Novel cholinesterase paralogs of Schistosoma mansoni have perceived roles in cholinergic signalling and drug detoxification and are essential for parasite survival

Abstract

Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite neurotransmission but has been poorly defined with respect to the molecules responsible. Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-containing genes (Smche)s, which we have shown to encode two functional acetylcholinesterases (AChE)s (Smache1 –smp_154600 and Smache2 –smp_136690) and a butyrylcholinesterase (BChE) (Smbche1 –smp_125350). Antibodies to recombinant forms of each SmChE localized the proteins to the tegument of adults and schistosomula and developmental expression profiling differed among the three molecules, suggestive of functions extending beyond traditional cholinergic signaling. For the first time in schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and SmBChE1. Parasite survival in vitro and in vivo was significantly impaired by silencing of each smche, either individually or in combination, attesting to the essential roles of these molecules. 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-schistosome AChE inhibitor 2,2- dichlorovinyl dimethyl phosphate—dichlorvos (DDVP), whereas smbche1-silenced parasites displayed increased sensitivity to DDVP.

Vaccination withSchistosoma mansonicholinesterases reduces parasite burden and egg viability in a mouse model of schistosomiasis

“…Surface-exposed proteins and secreted proteins are effective targets for vaccine development in schistosomes due to their capacity for interaction with host antibodies [17,18]. In this regard, SmChEs are promising candidates as we have shown in previous immunolocalization studies in S. mansoni that SmAChE1, SmBChE1 and SmAChE2 are expressed in the tegument of adult worms and schistosomula and proteomic analysis of S. mansoni ES products has confirmed the presence of SmAChE1 and SmBChE1 [6]. Further, RNAi-mediated silencing of all 3 SmChE genes, both individually and in combination, significantly decreased schistosomula viability in vitro and parasite survival in vivo [6], implying that these genes are essential for proper worm development and function.…”

“…A recent study by us [6] has documented the existence of three SmChE paralogs (two acetylcholinesterases -smache1 and smache2 -and one butyrylcholinesterase -smbche1) and we showed that each molecule localized to the tegument of adults and schistosomula and demonstrated, through RNAi-mediated suppression in vitro and in vivo, that each paralog was essential to parasite survival. We also reported a significant reduction in the glucose-scavenging ability of silenced parasites, providing evidence for the involvement of tegumental AChE in the mediation of exogenous glucose uptake, which has also been documented by other studies [10][11][12].…”

“…In this regard, SmChEs are promising candidates as we have shown in previous immunolocalization studies in S. mansoni that SmAChE1, SmBChE1 and SmAChE2 are expressed in the tegument of adult worms and schistosomula and proteomic analysis of S. mansoni ES products has confirmed the presence of SmAChE1 and SmBChE1 [6]. Further, RNAi-mediated silencing of all 3 SmChE genes, both individually and in combination, significantly decreased schistosomula viability in vitro and parasite survival in vivo [6], implying that these genes are essential for proper worm development and function. Moreover, recent protein array studies have demonstrated high levels of circulating antibodies to SmBChE1 in individuals exhibiting drug-induced resistance and a low pathology reaction to schistosomiasis, implicating these antibodies in a protective anti-schistosomal response [8,9].…”

“…Complete ORFs for smache1, smbche1 and smache2 were synthesized by Genewiz. Attempts to express full-length sequences in E. coli were unsuccessful, so primer sets incorporating NdeI (forward primer) and XhoI restriction enzyme sites (reverse primer) were designed to amplify partial, non-conserved regions of each smche [6], which might prove more amenable to expression. Sequences (containing NdeI/ XhoI sites) for each SmChE were amplified from each full-length template by PCR and cloned into the pET41a expression vector (Novagen) such that the N-terminal GST tag was removed.…”

“…Due to the fundamental roles they play in parasite biology (reviewed in [4], schistosome cholinesterases (SmChEs) have been posited as intervention targets against schistosomiasis and there are several indications to support the feasibility of their use as vaccines. Firstly, SmChEs have been localized to the tegument of schistosomula and adult worms [5,6] and anti-SmChE antibodies have been shown to bind to and kill schistosomula [7], suggesting that the enzymes are accessible to immune attack. Anti-SmChE antibodies also showed no cross-reactivity against human AChE [5], indicating that a vaccine safe for human use could be designed.…”