Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase is a lung-stage schistosomula surface membrane antigen

Abstract

Abstract. We have previously reported that Schistosoma mansoni larvae emerging from host lung at pH 7.5-7.8 and then fixed with diluted formaldehyde (HCHO) readily bind radiation-attenuated cercariae (RA) vaccine serum antibodies, as assessed by indirect membrane immunofluorescence (IF). Here we show that S. mansoni schistosomula emerging from lung pieces under 5% CO 2 (pH ≤7.3) readily bind RA vaccine serum antibodies, provided they have been incubated for 12 h at pH 7.5-7.8 in foetal calf serum-free RPMI medium, and fixed with diluted HCHO. Ex vivo larvae exposed during incubation to GW4869, a specific inhibitor of tegument-bound, neutral sphingomyelinase (nSMase) displayed significantly diminished binding of RA vaccine serum antibodies, thus suggesting that nSMase activity at pH ≥7.5 leads to exposure of lung-stage larvae surface membrane antigens to specific antibody detection. More importantly, ex vivo larvae readily bound antibodies directed to dipeptidic multiple antigen peptide constructs, based on S. mansoni-specific sequences in S. mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH). Lung-stage schistosomula IF reactivity was diminished following antiserum absorption with recombinant SG3PDH. The data together indicate that intact ex vivo, as well as, 5-day-old in vitro-grown larvae express SG3PDH on their surface membrane. The findings are discussed in relation to the importance of surface membrane proteins as candidate vaccine antigens.

In Vitro and In Vivo Activities of Arachidonic Acid against Schistosoma mansoni and Schistosoma haematobium

“…No adverse effects were noted in any treated mice, supporting the data showing that administration of 2,000 mg/kg ARA oil for a period of 4 weeks did not induce obvious signs of toxicity in Wistar rats (14). It is not clear whether in vivo ARA-mediated worm killing is predominantly due to production of ceramide (12,21) or to exposure of the parasite to the host effector immune cells, antibodies, and toxic molecules and radicals (8,29,30). Of note, S. haematobium, which displayed higher in vivo sensitivity to ARA than S. mansoni, was shown to be more sensitive than S. mansoni to in vitro ARA-mediated exposure of otherwise concealed surface membrane antigens (29).…”

“…Second, while dead parasites did not show spasmodic body contractions as they did after PZQ treatment, electron microscopy studies revealed entire disruption of the outer lipid bilayers, the strength of which was correlated with the ARA concentration. Third, addition of the nSMase inhibitors CaCl 2 (15,23,29) and GW4869 (19,20,30) entirely protected the parasite from the lethal ARA actions, and nearly all worms remained viable beyond the 5-h exposure period.…”

“…Lung pieces were incubated in RPMI 1640 medium supplemented with 200 U/ml penicillin, 200 g/ml streptomycin, 25 mM HEPES (RPMI medium), 5 U/ml heparin, and 10% fetal calf serum (all from BioWhittaker Europe, Verviers, Belgium) for 4 h at 37°C in a humidified atmosphere containing 5.0% CO 2 , to allow the schistosomula to emerge. The larval suspension was then poured through a sterile Nitex 132-mesh nylon screen; treated for 10 min at 4°C with sterile 0.16 M ammonium chloride-0.017 M Tris buffer, pH 8.0, to lyse the erythrocytes; and isolated from contaminant lung cells by centrifugation over 40% Percoll (Pharmacia, Uppsala, Sweden) in RPMI medium as described previously (29,30). All larvae were viable, transparent, elongated, and highly motile.…”

“…GW4869 (Calbiochem), a potent, specific, and noncompetitive inhibitor of nSMase (19,20,30), was dissolved in dimethyl sulfoxide (DMSO) and used at the preselected concentration of 12.5 M (30). Calcium chloride (CaCl 2 ), a potent inhibitor of nSMase activity at mM concentrations (15,23,29), was used at a final concentration of 2 mM.…”

“…ARA is a powerful activator of the parasite tegumentassociated, magnesium-dependent neutral sphingomyelinase (nSmase) (http://www.genedb.org/gene/Smp_162880). Subsequent sphingomyelin hydrolysis elicits an increase in membrane permeability, bending, and aggregation and dramatic perturbations in lipid content and rigidity, allowing the free movement of antigenic molecules in the plane of the surface membranes and avid binding of specific antibodies (8,29,30).…”