Fructose-bisphosphate aldolase and enolase from Echinococcus granulosus: Genes, expression patterns and protein interactions of two potential moonlighting proteins

Abstract

Glycolytic enzymes, such as fructose-bisphosphate aldolase (FBA) and enolase, have been described as complex multifunctional proteins that may perform non-glycolytic moonlighting functions, but little is known about such functions, especially in parasites. We have carried out in silico genomic searches in order to identify FBA and enolase coding sequences in Echinococcus granulosus, the causative agent of cystic hydatid disease. Four FBA genes and 3 enolase genes were found, and their sequences and exon-intron structures were characterized and compared to those of their orthologs in Echinococcus multilocularis, the causative agent of alveolar hydatid disease. To gather evidence of possible non-glycolytic functions, the expression profile of FBA and enolase isoforms detected in the E. granulosus pathogenic larval form (hydatid cyst) (EgFBA1 and EgEno1) was assessed. Using specific antibodies, EgFBA1 and EgEno1 were detected in protoscolex and germinal layer cells, as expected, but they were also found in the hydatid fluid, which contains parasite's excretory-secretory (ES) products. Besides, both proteins were found in protoscolex tegument and in vitro ES products, further suggesting possible non-glycolytic functions in the host-parasite interface. EgFBA1 modeled 3D structure predicted a F-actin binding site, and the ability of EgFBA1 to bind actin was confirmed experimentally, which was taken as an additional evidence of FBA multifunctionality in E. granulosus. Overall, our results represent the first experimental evidences of alternative functions performed by glycolytic enzymes in E. granulosus and provide relevant information for the understanding of their roles in host-parasite interplay.

The secreted protein signature of hydatid fluid from pulmonary cystic echinococcosis

“…Previous studies indicated that some carbohydrate-metabolizing enzymes exerted moonlighting effects along with their primary biochemical roles [40]. These molecules did not bear signal peptide, but significant amounts appeared to be secreted, probably through specific mechanisms, such as extracellular vesicles, to play a role extracellularly [37,41].…”

Systems biology approach reveals possible evolutionarily conserved moonlighting functions for enolase

“…4B), shared orthologs between networks were predominantly assigned to the cell (40%) and organelle (30%) categories, which is in line with the idea of enolase as a typical cytoplasmic protein. However, extracellular (1%) and membrane-enclosed lumen (11%) proteins were also identified, which may indicate ectopic enolase localization and interactions, already described for this protein and suggestive of moonlight (Lorenzatto et al, 2012;Mouveaux et al, 2014;Falabella et al, 2009). Regarding molecular functions (Fig.…”

“…Several studies have shown that enolase is a multifunctional protein (Georges et al, 2011;Lorenzatto et al, 2012;Piast et al, 2005;Ryu et al, 2012) and the use of enolase (and other proteins) for additional tasks in the cell seems to be recurrent. However, it is not so clear whether these functions were evolutionarily conserved.…”

Parasite molecules and host responses in cystic echinococcosis

“…However, this important aspect of Echinococcus biology has not been studied. In addition, as HCF (and also in vitro PSC secretions) is repeatedly found to contain certain parasite cytosolic proteins, nonconventional secretion mechanisms may be in operation . Some of these proteins are glycolytic enzymes, and hence, functions unrelated to their basic ones, related instead to influencing host processes, are proposed .…”

“…In addition, as HCF (and also in vitro PSC secretions) is repeatedly found to contain certain parasite cytosolic proteins, nonconventional secretion mechanisms may be in operation . Some of these proteins are glycolytic enzymes, and hence, functions unrelated to their basic ones, related instead to influencing host processes, are proposed . It would be desirable to compare quantitatively the protein compositions of HCF vs. GL and PSC, to gain an insight as to whether a subset of intracellular proteins is selectively released.…”