Why Do Intravascular Schistosomes Coat Themselves in Glycolytic Enzymes?

Abstract

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

1. Introduction

Schistosomes are snail-transmitted, water-borne parasitic platyhelminths (blood flukes) that cause the disease schistosomiasis. Over 200 million people are estimated to be infected with these parasites, 90% of whom live in sub-Saharan Africa.^[1] Three schistosome species are responsible for almost all human infections. These are: Schistosoma haematobium (the cause of urogenital schistosomiasis in Africa), S. mansoni, and S. japonicum (the causes of hepato-intestinal schistosomiasis in Africa/South America and East Asia, respectively).

Schistosomes can live for many years in their human hosts.^[2] The relatively large adult worms can be found in the bloodstream apparently undisturbed by the elements of the host’s immune and coagulation systems.^[3] Host-interactive proteins found in the parasite’s tegument (skin) have been hypothesized to be critical to the worm’s ability to dampen host immunity as well as to hamper blood clot formation around them.^[4,5] For instance, an S. mansoni tegumental adenosine triphosphate (ATP) diphosphohydrolase enzyme, SmATPDase1,^[6] can cleave the proinflammatory mediator ATP as well as adenosine diphosphate (ADP)—a powerful prothrombotic molecule.^[7,8] The worms possess an ectonucleotide pyrophosphatase/phosphodiesterase, SmNPP5, that can also cleave ADP and can impede platelet aggregation in vitro.^[9] Another schistosome tegumental ectoenzyme—the alkaline phosphatase SmAP—can degrade sphingosine-1-phosphate, a bioactive lipid that plays key roles in controlling inflammation and platelet aggregation.^[10] SmAP can additionally 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.^[11] Tegumental proteases can hydrolyze the blood-clotting proteins fibronectin and high molecular weight kininogen.^[12,13] Other tegumental proteins have been shown to be important for the intake of water and nutrients like glucose and amino acids.^[14–18] Perhaps the greatest surprise arising from proteomic exploration of the schistosome tegument are reports of the presence of several glycolytic enzymes. In this paper, we review the evidence for this, and we consider what functions extracellular glycolytic enzymes might have for intravascular schistosomes.

2. Evidence of Glycolytic Enzymes at the Schistosome Surface

As depicted in Figure 1, glycolysis is a sequence of ten enzyme-catalyzed reactions that facilitate the conversion of glucose into pyruvate, ultimately resulting in the generation of the high-energy molecule ATP and NADH—a reduced form of nicotinamide adenine dinucleotide.

Figure 1.

A depiction of glycolysis—the biochemical pathway in which glucose (top left) is converted through a series of ten enzyme-catalyzed reactions (blue circles, 1–10) into pyruvate (bottom left). The enzymes catalyzing steps 1 through 10 are indicated in yellow boxes and in bold font. The pathway results in the net generation per cycle of two molecules of the high-energy molecule adenosine triphosphate (ATP, from adenosine diphosphate, ADP, at steps 7 and 10) and NADH—a reduced form of nicotinamide adenine dinucleotide (NAD, at step 6). The photograph depicts an adult male Schistosoma mansoni. The scale bar = 0.5 mm.

Table 1 summarizes data from a series of 11 experiments undertaken by different laboratories that explore the tegumental proteome of the intravascular life stages (schistosomula (juveniles) and adult worms) of a variety of schistosome species, here focusing just on the ten glycolytic enzymes. Studies 1 through 3 examined the proteome of isolated tegumental preparations from adult S. mansoni (studies 1 and 2) or S. bovis (a parasite of ruminants, study 3) in which five to eight of the ten glycolytic enzymes were detected.^[19–21] There is less of a surprise in finding most of the enzymes in these preparations since the tegument (like all parasite tissues) is expected to engage in glycolysis to generate ATP and NADH, as mentioned earlier. Perhaps of greater surprise are data from other studies that suggest that glycolytic enzymes are located at the exterior of the parasites, exposed at the host–parasite interface. Study 4 shows that many glycolytic enzymes can be detected following trypsin treatment of live S. mansoni worms.^[22] This demonstrates that these enzymes were accessible to trypsin proteolysis from the outside. Four different trypsin experiments were conducted and the numbers within column 4 reflect how many times (out of four) each enzyme was detected in subsequent proteomic analyses. No enzyme was detected in all four replicates; several were detected two to three times. Study 5 shows the outcome of similar trypsin treatment but this time using S. bovis males.^[23] In this case, experiments were conducted three times and the numbers given (0–3) indicate the number of times (out of three) that enzyme was detected by proteomics following trypsin treatment of the worms. As with the S. mansoni work, different S. bovis glycolytic enzymes are also detected a variable number of times. Female S. bovis adults were tested once using a relatively gentle trypsin treatment and, of the glycolytic enzymes, only glyceraldehyde 3-phosphae dehydrogenase (GAPDH) was detected.^[23]

Table 1.

Detection of the ten glycolytic enzymes in schistosome tegumental proteomic studies (numbered 1–11). Studies 1–3 involved analysis of total tegumental membrane preparations; studies 4 and 5 involved analysis of protein released from live parasites following exposure to the protease trypsin; study 6 involved analysis of protein released from live parasites following exposure to phosphatidylinositol phospholipase C (PIPLC); studies 7–11 involved analysis of proteins that were labeled following live parasite biotinylation. “−” indicates that the protein was not detected in a study, “+” indicates that the protein was detected in a study; Sm, Schistosoma mansoni; Sb, Schistosoma bovis, Sj, Schistosoma japonicum; *numbers in the “study 4” column refer to how many times an enzyme was detected out of four S. mansoni tegument trypsin experiments; ^#numbers in the “study 5” column refer to how many times an enzyme was detected out of three S. bovis tegument trypsin experiments; the extreme right column (“+/11”) tallies the number of studies in which a particular enzyme was detected (of 11 total studies). All studies used adult worms except study 8, which used schistosomula (juveniles).

		Total tegument membrane prep.			Tegument trypsin prep.		PIPLC prep.	Tegument surface biotinylation
	Study No.	1	2	3	4*	5#	6	7	8	9	10	11
		Sm	Sm	Sb	Sm	Sb	Sm	Sm	Sm	Sj	Sj	Sb	+/11
1	Hexokinase	−	−	−	0	0	−	−	+	−	−	−	1
2	Phosphoglucose isomerase (PGI)	+	−	−	0	0	−	−	+	−	+	−	3
3	Phosphofructokinase	+	−	−	0	1	−	−	+	−	+	−	4
4	Fructose biophosphate aldolase (FBA)	+	+	+	2	3	+	−	+	−	+	+	9
5	Triose phosphate isomerase (TPI)	+	+	+	0	1	+	−	+	−	+	+	8
6	Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)	+	+	+	3	3	−	+	+	+	+	+	10
7	Phosphogylcerate kinase (PGK)	−	−	+	3	3	−	−	+	−	+	+	6
8	Phosphoglycerate mutase (PGM)	+	+	+	3	1	−	−	+	+	+	+	9
9	Enolase	+	+	+	2	3	+	-	+	+	+	+	10
10	Pyruvate kinase	+	−	+	3	1	−	−	+	−	+	+	7
	References	[19]	[20]	[21]	[22]	[23]	[22]	[24]	[25]	[26]	[27]	[28]

Using a different approach, three glycolytic enzymes (fructosebisphosphate aldolase (FBA), triosephosphate isomerase (TPI), and enolase) were detected following treatment of living S. mansoni worms with phosphatidyl inositol phospholipase C—an enzyme that cleaves glycosylphosphatidylinositol (GPI)-linkages (study 6).^[22] Since none of the three enzymes is predicted to be GPI-linked based on its amino acid sequence, this result suggests that the three are associated with other, bona-fide GPI-linked surface proteins (of which several have been identified^[14]).

Surface biotinylation of adult worms led to the detection of one glycolytic enzyme (GAPDH) in one study involving S. mansoni adults by one group (study 7, Table 1^[24]) but in a study of S. mansoni schistosomula by another group (study 8^[25]) all ten glycolytic enzymes were detected. This last study is the only schistosome tegumental proteomic work to detect hexokinase—the enzyme that drives the first step in glycolysis. Biotinylation of adults of another schistosome species (S. japonicum) led to the detection of three glycolytic enzymes (GAPDH, phosphoglycerate mutase (PGM), and enolase, study 9^[26]) while other biotinylation work involving these parasites detected nine of the ten glycolytic enzymes (study 10^[27]; hexokinase was not seen). Finally, in an innovative study in which the surface of S. bovis adults was biotinylated while the worms were inside the vasculature of their hosts, seven of the ten glycolytic enzymes were labelled (study 11).^[28] Presumably different methodologies and modes of handling the worms in different biotinylation experiments are the cause of the variable detection results. Despite different outcomes, several proteomic studies suggest that some, and perhaps most, glycolytic enzymes are exposed to a greater or lesser degree at the exterior surface of intravascular schistosomes. The final column in Table 1 (marked “+/11”) tallies the number of times each enzyme was detected in the 11 studies reviewed here; enolase and GAPDH are detected most often, that is in 10 of the 11 studies, whereas FBA and PGM were detected in 9 of 11 and TPI in 8 of 11 studies.

The proteomic data described are remarkable as glycolysis is an intracellular cytosolic process and the enzymes involved lack clear secretion/signal motifs that would transfer them conventionally to the tegument surface. However, these data on schistosome surface-associated glycolytic enzymes are in keeping with several reports that some bacteria and protists also have glycolytic enzymes associated with the outer aspect of their cells. Currently, Gram-positive organisms such as streptococci and staphylococci and the cell wall-deficient mycoplasmas are said to have most of the enzymes of the glycolytic pathway on their cell surfaces,^[29–33] and glycolytic enzymes have been detected at the surface of parasitic protists like Entamoeba histolytica and Plasmodium spp.^[34–37] Indeed, some yeast cells and mammalian cells are also reported to express some glycolytic enzymes at their surfaces.^[38–40]

In schistosomes, immunolocalization work has additionally shown that several glycolytic enzymes are expressed in the parasite tegument (in addition to in the internal tissues).^[41–43] More definitive evidence for the external location of these enzymes would be a demonstration of specific enzyme activity by intact, live worms. In the case of enolase, it has been shown that living S. mansoni schistosomula as well as adult males and females can drive the interconversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) (glycolysis step 9, Figure 1).^[43] This means that the active enzyme is expressed at the host/parasite interface. Importantly, culture medium in which the parasites were incubated does not display this activity, meaning that the enolase enzyme was not released by the worms to any measurable degree during culture but remained associated with the outer worm membrane. The enolase substrate 2PG is also efficiently converted to PEP by adult S. haematobium and S. japonicum worms in culture.^[43]

Evidence for the existence of bona-fide glycolytic enzymes on the outer surface of schistosomes leads to the question as to whether there is a selective advantage for the parasites to express these proteins there. It seems unthinkable that the worms would engage in some form of extracellular glycolysis—especially given that an important product of glycolysis—ATP—could then be hydrolyzed via, e.g., the SmATPDase1 ectoenzyme noted earlier. Instead, the extracellular glycolytic enzymes may be engaged in other nontraditional functions on the worm surface. Such functions outside of, or in addition to, their well-known activity in glycolysis has resulted in such enzymes being described as “moonlighting proteins.” There is an increasing amount of evidence that these enzymes on pathogen surfaces can act in a variety of novel ways, for instance in the processes of immune modulation and blood clot dissolution.

3. Moonlighting Functions of Tegumental Glycolytic Enzymes: Prothrombolysis

Under normal conditions, blood clot breakdown begins when the zymogen plasminogen (PLMG) is converted by, e.g., tissue plasminogen activator (tPA) into its enzymatically active form, plasmin. This enzyme hydrolyses cross-linked fibrin to dissolve blood clots. In schistosomes, enolase (produced in recombinant form) from S. mansoni, S. japonicum, and S. bovis have all been shown to bind to PLMG.^[43–45] Furthermore, these proteins can enhance PLMG activation in the presence of tPA, an ability that likely allows the worms to minimize clot buildup, and so control hemostasis around them within the blood vessels of their hosts.^[43–45] Analysis of PLMG binding to a series of peptides derived from the S. bovis enolase protein revealed that C-terminal lysine residues were not involved; the only interacting motif was ²⁵⁰EFHKNGKYDLDF²⁶¹.^[46] Unlike in S. bovis where PLMG binds only to adult male worms and not to adult females,^[44] in the case of S. mansoni, PLMG binds to extracts of both adult females and males (and schistosomula).^[43] All intravascular S. mansoni life stages (schistosomula and adult males and females) expressed enolase at the host-exposed surface as determined by enzyme activity assays. In addition, all life stages (including females) were able to enhance the activation of PLMG to generate plasmin, in the presence of tPA.^[43] These data indicate differences in the biology of S. mansoni compared to S. bovis; the task of enolasemediated PLMG binding and activation in S. bovis may be confined to the male worms only, whereas this is not the case for S. mansoni.

In other helminths too, enolase has been identified as a PLMG-binding protein, for instance in the secretory products of Fasciola hepatica^[47] and Taenia pisiformis,^[48] and in multiple tissues of Onchocerca volvulus.^[49] Extracellular enolase can also act as a PLMG receptor in many pathogenic bacteria and protists and in mammalian tumor cells where, as in helminths, the enzyme has been proposed to facilitate unrestricted movement and increase virulence.^[50]

The ability to bind to PLMG (with potential importance in controlling thrombus dissolution) is a common feature of other glycolytic enzymes, beyond enolase. For instance, in tegument extracts of S. bovis additional glycolytic enzymes—FBA, TPI, GAPDH, and PGM—were all found to bind PLMG.^[51] S. bovis GAPDH and FBA proteins were subsequently reported to be able to bind PLMG in vitro, but only FBA potentiated its conversion to plasmin in the presence of tPA.^[46] The ability of glycolytic enzymes to interact with PLMG is reported in the case of other parasitic worms as well. For instance, GAPDH from the Chinese liver fluke Clonorchis sinensis was shown to both be present at the parasite surface and, in recombinant form, to bind human PLMG in a dose-dependent manner, as measured by ELISA.^[52] Among the surface-associated antigens of the intravascular nematode parasite Dirofilaria immitis are three glycolytic enzymes: enolase, GAPDH, and FBA, and all three bind PLMG.^[53] Recombinant D. immitis GAPDH has been found to both bind PLMG and stimulate plasmin generation by tPA.^[54] Taken together, these data suggest that a number of glycolytic enzymes from a variety of parasitic helminths can impinge on the processes of blood clot formation and dissolution.

4. Moonlighting Functions of Tegumental Glycolytic Enzymes: Immune Modulation

Besides their suggested involvement in controlling coagulation, pathogen glycolytic proteins have also been reported to be potentially involved in modulating the immune response. GAPDH from Haemonchus contortus, a blood-feeding parasitic nematode of ruminants, can bind to complement factors C1q and C3 and inhibit complement function, as measured by in vitro assay.^[55] C1q is important in the initiation of the classical complement pathway and C3 is a key activator of all three complement pathways—classical, alternative, and lectin dependent. Binding activity was mapped to the N-terminal region of the parasite enzyme.^[56] It is hypothesized that parasite protein binding to host complement components could limit membrane attack complex formation and any resulting damage to the worm. In the Gram-positive bacterium Streptococcus pneumonia, the glycolytic enzyme phosphoglycerate kinase (PGK) is found at the bacterial surface; recombinant S. pneumoniae PGK interacts with complement membrane attack complex components C5, C7, and C9, thereby blocking the assembly and membrane insertion of the attach complex and this results in significant inhibition of the hemolytic activity of human serum.^[57]

Another glycolytic enzyme with moonlighting functions is phosphoglucose isomerase (PGI); in the parasitic helminth Echinococcus multilocularis, PGI can stimulate division of endothelial cells in vitro.^[58] In tumor cells, extracellular PGI has been shown to be involved in cell proliferation and angiogenesis.^[59,60]

Ongoing study of the glycolytic enzymes reveals that they express a plethora of uncommon functions independent of their roles in energy metabolism. For instance, GAPDH can bind nucleic acids with an impact on, e.g., mRNA stability, tRNA export, gene expression, and telomer biochemistry and the protein can also play a role in intracellular membrane trafficking as well as heme metabolism (reviewed by Sirover^[61]). While it is unclear whether these various intracellular functions of GAPDH are relevant in the context of extracellular glycolytic enzymes expressed by schistosomes, extracellular moonlighting functions of GAPDH have also been described. For instance, GAPDH is a major surface protein of streptococci bacteria that has been shown to act as an ADP-ribosylating enzyme.^[62] Recombinant GAPDH from Streptococcus agalactiae has been shown to induce the proliferation and differentiation of B cells in vitro and give rise to a rapid increase in serum levels of IL-10 in mice injected intraperitoneally with the protein.^[63] In addition, an S. agalactiae strain overexpressing GAPDH showed increased virulence in mice as compared with wild type.^[63] Extracellular GAPDH from the related bacterial pathogen Streptococcus pyogenes can also bind to multiple host proteins (fibronectin, actin, and lysosome).^[64] In addition, recombinant streptococcus GAPDH inhibits complement component C5a-activated chemotaxis and H₂O₂ production in isolated human neutrophils^[65] and can induce apoptosis in murine macrophages.^[66] Finally, S. pyogenes mutants unable to export GAPDH to their surface are completely attenuated.^[64] Thus, streptococcal GAPDH is increasingly recognized as an important virulence factor for these bacteria.^[67] These findings raise the possibility that schistosome surface-bound GAPDH might exert similar functions. Figure 2 summarizes the major moonlighting functions ascribed to extracellular glycolytic enzymes of a variety of organisms, including schistosomes.

Figure 2.

A list of the ten glycolytic enzymes with a summary of the major moonlighting functions ascribed to them. The species expressing the enzyme with the described function is listed in the right-hand column and the life form of that species (whether worm (platyhelminth or nematode) or bacterium) is noted in brackets.

5. Getting Glycolytic Enzymes to the Tegument Surface

The question arises as to how normally cytosolic glycolytic enzymes that lack signal sequences can, nonetheless, also be found at an organism’s exterior surface. The schistosome tegument is bounded by two lipid bilayers; an inner plasma membrane that is overlain by a second bilayer known as the membranocalyx.^[14] This unusual covering is formed and maintained by the fusion of organelles called multilamellar bodies (MLBs) with the plasma membrane. This leads to the release of the MLBs’ membranous contents which coalesce to form the membranocalyx.^[14] Therefore, targeting the glycolytic enzymes to the interior of the MLBs in tegumental cell bodies would be one method to ensure their eventual efficient delivery to the host–parasite interface at the worm surface. The chemistry involved in retaining these enzymes at the surface in the absence of transmembrane or conventional anchoring domains is not known.

The presence of glycolytic enzymes associated with the intravascular schistosome surface provides rationale for the many reports showing that immunization with some of these enzymes can generate protective immunity in vaccine trials.^[68–76] Heretofore, how an intracellular cytosolic enzyme could be a focus for a useful immunological attack was a conundrum. Given that these enzymes are now known to be additionally exposed and host-interactive, realistic models of protective immunity can be formulated. Given the generally high degree of sequence conservation between the glycolytic enzymes of parasite and host, it bears noting that an immune response targeting parasite glycolytic enzymes could potentially also impact any moonlighting functions of host homologs.

6. Conclusions

In summary, it is clear that intravascular schistosomes express a collection of glycolytic enzymes at their host-exposed surface. It seems likely that these enzymes are engaged here in still-undescribed, nonglycolytic functions such as immune modulation and hemostasis control. This is in agreement with work in other systems indicating an involvement of glycolytic enzymes in the control of blood flow and, in particular, in the efficient conversion of the inactive enzyme plasminogen into its active form plasmin—a key component in fibrinolysis. Beyond hemostasis, glycolytic enzymes can also be immunomodulatory; some inhibit the complement cascades, others impinge on immune cell signaling to potentially dampen immunological responses. These external tegumental glycolytic enzymes present a novel and underrecognized mode of host–parasite communication. Ultimately, the ability of the glycolytic enzymes to alter immune outcomes and to impinge on blood flow represent additional modes of communication between parasite and host that likely help prevent parasite expulsion and ensure the long-term survival of the intravascular worms.

Several schistosome glycolytic enzyme cDNAs have already been cloned and expressed and the glycolytic capabilities of these proteins have been documented.^[41–43] Thus, the ready availability of functionally active, recombinant schistosome glycolytic enzymes should facilitate rapid progress in an assessment of any nonglycolytic, novel, moonlighting functions of these proteins; functions that likely contribute to the survival of schistosomes and to their longevity. Impeding such functions by, e.g., vaccination with these proteins (alone or in combination) and/or by using specific chemical inhibitors of these enzymes may represent new control strategies for these globally important parasites.