Schistosoma mansoni Stomatin Like Protein-2 Is Located in the Tegument and Induces Partial Protection against Challenge Infection

Schistosomiasis is a parasitic disease causing serious chronic morbidity in tropical countries. Together with the publication of the transcriptome database, a series of new vaccine candidates were proposed based on their functional classification. However, the prediction of vaccine candidates from sequence information or even by proteomics or microarrays data is somewhat speculative and there remains the considerable task of functional analysis of each new gene/protein. In this study, we present the characterization of one of these molecules, a stomatin like protein 2 (SmStoLP-2). Sequence analysis predicts signals that could contribute to protein membrane association and mitochondrial targeting, which was confirmed by differential extractions of schistosome tegument membranes and mitochondria. Additionally, confocal microscope analysis showed SmStoLP-2 present in the tegument of 7-day-old schistosomula and adult worms. Studies in patients living in endemic areas for schistosomiasis revealed high levels of IgG1, IgG2, IgG3 and IgA anti-SmStoLP-2 antibodies in individuals resistant to reinfection. Recombinant SmStoLP-2 protein, when used as vaccine, induced significant levels of protection in mice. This reduction in worm burden was associated with a typical Th1-type immune response. These results indicate that SmStoLP-2 could be useful in association with other antigens for the composition of a vaccine against schistosomiasis

On the three-finger protein domain fold and CD59-like proteins in Schistosoma mansoni

Background: It is believed that schistosomes evade complement-mediated killing by expressing regulatory proteins on their surface. Recently, six homologues of human CD59, an important inhibitor of the complement system membrane attack complex, were identified in the schistosome genome. Therefore, it is important to investigate whether these molecules could act as CD59-like complement inhibitors in schistosomes as part of an immune evasion strategy. Methodology/Principal Findings: Herein, we describe the molecular characterization of seven putative SmCD59-like genes and attempt to address the putative biological function of two isoforms. Superimposition analysis of the 3D structure of hCD59 and schistosome sequences revealed that they contain the three-fingered protein domain (TFPD). However, the conserved amino acid residues involved in complement recognition in mammals could not be identified. Real-time RT-PCR and Western blot analysis determined that most of these genes are up-regulated in the transition from free-living cercaria to adult worm stage. Immunolocalization experiments and tegument preparations confirm that at least some of the SmCD59-like proteins are surface-localized; however, significant expression was also detected in internal tissues of adult worms. Finally, the involvement of two SmCD59 proteins in complement inhibition was evaluated by three different approaches: (i) a hemolytic assay using recombinant soluble forms expressed in Pichia pastoris and E. coli; (ii) complement-resistance of CHO cells expressing the respective membrane-anchored proteins; and (iii) the complement killing of schistosomula after gene suppression by RNAi. Our data indicated that these proteins are not involved in the regulation of complement activation. Conclusions: Our results suggest that this group of proteins belongs to the TFPD superfamily. Their expression is associated to intra-host stages, present in the tegument surface, and also in intra-parasite tissues. Three distinct approaches using SmCD59 proteins to inhibit complement strongly suggested that these proteins are not complement inhibitors and their function in schistosomes remains to be determined.Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Grant Number:04/12872-3)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institute of Health, National Institute of Allergy and Infectious Diseases (NIH-NIAID), Grant AI-095893NIH-NIAID Grant AI-056273FAPESP 00/11624-

Fluorescence confocal microscopy images showing immunolocalization of SmCD59.1 and SmCD59.2 in whole mount and in transverse sections of <i>S. mansoni</i> adult worms.

<p>(A and B) Confocal projections of SmCD59.1 and SmCD59.2 protein in the tegument of whole mount adult male worms. (D and E) Fluorescence detection of SmCD59.1 and SmCD59.2 in transverse sections of <i>S. mansoni</i> adult worms. (C and F) Negative control, serum from naïve rat. Secondary antibody coupled to Alexa 488 (green) was used for SmCD59 localization. DAPI (blue) was used for nucleus localization (E and F) and Rhodamine Phalloidin (red) was used for actin localization (A, B and C). Arrows – tegument tubercules; M – male; F – female; p – parenchyma; mc – muscle cells.</p

ClustalX multiple sequence alignment of the mature protein sequence (excluding the signal peptide) of TFPDs from platyhelminthes, CD59 and Ly6.

<p>The regions with high identity and similarity between sequences are shown as black and gray columns, according to the ClustalX algorithm. Arrows indicate highly conserved Cysteines and Asparagines with a C and an N, respectively. Dashed lines represent pairs of cysteine residues forming disulfide bonds determined from Hs-CD59 (red) and predicted for SmCD59.2 (black). Only for SmCD59 sequences, potential sites for N-glycosylation are shown in blue with asparagine (N) in red, and potential sites for GPI anchor are shown in yellow. Human CD59 active sites are shaded in red. The sequences abbreviation are: <i>Schistosoma mansoni</i> (SmCD59.1-7), <i>Schistosoma japonicum</i> (Sj1, Sj2.3, Sj3, Sj4.1, Sj6), <i>Schistosoma hematobium</i> (Sh1-3 and Sh5-7), <i>Clonorchis sinensis</i> (Cs-757, Cs-8328, Cs-8627), <i>Opisthorchis viverrini</i> (Ov-8524, Ov-3995 and Ov-6738), <i>Fasciola hepatica</i> (Fh-6273), <i>Fasciola gigantica</i> (Fg-25430 and Fg-15245), <i>Schmidtea mediterranea</i> (Smed), <i>Equus caballus</i> (Ec-Ly6), <i>Pongo abelii</i> (Pa-Ly6 and Pa-CD59), <i>Macaca mulatta</i> (Mam-Ly6), <i>Mus musculus</i> (Mm-Ly6 and Mm-CD59), <i>Monodelphis domestica</i> (Md-Ly6), <i>Ornithorhynchus anatinus</i> (Oa-Ly6), <i>Homo sapiens</i> (Hs-Ly6 and Hs-CD59), <i>Saimiriine herpesvirus</i> (Sah-CD59), <i>Rattus norvegicus</i> (Rn-CD59) (the accession numbers are listed in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002482#pntd.0002482.s006" target="_blank">Table S2</a>).</p

Evaluation of the ability of SmCD59.1 and SmCD59.2 to modulate complement activity.

<p>Hemolytic assays were performed after incubating normal human serum (NHS) with different amounts of SmCD59.1 (produced in <i>P. pastoris</i>), SmCD59.2 (produced in <i>P. pastoris</i> or <i>E. coli</i>), or BSA (negative control). (A) Treated NHS was then incubated with rabbit erythrocytes (Alternative Pathway) or (B) antibody-sensitized sheep erythrocytes (Classical Pathway). The percentage of hemolysis was calculated in comparison with erythrocytes suspensions completely lysed with water (100% lysis). The volume of NHS used in these assays corresponds to the amount that promotes 50% lysis of erythrocytes. Each column represents the mean of three independent experiments ± SD.</p

Complement resistance of CHO cells expressing SmCD59.1, SmCD59.2 and hCD59 (positive control).

<p>Viable cells were quantified by flow cytometry following exposure to anti-CHO antibodies and normal human serum (NHS) or heat-inactivated serum (iNHS). Black histograms are CHO cells transfected with hCD59 (A), SmCD59.1 (B) or SmCD59.2 (C), respectively. Red histograms are control cells transfected with empty pcDNA vector (A, B and C). The first peak in both black and red histograms represents viable cells and the second peak represents dead cells stained by propidium iodide (PI). (D) Cell viability of CHO cells transfected with hCD59 or pcDNA treated with anti-CHO antibodies and NHS or iNHS in three independent experiments (mean ± SD). P values are indicated.</p

Immunoblotting of protein extracts from <i>S. mansoni</i> stages using anti-rSmCD59.1 or anti-rSmCD59.2 polyclonal antibodies.

<p>Protein extracts (20 µg) from different <i>S. mansoni</i> stages: EGG (eggs), MIR (miracidia), CER (cercariae), SCH (<i>in vitro</i> 7-day-old schistosomula), ♂ (male adult worm), ♀ (female adult worm) and P (positive control, 100 ng of rSmCD59.1) were analyzed using (A) anti-rSmCD59.1 antiserum; or (B) anti-rSmCD59.2 antiserum, P (positive control, 100 ng of rSmCD59.2 expressed in <i>E. coli</i>). Extracts of stripped worms (Strip) and Tegument (Teg) of adult worms were probed with (C) anti-rSmCD59.1 or (D) anti-rSmCD59.2 antisera. Insoluble (Ins) and soluble (Sol) protein extracts of stripped worms; (Tsm) enriched tegument surface membranes fraction; (Twm) tegument extract without surface membranes. Positions of molecular mass standard are indicated.</p

Homology modeling of SmCD59.2.

<p>Ribbon display, side view (A) and top view (B). The models were generated with Modeller 5.1 using 3-D structure of human CD59 as a template (2UWR). The quality of the model was assessed with Procheck. Disulfide bridges are visualized as magenta sticks, and the three finger-shaped backbone is visualized as projections emerging from the disulfide bridges.</p