The blood fluke Schistosoma mansoni cleaves the coagulation protein high molecular weight kininogen (HK) but does not generate the vasodilator bradykinin

Abstract Background Schistosomes are blood dwelling parasitic worms that cause the debilitating disease schistosomiasis. Here we examined the influence of the parasites on their external environment by monitoring the impact of adult Schistosoma mansoni worms on the murine plasma proteome in vitro and, in particular, on how the worms affect the blood coagulation protein high molecular weight kininogen (HK). Methods Following the incubation of adult schistosomes in murine plasma, two-dimensional differential in-gel electrophoresis (2D-DIGE) was conducted to look for changes in the plasma proteome compared with control plasma. A major change to the blood protein kininogen (HK) was observed, and the interaction of Schistosoma mansoni parasite with this protein alone was then investigated by western blot analysis and activity assays. Finally, the generation of bradykinin from HK was monitored using a bradykinin detection kit. Results The most striking change to the plasma proteome concerned HK; while the full-length protein was more abundant in control plasma, carboxyl-terminal truncated forms were more abundant in plasma that contained schistosomes. Incubating parasites in buffer with pure HK followed by Western blot analysis confirmed that human HK is degraded by the worms. The resulting digestion pattern differed from that brought about by kallikrein, a host serine protease that normally acts on HK to release the vasodilator bradykinin. We found that live schistosomes, while digesting HK, do not generate bradykinin nor do they cleave a chromogenic kallikrein substrate. Since the cleavage of HK by the worms is not impeded by the serine protease inhibitor PMSF but is blocked by the cysteine protease inhibitor E64c, we hypothesize that schistosome tegumental cysteine proteases are responsible for HK cleavage. Conclusions Since proteomic and biochemical studies have revealed that the schistosome tegument contains two cysteine proteases belonging to the calpain family (SmCalp1 and SmCalp2) we conclude that these are likely responsible for the HK cleavage reported here. Schistosome cleavage of HK should help impede blood clotting and inflammation around the worms in vivo and so promote their ease of movement within the vasculature of their hosts

DataSheet_1_A novel, non-neuronal acetylcholinesterase of schistosome parasites is essential for definitive host infection.pdf

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.</p

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

Dendritic cells activated by an anti-inflammatory agent induce CD4+ T helper type 2 responses without impairing CD8+ memory and effector cytotoxic T-lymphocyte responses

Prevalence of pro-inflammatory diseases is rising in developed country populations. The increase in these diseases has fuelled the search for new, immune suppressive, anti-inflammatory therapies, which do not impact, or minimally impact, CD4+ and/or CD8+ T-cell-mediated immunity. The goal of this study was to determine if antigen-presenting cells (APCs) activated by the anti-inflammatory oligosaccharide, lacto-N-fucopentaose III (LNFPIII), would have an impaired ability to drive CD4+ T helper (Th) or CD8+ memory and effector T-cell responses. To investigate this we activated splenic dendritic cells (SDCs) with LNFPIII and examined their ability to drive antigen-specific CD4+ Th, and CD8+ memory and cytotoxic T-cell (CTL) responses compared with lipopolysaccharide (LPS) -stimulated SDCs. The LNFPIII-activated SDCs had altered co-stimulatory molecule expression compared with LPS-stimulated SDCs, while the levels of SDC chemokines following activation by either compound were similar. LNFPIII-activated SDCs produced significantly lower levels of interleukin-12 but surprisingly higher levels of interleukin-6 than LPS-activated SDCs. Similar to previous studies using bone-marrow-derived DCs, LNFPIII-activated SDCs induced strong Th2 responses in vivo and ex vivo. LNFPIII activation of APCs was independent of the Toll-interleukin-1 receptor adaptor myeloid differentiating factor 88. Importantly, LNFPIII-matured DCs induced CD8+ memory and effector CTL responses similar to those driven by LPS-matured DCs, including the frequency of interferon-γ-producing CD8+ T cells and induction of CTL effectors. Treatment of APCs by the anti-inflammatory glycan LNFPIII did not impair their ability to drive CD8+ effector and memory cell-mediated immunity

Schistosome tegumental ecto-apyrase (SmATPDase1) degrades exogenous pro-inflammatory and pro-thrombotic nucleotides

Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs) and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP). Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5) and ATP diphosphohydrolase (SmATPDase1). In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms’ ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, full-length recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The Km of SmATPDase1 for ATP is 0.4 ± 0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals like ATP, and pro-thrombotic signals like ADP, these parasite enzymes may minimize host immune responses, inhibit blood coagulation and promote schistosome survival

DNA-based vaccines protect against zoonotic schistosomiasis in water buffalo

Schistosomiasis japonica is an endemic, zoonotic disease of major public health importance in China where water buffaloes account for approximately 75% of disease transmission. Interventions that reduce schistosome infection in water buffaloes will enhance their health simultaneously reducing disease transmission to humans. While chemotherapy has proved successful, it requires continued time consuming and expensive mass treatments. A more sustainable option would be development of vaccines that reduce transmission of S. japonicum from bovines to replace bovine chemotherapy. We performed two randomized double blind trials in water buffaloes to determine if DNA vaccines encoding triose-phosphate isomerase (SjCTPI), or the tetraspanin 23 kDa integral membrane protein (SjC23), alone or fused to bovine heat shock protein 70 (Hsp70) could induce a level of immunity conducive to long-term sustainable control. Groups of water buffaloes (15/group) received three intramuscular injections, 4 weeks apart. Booster immunizations were co-administered with a plasmid DNA encoding IL-12. Four weeks after the last injection, water buffaloes were challenged with 1000 cercariae, and vaccine efficacy analyzed 8 weeks later. Water buffaloes vaccinated with SjCTPI-Hsp70 or SjCTPI plasmids had worm burdens reduced by 51.2% and 41.5%, respectively. Importantly, fecal miracidial hatching was reduced by 52.1% and 33.2% respectively compared to control vaccinated water buffaloes. Vaccination with SjC23-Hsp70 and SjC23 plasmids reduced worm burdens by 50.9% and 45.5%, respectively, and fecal miracidial hatching by 52.0% and 47.4%. A mathematical model of schistosome transmission predicts that schistosome vaccines capable of reducing water buffaloes’ fecal egg output by 45%, alone or in conjunction with praziquantel treatment, will lead to a significant reduction in transmission of schistosomiasis. Both DNA vaccines tested here exceed this hypothetical level. Indeed, mathematical modeling of SjCTPI-Hsp70 and SjC23-Hsp70 alone and in conjunction with human chemotherapy showed a significant reduction in transmission almost to the point of elimination

Anti-Transmission DNA Vaccine for Schistosomiasis japonica in China

Despite intensive control efforts, schistosomiasis remains an endemic, zoonotic disease of major public health importance in China. In the marsh and lake regions of China, water buffalo account for approximately 75% of disease transmission. In addition to acting as the major reservoir, infected water buffalo often experience poor growth and weight gain compared to non-infected animals. Thus, interventions which reduce schistosome infection in buffalo will be beneficial to buffalo health and aid in reducing disease prevalence in humans. In this regard, a mathematical model predicted that an anti-fecundity vaccine which reduces fecal egg output in water buffalo by 40-45% in conjunction with praziquantel treatment will significantly lead to reduction in transmission of schistosomiasis. In this study, we tested the ability of four schistosome- DNA vaccine constructs to reach these levels in water buffalo. The DNA vaccine constructs encode the glycolytic enzyme triose phosphate isomerase (SjCTPI) or the tetraspanin 23 kDa integral membrane protein (SjC23) or the same antigens fused to the N-terminus end of the bovine heat shock protein 70 (SjCTPI-Hsp70 and SjC23-Hsp70). We found that compared to buffalo vaccinated with the control plasmid DNA (pVAX), vaccination with SjCTPI-Hsp70 or SjCTPI plasmids reduced worm burdens by 51.2% and 41.5% respectively and importantly, fecal miracidialhatching was reduced by 52.1% and 33.2% respectively. Vaccination with SjC23-Hsp70 and SjC23 plasmids reduced worm burdens by 50.9% and 45.5% respectively and fecal miracidial-hatching by 52.0% and 47.4%. Thus both the SjCTPI-Hsp70 and SjC23-Hsp70 plasmid DNA vaccines exceeded the level of protection predicted by the mathematical model to significantly reduce transmission of schistosomiasis in the lakes and marsh regions of China. These data support the use of either of these two vaccines in a field-based intervention to determine if vaccination of buffalo will reduce transmission of schistosomiasis in China. (ACMCIP Abstract