Schistosoma mansoni Stomatin like protein-2 is located in the tegument and induces partial protection against challenge infection

Abstract

Background: Schistosomiasis affects more than 200 million individuals worldwide, with a further 650 million living at risk of infection, constituting a severe health problem in developing countries. Even though an effective treatment exists, it does not prevent re-infection, and the development of an effective vaccine still remains the most desirable means of control for this disease.

Methodology/principal findings: Herein, we report the cloning and characterization of a S. mansoni Stomatin-like protein 2 (SmStoLP-2). In silico analysis predicts three putative sites for palmitoylation (Cys11, Cys61 and Cys330), which could contribute to protein membrane association; and a putative mitochondrial targeting sequence, similar to that described for human Stomatin-like protein 2 (HuSLP-2). The protein was detected by Western blot with comparable levels in all stages across the parasite life cycle. Fractionation by differential centrifugation of schistosome tegument suggested that SmStoLP-2 displays a dual targeting to the tegument membranes and mitochondria; additionally, immunolocalization experiments confirm its localization in the tegument of the adult worms and, more importantly, in 7-day-old schistosomula. Analysis of the antibody isotype profile to rSmStoLP-2 in the sera of patients living in endemic areas for schistosomiasis revealed that IgG1, IgG2, IgG3 and IgA antibodies were predominant in sera of individuals resistant to reinfection as compared to those susceptible. Next, immunization of mice with rSmStoLP-2 engendered a 30%-32% reduction in adult worm burden. Protective immunity in mice was associated with specific anti-rSmStoLP-2 IgG1 and IgG2a antibodies and elevated production of IFN-gamma and TNF-alpha, while no IL-4 production was detected, suggesting a Th1-predominant immune response.

Conclusions/significance: Data presented here demonstrate that SmStoLP-2 is a novel tegument protein located in the host-parasite interface. It is recognized by different subclasses of antibodies in patients resistant and susceptible to reinfection and, based on the data from murine studies, shows protective potential against schistosomiasis. These results indicate that SmStoLP-2 could be useful in a combination vaccine.

Figure 1. The complete protein sequence of SmStoLP-2 in relation to other members of the stomatin gene family.

ClustalX alignment of the derived amino acid sequence of SmStoLP-2 (EU531730) with S. japonicum StoLP-2 (AAX30477), D. rerio Hypothetical (NP_957325), H. sapiens SLP-2 (NP_038470), S. mansoni stomatin excluding the first 120 amino acids (GeneDB accession no. Smp_162440), C. elegans Mec-2 excluding the first 50 amino acids (NP_741797), S. mansoni Mec-2 (GeneDB accession no. Smp_122810), H. sapiens stomatin (NP_004090), H. sapiens SLP-3 (NP_660329), M. musculus SLP-3 (NP_694796), H. sapiens SLP-1 (NP_004800) and C. elegans Unc-24 (NP_501335). Highlighted are the putative transmembrane domains as predicted by TMHMM (continuous box) absent in all SLP-2, the stomatin signature sequence (dashed box), mitochondrial targeting sequence as predicted by MitoProt II (dashed and dotted box), sites for palmitoylation (underlined). The regions with high identity and similarity between stomatin sequences are shown as black and gray columns, according to the Clustal X algorithm.

Figure 2. SDS-PAGE (4%–12%) analysis of cell extracts and fractions from E. coli (BL21DE3) transformed with the pDEST17-SmStoLP-2, and immunoblotting of protein extracts from S. mansoni stages and fractions using anti-rSmStoLP-2 polyclonal antibodies.

(A) Lanes 1 and 2 represent a clone before and after induction with 1 mM IPTG, respectively; (B) Inclusion bodies were extracted with urea and denatured protein was refolded by dilution before being purified through Ni²⁺-charged column chromatography. Lanes 1 and 2 show the soluble fraction after lysis and the inclusion bodies after solubilization with 8 M urea, respectively. Lanes 3–5 and 7–9 show the fractions of rSmStoLP-2-6xHIS-tag fusion protein eluted after Ni²⁺ chromatography, Lane 6, MW ladder (kDa); (C) Immunoblotting of S. mansoni extracts from different stages using anti-rSmStoLP-2 polyclonal antibodies (20 µg of protein were loaded in each lane). Cer – cercariae, Sch – 7-day-old schistosomula, Ad – adult worms, Egg – eggs, Mir – miracidia. (D) Western blot of soluble (S) and insoluble (I) protein extracts of 10-day-old schistosomula (Sch) and adult worms (Ad); (E) Detection of SmStoLP-2 in the tegument of S. mansoni adult worms, (1) proteins soluble in urea and thiourea, (2) proteins soluble in urea, thiourea, CHAPS and SB 3–10, (3) proteins soluble in 2% SDS. (F) Dual targeting of SmStoLP-2 to tegumental membranes and tegumental mitochondria, TWM, tegument extract without surface membranes, TSM, tegument enriched in surface membranes and TFM, tegument fraction enriched in mitochondria (20 µg of protein were loaded in each lane), Mfn-1 – is the Mitofusin-1 mitochondrial marker. Arrows indicate the rSmStoLP-2 and the most reactive bands of native SmStoLP-2 detected in each experiment. Positions of molecular mass standards (kDa) are indicated on the right or in the center. Positive control (P) contains 50–60 ng of rSmStoLP-2.

Figure 3. Immunolocalization of SmStoLP-2 in S. mansoni tegument.

Fluorescence confocal microscopy images (Fluor) and corresponding differential interface contrast (DIC) images of male (A–D, O, and P), female adult worms (E–H), and schistosomulum (I–N and R–T) of S. mansoni are shown. Polyclonal anti-rSmStoLP-2 and secondary antibody coupled to Alexa 488 (green) were used for fluorescence detection of SmStoLP-2. Serum from naive rat was used as negative control for male (C), female (G) and schistosomulum (L). DAPI (blue) was used for nucleus localization (E, G, O, and P), and phalloidin rhodamine (red) was used for actin localization (J, K, M, O, P and T); (M) Diagram of the S. mansoni tegument and an associated cell body (not to scale). Cy: cytoskeleton; bm: basal membrane; n: nucleus; s: spine (extracted and modified from [43]).

Figure 4. Isotype profile of sera from schistosomiasis patients reactive to rSmStoLP-2.

Analysis of (A) IgG, (B) IgA and (C) IgG subclass antibody responses in sera of infected patients (INF), individuals susceptible to reinfection (SR), resistant to reinfection (RR) and non-infected individuals (NI). Results are expressed as means of individual measurements. Error bars indicate S.D. of the means. # Statistically significant as compared to the non-infected group (P<0.05). * Statistically significant as compared to the group susceptible to reinfection.

Figure 5. Cytokine profile of mice immunized with rSmStoLP-2.

Ten days after the last immunization, splenocytes were isolated and assayed for (A) IFN-γ, (B) TNF-α and (C) IL-10 production in response to rSmStoLP-2 (25 µg/ml) or medium as control. The results are presented as mean ± S.D. for each group. Significant differences of secreted cytokines after rSmStoLP-2 stimulation or non-stimulated splenocytes are denoted by an asterisk (p<0.05).

Figure 6. Scattergram of worm burden from two independent experiments of mice immunized with rSmStoLP-2 and challenged with live S. mansoni cercariae.

Statistical analyses were performed with Student's t-test, *statistically significant (p<0.05) compared to control group (CFA/IFA).