Proteomic analysis of membrane proteins involved in Leishmania ‐ macrophage interaction

Abstract

IntroductionVisceral leishmaniasis (VL) is a potentially fatal infection caused by Leishmania donovani, treatment of which relies primarily on chemotherapy. Emergence of parasites resistant towards current antileishmanial agents and increasing number of relapses are major concerns. Membrane proteins play a crucial role during internalization of the parasite into host cell, therefore understanding of disease pathogenesis at host‐parasite interface becomes crucial. The aim of this study was to identify membrane proteins of host and parasite origin during interaction of L. donovani and macrophage.MethodologyL. donovani promastigotes and THP‐1 macrophages were lysed by sonication and membrane proteins isolated by ultracentrifugation. Integrity and purity of membrane fraction was verified by Western blotting using Membrane Fraction WB Cocktail ab. Cy5 labeled parasite membrane proteins were incubated with macrophages, while, Cy5 labeled membrane protein isolated from macrophages were interacted with L. donovani parasites. Membrane proteins involved in host‐parasite interaction were isolated and resolved by two‐dimensional gel electrophoresis (2‐DE) with minimal streaking. Twenty major spots from parasite and 10 spots from macrophage membrane fraction, primarily in the pH range of 4–7, were observed by scanning 2‐DE gel using Typhoon trio scanner. Consistently expressed and well resolved spots (n=4 from parasite and n=2 from macrophages) were excised, trypsin digested and analyzed by mass spectrometry (MALDI‐TOF/MS) for identification of interacting proteins. The macrophage‐parasite interaction was evaluated in presence or absence of Withaferin A (an inhibitor of activated C kinase) in two different macrophage cell lines (THP‐1 and RAW 264.7).ResultsActivated C kinase, peroxidoxin, small myristoylated protein 1 (SMP‐1) and cytochrome C oxidase from parasite while FILIP‐1 and beta actin from macrophages were identified to be involved in host‐parasite interaction. The role of activated protein C kinase in parasite replication within host macrophages was investigated using its inhibitor Withaferin A (WA). In the presence of 0.1 μM WA, the infectivity of L. donovani parasites to both THP‐1 macrophages as well as RAW 264.7 macrophages was reduced significantly (p<0.01), indicating a significant role of activated C kinase protein in parasite interaction with macrophages.ConclusionIn this study we have identified certain membrane proteins of macrophage and L. donovani origin, involved in the host parasite interaction. Further, we established the role of activated protein C kinase in parasite replication within host macrophages following inhibitor based assay. The proteins identified here may be explored as targets for chemotherapeutic interventions.Support or Funding InformationFunding by Indian Council of Medical Research (ICMR), India, is gratefully acknowledged. PS is a JC Bose Fellow under DST‐SERB.Withaferin A inhibits proliferation of L. donovani in host macrophages. Amastigote levels in THP‐1 (A) and RAW 264.7 (B) macrophage upon infection with L. donovani AG83 and K133 parasites in the presence or absence of Withaferin A (WA), an inhibitor of Activated C kinase protein. Error bars show standard deviation. Asterisks show level of significance (* p<0.05; ** p<0.001).Figure 1