Excreted/secreted Schistosoma mansoni venom allergen-like 9 (SmVAL9) modulates host extracellular matrix remodelling gene expression

Abstract

The Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-β-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration.

Keywords: Biomphalaria glabrata; Matrix metalloproteinase; Schistosoma mansoni; Venom allergen-like.

Graphical abstract

Fig. 1

Schistosoma mansoni venom allergen-like 9 (SmVAL9) undergoes transcriptional regulation throughout schistosome development, is maximally expressed in miracidia and is post-translationally modified in eggs. (A) DNA microarray analysis of SmVAL9 expression throughout 15 lifecycle stages. Histogram represents normalised mean fluorescent intensities + S.D. (n = 3 replicates/lifecycle stage except adult female where n = 2) of SmVAL9 transcript abundance derived from oligonucleotide CD111828 as described previously (Fitzpatrick et al., 2009). Dashed box encloses schistosome lifecycle stages where SmVAL9 is maximally expressed. Inset drawing represents SmVAL9 (Smp_176180) gene organisation (five exons – black boxes; four introns – black lines) and localisation of oligonucleotide CD111828 to exon 5 (SchistoGeneDB v5.1). (B) Expression and purification of recombinant SmVAL9 (rSmVAL9) in Escherichia coli cells. The majority of rSmVAL9 is found in insoluble inclusion bodies after induction, allowing preparatory cell electrophoresis (Prep. Cell) to concentrate and purify large amounts of the protein for murine immunisations and other downstream studies. (C) Anti-rSmVAL9 recognises the 19.3 kDa rSmVAL9 immunogen and a ∼25 kDa post-translationally modified, native SmVAL9 (nSmVAL9) from soluble egg antigen. Pre-bleed normal mouse serum does not recognise either protein or the BSA control.

Fig. 2

Schistosoma mansoni venom allergen-like 9 (SmVAL9) is an N-glycosylated soluble egg antigen containing a schistosome-specific, difucosyl modification. (A) Electrospray ionisation-ion trap MS/MS with collision-induced dissociation and with electron transfer dissociation (B) of the tryptic glycopeptide K₇₁AQNLSDQCK₈₀ from SmVAL9. The [M+3H]³⁺ parent ion at m/z 954.0 of the glycopeptide carrying a glycan of composition F₃H₃N₃X₁ was selected. Fragment ions are indicated in the figure. Monoisotopic masses are given. Square, N-acetylglucosamine; circle, mannose; triangle, fucose; star, xylose; pep, peptide moiety.

Fig. 3

Schistosoma mansoni venom allergen-like 9 (SmVAL9) is translationally regulated in intra-molluscan S. mansoni lifecycle stages and is found throughout the parasite’s parenchyma, perikarya and putative germinal cells. (A) Quantitative assessment of SmVAL9 protein expression in free-swimming miracidia and primary sporocysts at different times post in vitro cultivation (0.5 h to 10 days). Histogram on the left represents the relative means + S.D. of steady-state sporocyst SmVAL9 protein normalised to miracidia, as a function of time in culture. Means were generated by densitometric analyses of western blots displaying immunoreactive native (n)SmVAL9 in miracidia and sporocysts (n = 3; representative figure is shown). All larval values were normalised to anti-SmActin reactivities (loading control) prior to statistical analysis (ANOVA; F_6,2 = 8.59; P = 0.0048). Mean values sharing the same letter are not significantly different as determined by Tukey’s multiple comparison test. (B) Immunocytochemical localisation of SmVAL9 in S. mansoni miracidia and 24 h cultured primary sporocysts by laser scanning confocal microscopy. Specific immunoreactivity was observed diffusely distributed in the parenchyma, perikarya and putative germinal cells (insert, 24 h sporocyst) of miracidia and sporocysts. Surface immunostaining in normal mouse serum and secondary antibody only controls (data not shown) likely represents non-specific bound mouse IgG acquired in ovo. Bars = 50 μm; insert bar = 10 μm. GC, germinal cells.

Fig. 4

Schistosoma mansoni venom allergen-like 9 (SmVAL9) released during the process of miracidia to sporocyst transformation induces Biomphalaria glabrata matrix metalloproteinase expression. (A) Native SmVAL9 is released into medium during early larval cultivation. Western blot showing immunoreactive native (n)SmVAL9 in culture supernatant containing larval transformation proteins and in whole body homogenates (Body), but not in shed epidermal plates. Absence of epidermal plates immunoreactivity is not due to differences in sample protein loads as indicated by intensities of Coomassie blue staining. (B) In vitro exposure of the B. glabrata embryonic (Bge) cell line to rSmVAL9 (0.8 μg/ml) stimulates matrix metalloproteinase 1 (BgMMP1) transcription. Using B. glabrata α (Bgα)-actinin as a reference, relative intensity of the BgMMP1 amplicon was greater in rSmVAL9-exposed Bge cells compared with cells treated with snail saline alone (CBSS) or rSmVAL9 Tris–glycine buffer alone (TG buffer). Results of two independent experiments are shown with inset histogram representing mean densitometric values of BgMMP1/Bgα-actinin expression + S.D.

Fig. 5

Schistosoma mansoni venom allergen-like 9 (SmVAL9) is an immunogenic IgG target recognised during experimental murine schistosomiasis. (A) Ten micrograms of recombinant (r)SmVAL9 or BSA were electrophoresed, blotted onto polyvinyl difluoride and probed with chronic mouse serum; obtained from mice experimentally infected for 14 weeks; 50 cercariae/mouse or normal mouse serum (both at 1:150). A 19.3 kDa, immunoreactive protein is specifically observed in the rSmVAL9 lane when probed with chronic mouse serum, but not normal mouse serum. (B) ELISA measurement of anti-rSmVAL9 IgG reactivity in mice experimentally infected (50 cercariae/mouse) with S. mansoni. Histograms represent mean (+S.D.) anti-rSmVAL9 total IgG absorbance (four to six animals/time-point) measured in mice at 2–14 weeks p.i. from two repeat experiments (black histograms, murine infection 1; white histograms, murine infection 2). Dashed line represents mean anti-rSmVAL9 reactivity + 3 S.D. derived from normal mouse serum.

Fig. 6

Schistosoma mansoni venom allergen-like 9 (SmVAL9) differentially activates matrix metalloproteinase and tissue inhibitors of metalloproteinase expression in murine bone marrow-derived macrophages. Bone marrow-derived macrophage cultures were stimulated with Tris–glycine buffer (recombinant (r)SmVAL9 solvent; white bars), rSmVAL9 (10 μg/ml; black bars) or rSmVAL9 (10 μg/ml) in the presence of polymyxin B (10 μg/ml; grey bars) for 20 h. The relative expression of MMP2, MMP9, MMP12, MMP13, MMP14 (MT-MMP1), MMP28, TIMP1 and TIMP2 was measured via quantitative reverse transcription-PCR and compared with untreated macrophage populations. Histograms represent mean MMP/TIMP expression (normalised fold change) ± S.E.M. (n = 3). Statistical significance was determined using ANOVA tests (P < 0.01) and post hoc t-tests where appropriate. Mean values sharing the same italicised letter are not significantly different.