The role of G protein coupled receptor-mediated signaling in the biological properties of Acanthamoeba castellanii of the T4 genotype Yousuf Aqeel, Ruqaiyyah Siddiqui, Zainab Manan, Naveed Ahmed Khan * Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan article info Article history: Received 3 December 2014 Received in revised form 7 March 2015 Accepted 9 March 2015 Available online 11 March 2015 Keywords: Acanthamoeba castellanii G protein couple receptors b adrenergic receptor Viability Zymographic assays Cytotoxicity abstract Despite advances in antimicrobial chemotherapy and supportive care, the prognosis of Acanthamoeba infections remains poor, suggesting that new targets are needed that can affect parasite survival and host epathogen interactions. G proteins and their coupled receptors are well known regulators of a variety of cellular functions. The overall aim of the present study was to study the role of G-protein coupled re- ceptor, b adrenergic receptor on the biology and pathogenesis of keratitis isolate of Acanthamoeba cas- tellanii of the T4 genotype. Inhibition of b adrenergic receptor using antagonist, propranolol had detrimental effects on the extracellular proteolytic activities A. castellanii as determined using zymo- graphic assays. Conversely, b adrenergic receptor agonist, isoprenaline showed increased proteases. Interestingly, b adrenergic receptor inhibition affected A. castellanii growth (using amoebistatic assays), viability (using amoebicidal assays by measuring uptake of Trypan blue) and encystation as determined by trophozoite transformation into the cyst form. Pre-treatment of parasites with propranolol hampered A. castellanii-mediated human brain microvascular endothelial cell cytotoxicity, as measured by the lacatate dehydrogenase release. The aforementioned findings suggest that G-protein coupled receptor, b adrenergic receptor-mediated signaling in A. castellanii biology and pathogenesis may offer new phar- macological targets. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Free-living opportunistic eukaryotic pathogen, Acanthamoeba exists in two different life forms, i.e., a reproducible trophozoite stage and a dormant cyst stage. It is responsible for causing en- cephalitis and a sight-threatening infection of the cornea [1e4]. Despite our advances in antimicrobial chemotherapy and sup- portive care, Acanthamoeba infections have remained significant. For example, the recommended treatment against Acanthamoeba encephalitis is a combination of ketoconazole, fluconazole, sulfa- diazine, pentamidine isethionate, rifampicin, amphotericin B, yet the mortality rate is more than 90% [1e4]. For Acanthamoeba keratitis, a combination of chlorhexidine, polyhexamethylene biguannide, propamidine isethionate and neomycin is prescribed but infection recurrence is reported in approximately 10% of the cases [5,6]. Additional concern is Acanthamoeba resistance to antimicrobial chemotherapy, and many of the aforementioned compounds are reported to produce host cell cytotoxicity [7e9].A complete understanding of the cell biology and the molecular ar- chitecture of Acanthamoeba, its life cycle, and mechanisms of pathogenesis of Acanthamoeba infections will undoubtedly lead to the identification of potential targets of therapeutic value. In particular, new targets may be found in the signal transduction pathways that can affect amoeba survival and hostepathogen interactions. GTP-binding proteins (G-proteins) are trans-membrane proteins that can modulate and regulate signaling pathways. G-proteins can be broadly classified into two main classes, i.e., monomeric small GTPases and heterotrimeric G-protein complexes. Heterotrimeric G-proteins are composed of three distinct subunits (Ga,Gb,Gg) and are coupled to seven transmembrane domain, known as G-protein coupled receptors (GPCRs). In its normal inactive state, Ga is bound to GDP and this complex is bound with high affinity to the heter- odimer segment Gbg. Once the receptor (GPCR) is activated, GDP is released from Ga, allowing binding of GTP instead [10]. The binding of GTP with Ga causes conformational change in the entire Gabg complex. This leads to release of Gbg heterodimer segment that activate downstream effectors such as adenylyl cyclases, * Corresponding author. Department of Biological and Biomedical Sciences, Aga Khan University, Stadium Road, Karachi, 74800, Pakistan. E-mail address: naveed5438@gmail.com (N.A. Khan). Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath http://dx.doi.org/10.1016/j.micpath.2015.03.006 0882-4010/© 2015 Elsevier Ltd. All rights reserved. Microbial Pathogenesis 81 (2015) 22e27