Glycotope analysis in miracidia and primary sporocysts of Schistosoma mansoni: differential expression during the miracidium-to-sporocyst transformation

Abstract

Fucosylated carbohydrate epitopes (glycotopes) expressed by larval and adult schistosomes are thought to modulate the host immune response and possibly mediate parasite evasion in intermediate and definitive hosts. While previous studies showed glycotope expression is developmentally and stage-specifically regulated, relatively little is known regarding their occurrence in miracidia and primary sporocysts. In this study, previously defined monoclonal antibodies were used in confocal laser scanning microscopy, standard epifluorescence microscopy and Western blot analyses to investigate the developmental expression of the following glycotopes in miracidia and primary sporocysts of Schistosoma mansoni: GalNAcbeta1-4GlcNAc (LDN), GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDN-F), Fucalpha1-3GalNAcbeta1-4GlcNAc (F-LDN), Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc (F-LDN-F), GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF), Fucalpha1-2Fucalpha1-3GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (DF-LDN-DF), Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis X) and the truncated trimannosyl N-glycan Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAcbeta1-Asn (TriMan). All but Lewis X were variously expressed by miracidia and sporocysts of S. mansoni. Most notably, alpha3-fucosylated LDN (F-LDN, F-LDN-F, LDN-F) was prominently expressed on the larval surface and amongst glycoproteins released during larval transformation and early sporocyst development, possibly implying a role for these glycotopes in snail-schistosome interactions. Interestingly, Fucalpha2Fucalpha3-subsituted LDN (LDN-DF, DF-LDN-DF) and LDN-F were heterogeneously surface-expressed on individuals of a given larval population, particularly amongst miracidia. In contrast, LDN and TriMan primarily localised in internal somatic tissues and exhibited only minor surface expression. Immunoblots indicate that glycotopes occur on overlapping but distinct protein sets in both larval stages, further demonstrating the underlying complexity of schistosome glycosylation. Additionally, sharing of specific larval glycotopes with Biomphalaria glabrata suggests an evolutionary convergence of carbohydrate expression between schistosomes and their snail host.

Fig. 1

Localisation of glycotope expression in miracidia and primary sporocysts of Schistosoma mansoni. Confocal laser scanning microscopy was used to assess the localisation of schistosome-associated carbohydrate terminal structures in miracidia and 2-day (2d) in vitro-cultivated primary sporocysts. Fixed and permeabilised larvae were immunostained with monoclonal antibodies recognising GalNAcβ1-4GlcNAc (LDN; (C and D)), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN; (E and F)), Fucα1-3GalNAcβ1- 4(Fucα1-3)GlcNAc (F-LDN-F; (G and H)), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F; (I and J)), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF; (K and L)), Fucα1-2Fucα1- 3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF; (M and N)), Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan; (O and P)) and Galβ1-4(Fucα1-3)GlcNAc (Lewis X; (Q and R)). Control larvae were exposed to secondary antibody without previous primary antibody treatment (A and B). Panels include paired micrographs depicting glycotope expression (green) alone and merged with counterstained actin (e.g., muscles, flame cells; red) and DNA (e.g., nuclei; blue). Approximate scale is represented in the lower right corner (bar = 50 μm). AG, apical gland; Cil, cilia; LG, lateral gland; M, sporocyst matrix; NM, neural mass; RC, interepidermal ridge cyton; SN, sensory nerve.

Fig. 1

Localisation of glycotope expression in miracidia and primary sporocysts of Schistosoma mansoni. Confocal laser scanning microscopy was used to assess the localisation of schistosome-associated carbohydrate terminal structures in miracidia and 2-day (2d) in vitro-cultivated primary sporocysts. Fixed and permeabilised larvae were immunostained with monoclonal antibodies recognising GalNAcβ1-4GlcNAc (LDN; (C and D)), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN; (E and F)), Fucα1-3GalNAcβ1- 4(Fucα1-3)GlcNAc (F-LDN-F; (G and H)), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F; (I and J)), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF; (K and L)), Fucα1-2Fucα1- 3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF; (M and N)), Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan; (O and P)) and Galβ1-4(Fucα1-3)GlcNAc (Lewis X; (Q and R)). Control larvae were exposed to secondary antibody without previous primary antibody treatment (A and B). Panels include paired micrographs depicting glycotope expression (green) alone and merged with counterstained actin (e.g., muscles, flame cells; red) and DNA (e.g., nuclei; blue). Approximate scale is represented in the lower right corner (bar = 50 μm). AG, apical gland; Cil, cilia; LG, lateral gland; M, sporocyst matrix; NM, neural mass; RC, interepidermal ridge cyton; SN, sensory nerve.

Fig. 2

Glycotope expression on the exposed surfaces of miracidia and primary sporocysts of Schistosoma mansoni. The exposed surfaces of miracidia and 2-day (2d) in vitrocultivated primary sporocysts were examined for the expression of schistosome-associated carbohydrate terminal structures using epifluorescence microscopy. Fixed but non-permeabilised larvae were treated with monoclonal antibodies that recognise GalNAcβ1-4GlcNAc (LDN; (C and D)), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN; (E and F)), Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F; (G and H)), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F; (I and J)), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF; (K and L)), Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF; (M and N)), Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan; (O and P)) and Galβ1- 4(Fucα1-3)GlcNAc (Lewis X; (Q and R)). Control larvae were exposed to secondary antibody without previous primary antibody treatment (A and B). Micrographs depict glycotope expression patterns that were generally observed for each treatment, however larvae exhibited considerable heterogeneity regarding the expression of several glycotopes, particularly LDN-F, LDN-DF and DF-LDN-DF (see Supplementary Fig. S2). Panels include paired brightfield and fluorescence micrographs of individual larvae. Approximate scale is represented in the lower right corner (bar = 50 μm). Cil, cilia; EP, epidermal plate; IR, interepidermal ridge; T, terebratorium; Teg, tegument.

Fig. 2

Glycotope expression on the exposed surfaces of miracidia and primary sporocysts of Schistosoma mansoni. The exposed surfaces of miracidia and 2-day (2d) in vitrocultivated primary sporocysts were examined for the expression of schistosome-associated carbohydrate terminal structures using epifluorescence microscopy. Fixed but non-permeabilised larvae were treated with monoclonal antibodies that recognise GalNAcβ1-4GlcNAc (LDN; (C and D)), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN; (E and F)), Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F; (G and H)), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F; (I and J)), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF; (K and L)), Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF; (M and N)), Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan; (O and P)) and Galβ1- 4(Fucα1-3)GlcNAc (Lewis X; (Q and R)). Control larvae were exposed to secondary antibody without previous primary antibody treatment (A and B). Micrographs depict glycotope expression patterns that were generally observed for each treatment, however larvae exhibited considerable heterogeneity regarding the expression of several glycotopes, particularly LDN-F, LDN-DF and DF-LDN-DF (see Supplementary Fig. S2). Panels include paired brightfield and fluorescence micrographs of individual larvae. Approximate scale is represented in the lower right corner (bar = 50 μm). Cil, cilia; EP, epidermal plate; IR, interepidermal ridge; T, terebratorium; Teg, tegument.

Fig. 3

Confocal laser scanning microscopy (CLSM) z-stack projections of immunoreactive glycotope-bearing structures in miracidia of Schistosoma mansoni. To better visualise immunoreactive structures, individual miracidia were optically sectioned, creating a series of CLSM micrographs (z-stack) depicting sequential focal planes (1 to n). (A) S.D. z-stack projection (images 45–82, n = 127) of anti-LDN-DF (GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc) immunoreactivity in miracidia; (B) S.D. projection (images 1–70, n = 118) of anti-TriMan (Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn) immunoreactivity in miracidia. Approximate scale is represented in the lower right corner (bar = 50 μm). LG, lateral gland; NM, neural mass; SN, sensory nerve.

Fig. 4

Western blots revealing glycotope expression amongst whole-body larval extracts, epidermal plate glycoproteins and parasite culture supernatants containing larval transformation proteins. Whole-body extracts of miracidia (Mir) and primary sporocysts cultivated 2 and 8 days (2dS and 8dS, respectively), as well as ciliated epidermal plate extracts (EP) and parasite culture supernatants containing larval transformation proteins (LTP), were SDS–PAGE-fractionated and immunoblotted using anti-glycotope monoclonal antibodies that recognise GalNAcβ1-4GlcNAc (LDN; (B)), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN; (C)), Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F; (D)), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F; (E)), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF; (F)), Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF; (G)), Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan; (H)) and Galβ1-4(Fucα1-3)GlcNAc (Lewis X; (I)). Control blots were exposed to the secondary antibody without previous primary antibody incubation (A), and total protein was visualised in-gel by silver stain (J).

Fig. 5

Two-dimensional (2D) Western blots demonstrating glycotope expression in surface-enriched protein extracts of miracidia and primary sporocysts. Gentle extraction of miracidia and 2-day in vitro-cultivated primary sporocysts yielded surface-enriched protein extracts that were subsequently 2D PAGE-fractionated and immunoblotted using anti-glycotope monoclonal antibodies. Panels include representative blot patterns for Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F; (A)), GalNAcβ1-4(Fucα1- 3)GlcNAc (LDN-F; (B)), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF; (C)) and Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan; (D)).

Fig. 6

Western blot analysis of Biomphalaria glabrata hemocyte and plasma glycoproteins. Hemocyte (Hc) and plasma (Pl) glycoproteins of uninfected B. glabrata were PAGE-fractionated and immunoblotted using anti-glycotope monoclonal antibodies against Fucα1-3GalNAcβ1-4GlcNAc (F-LDN; (B)) and Manα1- 3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan; (C)). Control blots were exposed to the secondary antibody without previous primary antibody treatment (A).