Metabolism of FAD, FMN and riboflavin (vitamin B2) in the human parasitic blood fluke Schistosoma mansoni.

Schistosomiasis is a parasitic disease caused by trematode worms of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. In this work we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni. We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while the levels of FMN increase. We show that live schistosomes can cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2 in the extracellular environment. Since schistosomes can be damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case, suggesting that covalently bound FAD on IL-4I1 is inaccessible to SmNPP5. We also report here that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM. Thus, the sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by, we hypothesize, the recently described schistosome riboflavin transporter SmaRT. In this work we also identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.

The riboflavin (vitamin B2) transporter protein (SmaRT) of the human intravascular parasitic trematode Schistosoma mansoni.

Schistosomes are intravascular parasitic worms infecting >200 million people globally. Here we examine how the worms acquire an essential nutrient - vitamin B2 (riboflavin). We demonstrate that all intravascular life stages (schistosomula, adult males and females) take up radiolabeled riboflavin. This process is impeded in the presence of excess unlabeled riboflavin and at 4 °C. We have identified a transporter homolog in worms designated SmaRT (Schistosoma mansoni riboflavin transporter) that localizes to the tegument and internal tissues of adults. CHO-S cells transfected with plasmid encoding SmaRT import significantly more radiolabeled riboflavin compared to controls. Uptake of radiolabel is impeded when SmaRT-expressing cells are incubated in an excess of unlabeled riboflavin but not by an excess of an irrelevant metabolite. Uptake is mediated in a sodium-independent manner and over a wide range of pH values (pH 5.5-9). This is the first identification of a bone fide riboflavin transporter in any platyhelminth.

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.

A novel, non-neuronal acetylcholinesterase of schistosome parasites is essential for definitive host infection.

Schistosomes are long-lived parasitic worms that infect >200 million people globally. The intravascular life stages are known to display acetylcholinesterase (AChE) activity internally as well as, somewhat surprisingly, on external tegumental membranes. Originally it was hypothesized that a single gene (SmAChE1 in Schistosoma mansoni) encoded both forms of the enzyme. Here, we demonstrate that a second gene, designated "S. mansoni tegumental acetylcholinesterase, SmTAChE", is responsible for surface, non-neuronal AChE activity. The SmTAChE protein is GPI-anchored and contains all essential amino acids necessary for function. AChE surface activity is significantly diminished following SmTAChE gene suppression using RNAi, but not following SmAChE1 gene suppression. Suppressing SmTAChE significantly impairs the ability of parasites to establish infection in mice, showing that SmTAChE performs an essential function for the worms in vivo. Living S. haematobium and S. japonicum parasites also display strong surface AChE activity, and we have cloned SmTAChE homologs from these two species. This work helps to clarify longstanding confusion regarding schistosome AChEs and paves the way for novel therapeutics for schistosomiasis.

Schistosoma mansoni and the purinergic halo.

Intravascular schistosomes may control immune and hemostatic responses by regulating the nature and amount of selected host purinergic signaling molecules - such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and nicotinamide adenine dinucleotide (NAD) - surrounding them. Such metabolites are collectively known as the worm's 'purinergic halo'. Host-interactive, membrane-bound, tegumental ectonucleotidases, notably SmATPDase1, SmNPP5, SmAP and SmNACE, can degrade proinflammatory, prothrombotic and immunomodulatory purinergic metabolites like those listed. A common catabolic product is the anti-inflammatory metabolite adenosine that can additionally be taken in by the worms as food. We envision the tegumental ectonucleotidases as having a twofold role at the worm surface: first, they degrade potentially harmful host signaling molecules, and second, they generate vital nutrients around the worms from where these can be conveniently imported.

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.

Schistosome secretomes.

Schistosomes are intravascular parasitic platyhelminths (blood flukes) that infect over 200 million people globally. Biomolecules secreted by the worms likely contribute to their ability to survive in the bloodstreams of immunocompetent hosts for many years. Here we review what is known about the protein composition of material released by the worms. Prominent among cercarial excretions/secretions (ES) is a ∼ 30 kDa serine protease called cercarial elastase (SmCE in Schistosoma mansoni), likely important in host invasion. Also prominent is a 117 amino acid non-glycosylated polypeptide (Sm16) that can impact several host cell-types to impinge on immunological outcomes. Similarly, components of the egg secretome (notably the 134 amino acid homodimeric glycoprotein "IL-4 inducing principle of schistosome eggs", IPSE, and the 225-amino acid monomeric T2 ribonuclease - omega-1) are capable of driving Th2-biased immune responses. A ∼36kDa chemokine binding glycoprotein SmCKBP, secreted by eggs, can negate the impact of several cytokines and can impede neutrophil migration. Of special interest is a disparate collection of classically cytosolic proteins that are surprisingly often identified in schistosome ES across life stages. These proteins, perhaps released as components of extracellular vesicles (EVs), include glycolytic enzymes, redox proteins, proteases and protease inhibitors, heat shock proteins, proteins involved in translation/turnover, histones, and others. Some such proteins may display "moonlighting" functions and, for example, impede blood clot formation around the worms. More prosaically, since several are particularly abundant soluble proteins, their appearance in the ES fraction may be indicative of worm damage ex vivo leading to protein leakage. Some bioactive schistosome ES proteins are in development as novel therapeutics against autoimmune, inflammatory, and other, non-parasitic, diseases.

NAD-catabolizing ectoenzymes of Schistosoma mansoni.

Infection with schistosomes (blood flukes) can result in the debilitating disease schistosomiasis. These parasites survive in their host for many years, and we hypothesize that proteins on their tegumental surface, interacting with the host microenvironment, facilitate longevity. One such ectoenzyme - the nucleotide pyrophosphatase/phosphodiesterase SmNPP5 can cleave ADP (to prevent platelet aggregation) and NAD (likely preventing Treg apoptosis). A second tegumental ectoenzyme, the glycohydrolase SmNACE, also catabolizes NAD. Here, we undertake a comparative biochemical characterization of these parasite ectoenzymes. Both are GPI-linked and exhibit different optimal pH ranges. While SmNPP5 requires divalent cations, SmNACE does not. The KM values of the two enzymes for NAD at physiological pH differ: SmNPP5, KM = 340 µM ± 44; SmNACE, KM = 49 µM ± 4. NAD cleavage by each enzyme yields different products. SmNPP5 cleaves NAD to form nicotinamide mononucleotide (NMN) and AMP, whereas SmNACE cleaves NAD to generate nicotinamide (NAM) and adenosine diphosphate ribose (ADPR). Each enzyme can process the other's reaction product. Thus, SmNACE cleaves NMN (to yield NAM and ribose phosphate) and SmNPP5 cleaves ADPR (yielding AMP and ribose phosphate). Metabolomic analysis of plasma containing adult worms supports the idea that these cleavage pathways are active in vivo. We hypothesize that a primary function of SmNPP5 is to cleave NAD to control host immune cell function and a primary function of SmNACE is to cleave NMN to generate the vital nutrient nicotinamide (vitamin B3) for convenient uptake by the worms. Chemical inhibition of one or both ectoenzymes could upset worm metabolism and control schistosome infection.

Schistosomes Impede ATP-Induced T Cell Apoptosis In Vitro: The Role of Ectoenzyme SmNPP5.

Schistosomes (blood flukes) can survive in the bloodstream of their hosts for many years. We hypothesize that proteins on their host-interactive surface impinge on host biochemistry to help ensure their long-term survival. Here, we focus on a surface ectoenzyme of Schistosoma mansoni, designated SmNPP5. This ~53 kDa glycoprotein is a nucleotide pyrophosphatase/phosphodiesterase that has been previously shown to: (1) cleave adenosine diphosphate (ADP) and block platelet aggregation; and (2) cleave nicotinamide adenine dinucleotide (NAD) and block NAD-induced T cell apoptosis in vitro. T cell apoptosis can additionally be driven by extracellular adenosine triphosphate (ATP). In this work, we show that adult S. mansoni parasites can inhibit this process. Further, we demonstrate that recombinant SmNPP5 alone can both cleave ATP and impede ATP-induced T cell killing. As immunomodulatory regulatory T cells (Tregs) are especially prone to the induction of these apoptotic pathways, we hypothesize that the schistosome cleavage of both NAD and ATP promotes Treg survival and this helps to create a less immunologically hostile environment for the worms in vivo.

Schistosome immunomodulators.

Schistosomes are long lived, intravascular parasitic platyhelminths that infect >200 million people globally. The molecular mechanisms used by these blood flukes to dampen host immune responses are described in this review. Adult worms express a collection of host-interactive tegumental ectoenzymes that can cleave host signaling molecules such as the "alarmin" ATP (cleaved by SmATPDase1), the platelet activator ADP (SmATPDase1, SmNPP5), and can convert AMP into the anti-inflammatory mediator adenosine (SmAP). SmAP can additionally cleave the lipid immunomodulator sphingosine-1-phosphate and the proinflammatory anionic polymer, polyP. In addition, the worms release a barrage of proteins (e.g., SmCB1, SjHSP70, cyclophilin A) that can impinge on immune cell function. Parasite eggs also release their own immunoregulatory proteins (e.g., IPSE/α1, omega1, SmCKBP) as do invasive cercariae (e.g., Sm16, Sj16). Some schistosome glycans (e.g., LNFPIII, LNnT) and lipids (e.g., Lyso-PS, LPC), produced by several life stages, likewise affect immune cell responses. The parasites not only produce eicosanoids (e.g., PGE2, PGD2-that can be anti-inflammatory) but can also induce host cells to release these metabolites. Finally, the worms release extracellular vesicles (EVs) containing microRNAs, and these too have been shown to skew host cell metabolism. Thus, schistosomes employ an array of biomolecules-protein, lipid, glycan, nucleic acid, and more, to bend host biochemistry to their liking. Many of the listed molecules have been individually shown capable of inducing aspects of the polarized Th2 response seen following infection (with the generation of regulatory T cells (Tregs), regulatory B cells (Bregs) and anti-inflammatory, alternatively activated (M2) macrophages). Precisely how host cells integrate the impact of these myriad parasite products following natural infection is not known. Several of the schistosome immunomodulators described here are in development as novel therapeutics against autoimmune, inflammatory, and other, nonparasitic, diseases.

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.

Structural Insights into Schistosoma mansoni Carbonic Anhydrase (SmCA) Inhibition by Selenoureido-Substituted Benzenesulfonamides.

Tegumental carbonic anhydrase from the worm Schistosoma mansoni (SmCA) is considered a new anti-parasitic target because suppressing its expression interferes with schistosome metabolism and virulence. Here, we present the inhibition profiles of selenoureido compounds on recombinant SmCA and resolution of the first X-ray crystal structures of SmCA in adduct with a selection of such inhibitors. The key molecular features of such compounds in adduct with SmCA were obtained and compared to the human isoform hCA II, in order to understand the main structural factors responsible for enzymatic affinity and selectivity. Compounds that more specifically inhibited the schistosome versus human enzymes were identified. The results expand current knowledge in the field and pave the way for the development of more potent antiparasitic agents in the near future.

The essential schistosome tegumental ectoenzyme SmNPP5 can block NAD-induced T cell apoptosis.

Infection with intravascular platyhelminths of the genus Schistosoma can result in the debilitating disease schistosomiasis. Schistosomes (blood flukes) can survive in the host for many years. We hypothesize that proteins on their host-interactive surface modify the worm's external environment to help insure worm survival. Previously, we have shown that a surface ectoenzyme of Schistosoma mansoni, SmNPP5 - a nucleotide pyrophosphatase/phosphodiesterase - can cleave ADP and block platelet aggregation in vitro. In this work, we show that both adult schistosomes and recombinant SmNPP5 can cleave the exogenous purinergic signaling molecule nicotinamide adenine dinucleotide (NAD). In doing so, worms and rSmNPP5 can prevent NAD-induced apoptosis of T cells in vitro. Since regulatory T cells (Tregs) are especially prone to such NAD-induced cell death (NICD), we hypothesize that schistosome cleavage of NAD promotes Treg survival which creates a more immunologically hospitable environment for the worms in vivo. In addition to SmNPP5, schistosomes express another host-interactive NAD-degrading enzyme, SmNACE. We successfully suppressed the expression of SmNPP5 and SmNACE (singly or together) using RNAi. Only SmNPP5-suppressed worms, and not SmNACE-suppressed worms, were significantly impaired in their ability to cleave exogenous NAD compared to controls. Therefore, we contend that ectoenzyme SmNPP5 on the surface of the worm is primarily responsible for extracellular NAD cleavage and that this helps modulate the host immune environment by preventing Treg cell death.

Schistosome migration in the definitive host.

Schistosomes are parasitic blood flukes that infect >200 million people around the world. Free-swimming larval stages penetrate the skin, invade a blood vessel, and migrate through the heart and lungs to the vasculature of the liver, where maturation and mating occurs. From here, the parasite couples migrate to their preferred egg laying sites. Here, we compare and contrast what is known about the migration patterns within the definitive host of the three major species of human schistosome: Schistosoma mansoni, S. japonicum, and S. haematobium. We conclude that intravascular schistosomes are inexorable colonizers whose migration and egg laying strategy is profligate; all three species (and their eggs) can be found throughout the mesenteric venules, the rectal venous plexus, and, to a greater or lesser extent, the urogenital venous plexuses. In addition, it is common for parasite eggs to be deposited in locations that lack easy access to the exterior, further demonstrating the relentless exploratory nature of these intravascular worms.

Sulfonamide Inhibition Studies of an α-Carbonic Anhydrase from Schistosoma mansoni, a Platyhelminth Parasite Responsible for Schistosomiasis.

Schistosomiasis is a debilitating infection provoked by parasitic flatworms called schistosomes. The species Schistosoma mansoni is endemic in Africa, where it causes intestinal schistosomiasis. Recently, an α-carbonic anhydrase (CA, EC 4.2.1.1) was cloned and characterized from this organism and designated as SmCA. The protein is expressed in the tegument (skin) of S. mansoni at the host-parasite interface. Recombinant SmCA possesses high catalytic activity in the CO2 hydration reaction, similar to that of human CA isoform II with a kcat of 1.2 × 106 s-1 and a kcat/KM of 1.3 × 108 M-1·s-1. It has been found that schistosomes whose SmCA gene is suppressed using RNA interference are unable to establish a robust infection in mice, suggesting that the chemicals that inhibit SmCA function should have the same debilitating effect on the parasites. In this study, a collection of aromatic/heterocyclic sulfonamides were investigated as possible SmCA inhibitors. Several sulfonamides inhibited SmCA with medium to weak potency (KI values of 737.2 nM-9.25 µM), whereas some heterocyclic compounds inhibited the enzyme with KI values in the range of 124-325 nM. The α-CA from S. mansoni, SmCA, is proposed as a new anti-schistosomiasis drug target.

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.

Vitamin B6 Acquisition and Metabolism in Schistosoma mansoni.

Schistosomes are parasitic platyhelminths that currently infect >200 million people globally. The adult worms can live within the vasculature of their hosts for many years where they acquire all nutrients necessary for their survival and growth. In this work we focus on how Schistosoma mansoni parasites acquire and metabolize vitamin B6, whose active form is pyridoxal phosphate (PLP). We show here that live intravascular stage parasites (schistosomula and adult males and females) can cleave exogenous PLP to liberate pyridoxal. Of the three characterized nucleotide-metabolizing ectoenzymes expressed at the schistosome surface (SmAP, SmNPP5, and SmATPDase1), only SmAP hydrolyzes PLP. Heat-inactivated recombinant SmAP can no longer cleave PLP. Further, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to cleave PLP compared to controls. When schistosomes are incubated in murine plasma, they alter its metabolomic profile-the levels of both pyridoxal and phosphate increase over time, a finding consistent with the action of host-exposed SmAP acting on PLP. We hypothesize that SmAP-mediated dephosphorylation of PLP generates a pool of pyridoxal around the worms that can be conveniently taken in by the parasites to participate in essential, vitamin B6-driven metabolism. In addition, since host PLP-dependent enzymes play active roles in inflammatory processes, parasite-mediated cleavage of this metabolite may serve to limit parasite-damaging inflammation. In this work we also identified schistosome homologs of enzymes that are involved in intracellular vitamin B6 metabolism. These are pyridoxal kinase (SmPK) as well as pyridoxal phosphate phosphatase (SmPLP-Ph) and pyridox(am)ine 5'-phosphate oxidase (SmPNPO) and cDNAs encoding these three enzymes were cloned and sequenced. The three genes encoding these enzymes all display high relative expression in schistosomula and adult worms suggestive of robust vitamin B6 metabolism in the intravascular life stages.

Why Do Intravascular Schistosomes Coat Themselves in Glycolytic Enzymes?

Schistosomes are intravascular parasitic helminths (blood flukes) that infect more than 200 million people globally. Proteomic analysis of the tegument (skin) of these worms has revealed the surprising presence of glycolytic enzymes on the parasite's external surface. Immunolocalization data as well as enzyme activity displayed by live worms confirm that functional glycolytic enzymes are indeed expressed at the host-parasite interface. Since these enzymes are traditionally considered to function intracellularly to drive glycolysis, in an extracellular location they are hypothesized to engage in novel "moonlighting" functions such as immune modulation and blood clot dissolution that promote parasite survival. For instance, several glycolytic enzymes can interact with plasminogen and promote its activation to the thrombolytic plasmin; some can inhibit complement function; some induce B cell proliferation or macrophage apoptosis. Several pathogenic bacteria and protists also express glycolytic enzymes externally, suggesting that moonlighting functions of extracellular glycolytic enzymes can contribute broadly to pathogen virulence. Also see the video abstract here https://youtu.be/njtWZ2y3k_I.

Crystal structure and chemical inhibition of essential schistosome host-interactive virulence factor carbonic anhydrase SmCA.

The intravascular parasitic worm Schistosoma mansoni is a causative agent of schistosomiasis, a disease of great global public health significance. Here we identify an α-carbonic anhydrase (SmCA) that is expressed at the schistosome surface as determined by activity assays and immunofluorescence/immunogold localization. Suppressing SmCA expression by RNAi significantly impairs the ability of larval parasites to infect mice, validating SmCA as a rational drug target. Purified, recombinant SmCA possesses extremely rapid CO2 hydration kinetics (kcat: 1.2 × 106 s-1; kcat/Km: 1.3 × 108 M-1s-1). The enzyme's crystal structure was determined at 1.75 Å resolution and a collection of sulfonamides and anions were tested for their ability to impede rSmCA action. Several compounds (phenylarsonic acid, phenylbaronic acid, sulfamide) exhibited favorable Kis for SmCA versus two human isoforms. Such selective rSmCA inhibitors could form the basis of urgently needed new drugs that block essential schistosome metabolism, blunt parasite virulence and debilitate these important global pathogens.

Schistosomes can hydrolyze proinflammatory and prothrombotic polyphosphate (polyP) via tegumental alkaline phosphatase, SmAP.

Schistosoma mansoni is a long-lived intravascular trematode parasite that can infect humans causing the chronic debilitating disease, schistosomiasis. We hypothesize that the action of host-interactive proteins found at the schistosome surface allows the worms to maintain a safe, anti-thrombotic and anti-inflammatory environment around them in the bloodstream. One such protein is the ˜60 kDa alkaline phosphatase SmAP which is known to be expressed in the outer tegument of all intravascular life stages. We demonstrate in this work that the parasites (schistosomula as well as adult males and females) can hydrolyze polyphosphate (polyP) - an anionic, linear polymer of inorganic phosphates that is produced and released by immune cells as well as by activated platelets and that induce proinflammatory and prothrombotic pathways. Purified recombinant SmAP can likewise cleave polyP and with a Km of 6.9 ±â€¯1 mM. Finally, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to hydrolyze polyP. SmAP-mediated cleavage of polyP may contribute to the armamentarium of schistosomes that promotes their survival in the hostile intravascular habitat. This is the first report of any pathogen cleaving this bioactive metabolite.