using a Z1 coulter counter. b, c Sb^III susceptibility was evaluated on amastigotes using THP-1-derived human macrophages. THP-1 macrophages infected with L. braziliensis lines were cultured in the absence or presence of increasing Sb^III concentrations (12.5 to 200 μM) for 72 h and the percentage of infected macrophages (b) and number of parasites per 100 macrophages (c) was determined. The EC₅₀ (μM) values were calculated for promastigotes (d) and amastigotes (e) of LbWT, LbGPI14C4 and LbGPI14C10 and the fold change in the resistance index of overexpressor clones relative to LbWT was determined. Statistical analysis of the curves was analyzed by Student’s t-test. ‘*’ represents a statistical difference between the wild-type lines and each overexpressor clone (*P < 0.05, **P < 0.01 and ***P < 0.001; see Tables 1 and 2 for details on pairwise comparisons)