Leishmania donovani depletes labile iron pool to exploit iron uptake capacity of macrophage for its intracellular growth

Abstract

Intracellular pathogens employ several strategies for iron acquisition from host macrophages for survival and growth, whereas macrophage resists infection by actively sequestering iron. Here, we show that instead of allowing macrophage to sequester iron, protozoan parasite Leishmania donovani (LD) uses a novel strategy to manipulate iron uptake mechanisms of the host and utilizes the taken up iron for its intracellular growth. To do so, intracellular LD directly scavenges iron from labile iron pool of macrophages. Depleted labile iron pool activates iron sensors iron-regulatory proteins IRP1 and IRP2. IRPs then bind to iron-responsive elements present in the 3' UTR of iron uptake gene transferrin receptor 1 by a post-transcriptional mRNA stability mechanism. Increased iron-responsive element-IRP interaction and transferrin receptor 1 expressions in spleen-derived macrophages from LD-infected mice confirm that LD employs similar mechanism to acquire iron during infection into mammalian hosts. Increased intracellular LD growth by holo-transferrin supplementation and inhibited growth by iron chelator treatment confirm the significance of this modulated iron uptake pathway of host in favour of the parasite.

Fig. 1

TfR1 expression in macrophages by LD infection. A. J774A.1 cells were infected with freshly isolated virulent LD (1:10) or treated with DFO (100 μM) for 12 h and Western blot analyses were performed with TfR1 (upper panel) or α-actin (lower panel) antibody. Densitometric analysis was shown in the side panel. B. Similarly, Western analyses for TfR1 (upper panel) and α-actin (lower panel) were performed in cell lysates after 0, 8, 16 and 24 h of infection. Relative expressions were determined by densitometric analysis (side panel). C. J774A.1 cells were infected with laboratory-maintained non-virulent LD (nV-LD) and freshly isolated virulent LD (V-LD) for 12 h and Western blot analyses were performed with TfR1 (upper panel) or α-actin (lower panel) antibody. Densitometric analysis was shown in the side panel. D. Macrophages isolated from spleen of uninfected mice were infected with LD for 12 h (10 multiplicity of infection) and Western analyses were performed with TfR1 antibody (upper panel), α-actin (lower panel) and densitometric analysis was shown in side panel. Results are represented as the mean of three observations and normalized to control, arbitrarily chosen as one unit.

Fig. 2

Leishmania increases TfR1 synthesis in macrophages by modulating mRNA expression. A. Total RNA was isolated after 10 h of LD-infected J774A.1 cells (10 multiplicity of infection) and abundance of TfR1 mRNA was determined after hybridizing with ³²P-labelled mouse TfR1 cDNA (upper panel). The abundance of loaded RNA was shown by ultraviolet (lower panel). Relative expression was determined by densitometric analysis (side panel). B. Similarly, total RNA was isolated from Leishmania major (LM)-infected J774A.1 cells after 0, 8, 16 and 24 h and RT-PCR was performed with specific primers of mouse TfR1 and β-actin. DFO (100 μM) was used as positive control. Relative expression was determined by densitometric analysis (side panel). C. To determine the mRNA stability of TfR1, uninfected and LD-infected J774 cells were added with actinomycin D (5 μg ml⁻¹) after 6 h of infection. Total RNA was isolated at 0, 1, 2 and 3 h of actinomycin D addition and RT-PCR was performed with specific primers of mouse TfR1 and β-actin. D. The relative stability of TfR1 mRNA in LD-infected and uninfected cells was determined by densitometric analysis. Results are represented as the mean of three observations and normalized to control, arbitrarily chosen as one unit.

Fig. 3

A. RNA gel-shift analysis was performed with in vitro transcribed gel-purified ³²P-labelled IRE containing transcript with cytosolic extracts isolated from J774A.1 macrophages after 0, 2, 4 and 8 h of infection with LD (10 multiplicity of infection). DFO (100 μM)-treated cytosolic extract was used as a positive control. Only probe without the incubation of cytosolic extract was run in the last lane. B. To test the specificity of the IRP–IRE interaction radiolabelled probe was incubated with 30× molar excess of unlabelled purified probe (lanes 3 and 4) or cytosolic extracts were incubated with 2% β-ME prior to incubation with radiolabelled probe and then gel-shift analysis was performed. C. Either IRP1, IRP2 or HIF-1α antibody (2 μg) was incubated for 30 min with the cytosolic extracts isolated from LD-infected or DFO (100 μM)-treated J774A.1 cells before the addition of radiolabelled IRE probe and gel-shift analysis was performed. D. Cytosolic extracts were isolated from LD-infected or uninfected J774 macrophages after 12 h of infection or DFO (100 μM) treatment and aconitase assay was performed. E. Expressions of IRP1 and IRP2 by LD infection were detected by Western analysis of cytosolic extracts isolated from J774A.1 macrophages after 6 and 12 h of infection. Results are represented as the mean of three observations.

Fig. 4

Detection of labile iron pool in host macrophages by LD infection. A. J774A.1 macrophages were infected with either LD (V-LD), non-virulent LD (nV-LD) or incubated with DFO (100 μM) for 2 h and then incubated with calcein-AM (0.5 μM). After 20 min fluorescence level was detected using a microscope (upper panel). Phase contrast pictures are shown in the bottom panel. B. Specificity of detection of LIP was tested by addition of apo-Tf (10 μM) and holo-Tf (10 μM) after 4 h of infection with LD or DFO treatment. C. Cytosolic extracts were prepared from L-NAME (1 mM)-pretreated (1 h) LD-infected J774A.1 cells and gel-shift analysis was performed using radiolabelled IRE probe as described earlier. D. ⁵⁵Fe uptake in intracellular LD from host J774A.1 was determined. Cells were initially incubated with ⁵⁵Fe–Tf for 6 h, washed to remove excess of ⁵⁵Fe–Tf and then infected with LD. After 30 min of infection excess LD was removed and fresh media were added. Then intracellular parasites were isolated at 2, 4 and 6 h of infection from macrophages and ⁵⁵Fe was detected in isolated LD in a scintillation counter. Results are represented as the mean of three observations.

Fig. 5

Alteration of iron uptake mechanism in spleen-derived macrophages in LD-infected Balb/c mice. A. One set of Balb/c mice (n = 3) were infected with LD and another set were kept uninfected in the similar condition. After 6 weeks splenic macrophages were isolated from both the groups, cytosolic extracts were prepared and RNA gel shift analysis was performed. To check the specificity 30× molar excess of cold probes (lanes 4 and 5) as well as 2% β-ME (lanes 6 and 7) were added with radiolabelled IRE containing in vitro transcribed probe. B. Similarly, total RNA was isolated from splenic macrophages of LD-infected and uninfected mice and RT-PCR was performed with primers specific to TfR1 (upper panel) and β-actin (lower panel). Results are representative as one of three independent experiments.

Fig. 6

Increased iron uptake by LD-infected macrophages influences intracellular parasite growth. A. J774A.1 macrophages were infected for 12 h with virulent LD or kept uninfected, and then ⁵⁵Fe–Tf was added to both the groups. After 30 min the intracellular ⁵⁵Fe was estimated using cell lysate in a scintillation counter. DFO (100 μM)-treated cells were used as positive control. B. LD-infected J774A.1 cells were supplemented after 4 h of infection either with holo-Tf (10 μM), apo-Tf (10 μM) or none. Total intracellular LD was counted after 2, 12 and 24 h of Tf addition. P < 0.001, anova. C. J774A.1 cells were infected with LD and supplemented with Tf like (B), washed, fixed, permeabilized and immunofluoroscence was performed using mAb to LAMP1 after 12 h. D. J774A.1 macrophages were initially treated with DFO (100 μM) for 4 h or none, washed thoroughly to remove DFO and infected with LD. Intracellular LD was counted after 12 h of infection. Results are represented as the mean of three observations and normalized to control.