GLYCOLYTIC ENZYMES AS VACCINES AGAINST SCHISTOSOMIASIS: TESTING SCHISTOSOMA MANSONI PHOSPHOGLYCERATE MUTASE IN MICE.

Schistosomiasis is a globally burdensome parasitic disease caused by flatworms (blood flukes) in the genus Schistosoma. The current standard treatment for schistosomiasis is the drug praziquantel, but there is an urgent need to advance novel interventions such as vaccines. Several glycolytic enzymes have been evaluated as vaccine targets for schistosomiasis, and data from these studies are reviewed here. Although these parasites are canonically considered to be intracellular, proteomic analysis has revealed that many schistosome glycolytic enzymes are additionally found at the host-interactive surface. We have recently found that the intravascular stage of Schistosoma mansoni (Sm) expresses the glycolytic enzyme phosphoglycerate mutase (PGM) on the tegumental surface. Live parasites display PGM activity, and suppression of PGM gene expression by RNA interference diminishes surface enzyme activity. Recombinant SmPGM (rSmPGM) can cleave its glycolytic substrate, 3-phosphoglycerate and can both bind to plasminogen and promote its conversion to an active form (plasmin) in vitro, suggesting a moonlighting role for this enzyme in regulating thrombosis in vivo. We found that antibodies in sera from chronically infected mice recognize rSmPGM. We also tested the protective efficacy of rSmPGM as a vaccine in the murine model. Although immunization generates high titers of anti-SmPGM antibodies (against both recombinant and native SmPGM), no significant differences in worm numbers were found between vaccinated and control animals.

Schistosoma mansoni phosphoglycerate mutase: a glycolytic ectoenzyme with thrombolytic potential.

Schistosomiasis is a debilitating parasitic disease caused by intravascular flatworms called schistosomes (blood flukes) that affects >200 million people worldwide. Proteomic analysis has revealed the surprising presence of classical glycolytic enzymes - typically cytosolic proteins - located on the extracellular surface of the parasite tegument (skin). Immunolocalization experiments show that phosphoglycerate mutase (PGM) is widely expressed in parasite tissues and is highly expressed in the tegument. We demonstrate that live Schistosoma mansoni parasites express enzymatically active PGM on their tegumental surface. Suppression of PGM using RNA interference (RNAi) diminishes S. mansoni PGM (SmPGM) gene expression, protein levels, and surface enzyme activity. Sequence comparisons place SmPGM in the cofactor (2,3-bisphosphoglycerate)-dependent PGM (dPGM) family. We have produced recombinant SmPGM (rSmPGM) in an enzymatically active form in Escherichia coli. The Michaelis-Menten constant (Km) of rSmPGM for its glycolytic substrate (3-phosphoglycerate) is 0.85 mM ± 0.02. rSmPGM activity is inhibited by the dPGM-specific inhibitor vanadate. Here, we show that rSmPGM not only binds to plasminogen but also promotes its conversion to an active form (plasmin) in vitro. This supports the hypothesis that host-interactive tegumental proteins (such as SmPGM), by enhancing plasmin formation, may help degrade blood clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo.

Title: La phosphoglycérate mutase de Schistosoma mansoni ­ une ectoenzyme glycolytique avec un potentiel thrombolytique. Abstract: La schistosomiase est une maladie parasitaire débilitante causée par des vers plats intravasculaires appelés schistosomes qui affecte plus de 200 millions de personnes dans le monde. L'analyse protéomique a révélé la présence surprenante d'enzymes glycolytiques classiques ­ typiquement des protéines cytosoliques ­ situées sur la surface extracellulaire du tégument du parasite. Des expériences d'immunolocalisation montrent que la phosphoglycérate mutase (PGM) est largement exprimée dans les tissus parasitaires et fortement exprimée dans le tégument. Nous démontrons que les parasites Schistosoma mansoni vivants expriment une PGM enzymatiquement active sur leur surface tégumentaire. La suppression de la PGM à l'aide de l'interférence ARN (ARNi) diminue l'expression du gène PGM de S. mansoni (SmPGM), les niveaux de protéines et l'activité enzymatique de surface. Les comparaisons de séquences placent la SmPGM dans la famille des PGM dépendantes du cofacteur (2,3-bisphosphoglycérate) (dPGM). Nous avons produit de la SmPGM recombinante (rSmPGM) sous une forme enzymatiquement active dans Escherichia coli. La constante de Michaelis-Menten (Km) de rSmPGM pour son substrat glycolytique (3-phosphoglycérate) est de 0,85 mM ± 0,02. L'activité de la rSmPGM est inhibée par le vanadate, un inhibiteur spécifique des dPGM. Ici, nous montrons que rSmPGM non seulement se lie au plasminogène mais favorise également sa conversion en une forme active (plasmine) in vitro. Cela soutient l'hypothèse selon laquelle les protéines tégumentaires interactives avec l'hôte (telles que SmPGM), en améliorant la formation de plasmine, peuvent aider à dégrader les caillots sanguins autour des vers dans le microenvironnement vasculaire et ainsi favoriser la survie du parasite in vivo.

Multifunctional Fructose 1,6-Bisphosphate Aldolase as a Therapeutic Target.

Fructose 1,6-bisphosphate aldolase is a ubiquitous cytosolic enzyme that catalyzes the fourth step of glycolysis. Aldolases are classified into three groups: Class-I, Class-IA, and Class-II; all classes share similar structural features but low amino acid identity. Apart from their conserved role in carbohydrate metabolism, aldolases have been reported to perform numerous non-enzymatic functions. Here we review the myriad "moonlighting" functions of this classical enzyme, many of which are centered on its ability to bind to an array of partner proteins that impact cellular scaffolding, signaling, transcription, and motility. In addition to the cytosolic location, aldolase has been found the extracellular surface of several pathogenic bacteria, fungi, protozoans, and metazoans. In the extracellular space, the enzyme has been reported to perform virulence-enhancing moonlighting functions e.g., plasminogen binding, host cell adhesion, and immunomodulation. Aldolase's importance has made it both a drug target and vaccine candidate. In this review, we note the several inhibitors that have been synthesized with high specificity for the aldolases of pathogens and cancer cells and have been shown to inhibit classical enzyme activity and moonlighting functions. We also review the many trials in which recombinant aldolases have been used as vaccine targets against a wide variety of pathogenic organisms including bacteria, fungi, and metazoan parasites. Most of such trials generated significant protection from challenge infection, correlated with antigen-specific cellular and humoral immune responses. We argue that refinement of aldolase antigen preparations and expansion of immunization trials should be encouraged to promote the advancement of promising, protective aldolase vaccines.

Schistosoma mansoni glyceraldehyde-3-phosphate dehydrogenase enhances formation of the blood-clot lysis protein plasmin.

Schistosomes are intravascular blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic analysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNA interference significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to ∼8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in Escherichiacoli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite's hosts, and has a Michaelis-Menten constant for glyceraldehyde-3-phosphate (GAP) of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thrombolysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade blood clots around the worms in the vascular microenvironment and thus promote parasite survival in vivoThis article has an associated First Person interview with the first author of the paper.

Fluorescent vinblastine probes for live cell imaging.

Herein we describe the synthesis of several fluorescent analogues of the clinically approved microtubule destabilizing agent vinblastine. The evaluated probes are the most potent described and provides the first example of uptake, distribution and live cell imaging using this well known antimitotic agent.