Targeting schistosome cholinesterases for vaccine and drug development

Abstract

The nervous system of schistosomes has been successfully targeted by anthelmintic drugs but the use of many of these has discontinued because of toxic side effects and so there is a need to better understand key neuronal processes at a molecular level to develop safer and more effective intervention strategies that target this vital system. Cholinesterases - acetylcholinesterases (AChE)s and butyrylcholinesterases (BChE)s - are key enzymes that play a pivotal role in the nervous system of schistosomes by regulating neurotransmission through acetylcholine hydrolysis and, accordingly, are an example of such an intervention target. The first results chapter (chapter two) of this thesis investigated the anti-schistosome efficacy of polypyridylruthenium (II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb₁₂-tri and Rubb₇-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb₁₂-tri) and 10 mg/kg (Rubb₇-tnl). Mice treated with Rubb₁₂-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb₁₂-tri - 68%, Rubb₇-tnl - 56%) and were significantly morphologically altered (Rubb₁₂-tri - 62% abnormal, Rubb₇-tnl - 35% abnormal). I hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway. Chapter three of this thesis provided the first comprehensive molecular characterization of three S. mansoni cholinesterases (SmChEs), designated as SmAChE1, SmBChE1 and SmAChE3, which were identified from the interrogation of the now wholly annotated S. mansoni genome. Anti-SmChE antibodies localized the proteins to the tegument and neuromusculature of adults and schistosomula and developmental expression profiling differed among the molecules, suggestive of functions extending beyond traditional cholinergic signaling for each of them. I also reported the presence of ChE activity in parasite ES products for the first time and proteomically identified the molecules responsible (SmAChE1 and SmBChE1). Functional recombinant versions of the three SmChEs were produced in Pichia pastoris and enzyme nomenclature (AChE or BChE) was verified based on substrate preference. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosomal AChE inhibitor dichlorvos whereas SmBChE1-silenced parasites displayed increased sensitivity to dichlorvos. SmChEs were further characterized by RNAi-based experiments in chapter four of this thesis. RNAi-mediated silencing of individual SmChEs, or simultaneous silencing of all three SmChEs, significantly suppressed transcript and protein expression levels and AChE activity in parasites. In a dissection of the hypothesis that tegumental AChE mediates exogenous glucose scavenging by the parasite, I showed that RNAi-mediated knockdown of SmAChE1 and SmAChE3, but not SmBChE1, significantly reduced glucose uptake by schistosomes. Parasite survivability in vitro and in vivo was significantly impaired with the silencing of SmChEs, either individually or in combination, attesting to the essentiality of these molecules. Chapter five of this thesis explored the vaccine potential of SmChEs. When treated in vitro with anti-SmChE IgG, parasites displayed significantly decreased ChE activity, which eventually resulted in death. Vaccination with individual SmChEs, or a combination of all three SmChEs, significantly reduced worm burdens (28% - 38%, averaged across two independent trials) compared to controls. Liver egg burdens were significantly decreased for all mice across both trials (13% - 46%) except those vaccinated with SmAChE1 in trial 1. Egg viability, as determined by egg hatching from liver homogenates, was significantly reduced in the groups vaccinated with the SmChE cocktail (40%) and SmAChE3 (46%). Surviving worms from each vaccinated group were significantly stunted and depleted of glycogen stores, compared to controls. In conclusion, this thesis has identified the burgeoning potential of a new class of antischistosome drugs that, at least in part, target the nervous system of the parasite and provided a comprehensive characterization of a family of ChEs from S. mansoni, giving compelling evidence for the essentiality of the proteins and their utility as intervention targets against schistosomiasis.