Identification and functional characterization of Leishmania donovani secretory peroxidase: delineating its role in NRAMP1 regulation

Abstract

Leishmania silently evades host immune system and establish in the hostile environment of host macrophage phagolysosomes. For differentiation, growth and division parasite acquires divalent cations especially iron from the host nutritive pool. Natural resistance associated with macrophage protein1 (NRAMP1), a cation transporter that effluxes out divalent cations specifically iron from phagosomal milieu to the cytosol, to create ions deprived status for pathogenic microorganisms. The mechanisms of NRAMP1 regulation are largely unknown in leishmanial infections. In the present study, we identified a secretory Leishmania donovani peroxidase (Prx) that showed peroxidoxin like peroxidase activity and significantly reduced H(2)O(2), O(2).(-) and NO levels in LPS activated macrophages. Further, we also observed down regulated Nramp1 expression and concomitantly declined labile iron pool in activated macrophages treated with identified peroxidase. Prx also decreased levels of TNF-α, IFN-γ and IL-12 in LPS activated macrophages. These observations indicate a bifunctional protective role of secretory Prx; first it reduces redox activation of macrophages, and secondly it allows iron access to Leishmania by down regulating NRAMP1 expression.

Figure 1. A silver stained gel showing various secretory proteins.

Lane M: molecular weight markers, A: Leishmanial secretory proteins (40 µg/well) on 12% SDS-PAGE ranging from 10–170 kDa, B: Secretory proteins on native gel, C: Identified peroxidase (25 kDa) on native gel showing peroxidase activity, D: Purified 25 kDa protein (20 µg) corresponding to peroxidase on 12% SDS-PAGE (CBB stained gel).

Figure 2. Complete sequence and Clustal W alignment of identified peroxidase.

CTP: cytosolic tryparedoxin peroxidase, P1: peroxidoxin1, SP: identified secretory peroxidase (Prx).

Figure 3. Production of O₂ ⁻, H₂O₂, NOx and phosphatase activity in various experimental conditions (B, LPS+B, LPS+PrxB).

The O₂ ⁻, H₂O₂ levels and phosphatase activities were estimated after 2 h and NOx was estimated after 24 h in their respective experimental conditions. LPS (100 ng/ml) was used as positive control and control beads have no Prx coating. A significant decrease in the levels of O₂ ⁻, H₂O₂, NOx was observed in peroxidase treated cells however, total phosphatase and PTPase activities were similar in both groups (student t-test was applied to get significance level between LPS+B and LPS+PrxB).

Figure 4. The mRNA expression levels of various proteins involved in macrophage oxidative burst in various experimental groups (B: only beads, LPS+B (beads), LPS+PrxB (peroxidase coated beads).

After 24 h of corresponding experimental conditions, total RNA was isolated and amplified using proteins mRNA specific primers (panel A). The relative mRNA expression is represented in the ratio of densitometric values of mRNAs to β-actin. The mRNA levels of all proteins except Cu-Zn Sod were significantly down regulated after Prx treatment (level of significance signifies comparison between LPS+B and LPS+PrxB; NS = not significant).

Figure 5. Western blot analysis of iNOS and NRAMP1 in peritoneal macrophages of various experimental groups.

Cell lysates were subjected to SDS-PAGE followed by blotting with proteins specific antibodies. Significant decrease in their levels was observed in LPS+PrxB as compared to LPS+B. The upper panel (A) depicts blots showing proteins expression. The lower panel (B) shows densitometric analysis of same with b-actin as control (level of significance is in between LPS+B and LPS+PrxB).

Figure 6. The western blot of signaling proteins involved in LPS induced activation of macrophages.

Cell lysates were subjected to SDS-PAGE followed by blotting with proteins specific antibodies. A: LPS induced maximum p38MAPK expression and phosphorylation. The peroxidase significantly reduced p38MAPK expression (p<0.035) and phosphorylation status (p<0.019) in macrophages. B: ERK1/2 expression and phosphorylation was almost similar (p = NS) in both LPS induced and peroxidase treated macrophages (level of significance is in between LPS+B and LPS+PrxB).

Figure 7. Levels of TNF-α, IFN-γ and IL-12 in cultured supernatant of peritoneal macrophages in various experimental conditions.

After 24 h, cell supernatants were collected and cytokines were estimated by cytokine ELISA and values were expressed in pg/ml. The cytokines level was decreased in the peroxidase treated group (LPS+PrxB) as compared to LPS induced cells (LPS+B). The lower picture depicts their relative mRNA expression levels after subsequent amplification with mRNA specific primers (ANOVA was done to obtain significance level between LPS+B and LPS+PrxB).

Figure 8. Total labile iron pool of activated cells presented as the difference of fluorescence intensity (ΔF in AU) in stimulated macrophages.

The secretory peroxidase decreased LIP status in LPS stimulated cells (comparison between LPS+B and LPS+PrxB).