%0 Journal Article
%T Restoration of IFN¦ÃR Subunit Assembly, IFN¦Ã Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol
%A Subha Sen
%A Koushik Roy
%A Sandip Mukherjee
%A Rupkatha Mukhopadhyay
%A Syamal Roy
%J PLOS Pathogens
%D 2011
%I Public Library of Science (PLoS)
%R 10.1371/journal.ppat.1002229
%X Despite the presence of significant levels of systemic Interferon gamma (IFN¦Ã), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFN¦Ã signaling in Leishmania donovani (LD) infected macrophages (LD-M£¿s); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFN¦Ã signaling in parasitized murine macrophages. Our study clearly shows that in LD-M£¿s IFN¦Ã receptor (IFN¦ÃR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-M£¿s as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-M£¿s are more fluid than normal M£¿s due to quenching of membrane cholesterol. The decreased rigidity in LD-M£¿s was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal M£¿s. IFN¦ÃR subunit 1 (IFN¦ÃR1) and subunit 2 (IFN¦ÃR2) colocalize in raft upon IFN¦Ã stimulation of normal M£¿s, but this was absent in LD-M£¿s. Oddly enough, such association of IFN¦ÃR1 and IFN¦ÃR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFN¦Ã allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-M£¿s, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1¨C5-Y-X1¨C5-(R/K)] in the transmembrane domain of IFN¦ÃR1 was also noted. The interaction of peptides representing this motif of IFN¦ÃR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27¡Á10£¿9 M versus 2.69¡Á10£¿7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFN¦ÃR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFN¦ÃR1 and IFN¦ÃR2 assembly and subsequent ligand driven signaling by quenching M£¿ membrane cholesterol.
%U http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002229