Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins

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Abstract

Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development.

Author Summary

Schistosomiasis is caused by blood flukes residing in the veins of infected individuals and afflicts millions of people in the developing world. The schistosome worms can remain healthy in the bloodstream for more than 10 years, implying an extraordinary ability to evade host immune damage. Scientists are seeking to understand immune evasion so as to find weaknesses in defenses that can be exploited in the development of effective vaccines. Here we investigate the normal antibody response to schistosomes during infection of mice, rats and humans, and show for the first time that this response is highly skewed to the recognition of a small number of proteins present at the worm surface. Surprisingly, these abundant antibodies recognize their targets only when the proteins retain their native conformations, stabilized by the presence of intramolecular disulfide bridges. Because of this conformational-dependence, these antibodies have remained undetected in prior studies in which antibody binding assays were routinely performed in a reducing environment that destroys disulfide bridges. The routine presence of these antibodies within the serum of schistosome infected patients and animals raises new and interesting questions as to their possible role in immune evasion, and has significant implications for schistosomiasis vaccine development.

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Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection

Fernanda Cardoso, Gilson C. Macedo, Elisandra Gava … (2008)

Introduction Schistosomiasis mainly occurs in developing countries and is the most important human helminth infection in terms of global mortality. This parasitic disease affects more than 200 million people worldwide causing more than 250,000 deaths per year [1]. Furthermore, schistosomiasis causes up to 4.5 million DALY (disability adjusted life year) losses annually [2]. Recently, King et al [3] have associated schistosomiasis with anemia, pain, diarrhea, exercise intolerance, and under-nutrition that results from chronic infection. Current schistosomiasis control strategies are mainly based on chemotherapy but, in spite of decades of mass treatment, the number of infected people remains constant [4]. Extensive endemic areas and constant reinfection of individuals together with poor sanitary conditions in developing countries make drug treatment alone inefficient [5]. Many consider that the best long-term strategy to control schistosomiasis is through immunization with an anti-schistosomiasis vaccine combined with drug treatment [6]. A vaccine that induces even a partial reduction in worm burdens could considerably reduce pathology and limit parasite transmission [7]. Sm29 protein has been characterized by our group [8], proving to be a membrane-bound antigen on adult worms that is strongly recognized by IgG1 and IgG3 antibodies of naturally resistant individuals and patients resistant to re-infection living in endemic areas for schistosomiasis in Brazil. Recent studies, using microarrays and RT-PCR, showed that Sm29 is among the 16% most highly expressed genes in S. mansoni, and probably serves an important function in the surface biology of this parasite [9],[10]. Proteomic analysis of the S. mansoni tegument composition identified Sm29 as one of the integral proteins to be consistently found in the outer surface [11]. Therefore, the next step would be to investigate humoral and cellular immune responses induced by Sm29 in vaccinated mice and protection studies. Herein, we determined that Sm29 is present on the tegument of lung-stage and male and female adult worms of S. mansoni by confocal microscopy. Besides, rSm29 induced a Th1-type of immune response in mice and reduction in worm burden and liver pathology. Methods Mice and parasites C57BL/6 and TLR4 KO female mice, 6–8 weeks old, were obtained from the Federal University of Minas Gerais (UFMG) animal facility. All procedures involving animals were approved by the local Ethics Committee on Animal Care (CETEA-UFMG). Cercariae of S. mansoni (LE strain) were maintained routinely in Biomphalaria glabrata snails at Rene Rachou Research Center (Fiocruz, Brazil) and prepared by exposing infected snails to light for 2 hrs to induce shedding of parasites. Cercarial numbers and viability were determined using a light microscope prior to infection. The protocols involving animals used in this study were approved by the Federal University of Minas Gerais Ethics Committee in Animal Experimentation (CETEA No. 023/2005). Antigen preparation The recombinant Sm29 was produced and purified as described previously [8]. Briefly, the Sm29 cDNA fused with a C-terminal 6x histidine was produced in E.coli using the pET21a expression vector (Novagen, NJ, USA). The recombinant Sm29 was purified in an affinity column and dialyzed against PBS pH 7.0. Immunolocalization of Sm29 in male, female and lung-stage schistosomula of S. mansoni Adult worms used in confocal microscopy studies were recovered from perfused mice and lung-stage schistosomula were prepared according to the method described by Harrop & Wilson [12]. Parasites fixed in Omnifix II (Ancon Genetics, St Petersburg, FL, USA) were used in whole mount or in section assays. For sections assays, 7 μm slices were deparaffinized with xylol series. Parasites were blocked with 1% BSA in PBST (Tween 20 0.05%) for 1 hr and incubated with anti-rSm29 serum diluted 1∶20 in blocking buffer. Serum from non-immunized mice was used as a negative control. Samples were washed three times with PBST and incubated with anti-mouse IgG antibody conjugated to Alexa Fluor 594 (Molecular Probes, CA, USA) diluted 1∶100 in blocking buffer containing Phalloidin Alexa Fluor 488 (Molecular Probes) to stain actin microfilaments. The samples were washed four times and mounted in antifade reagent (Prolong gold–Molecular Probes). For whole mount assays, parasites were blocked with 1% BSA, 0.1% Triton X-100, 0.1% azide in PBS for two hours at 4°C under agitation. Blocked parasites were incubated with anti-Sm29 serum diluted 1∶80 in the blocking buffer during 16hrs at 4°C under agitation. Samples were washed six times with PBST and incubated with anti-mouse and phalloidin, both diluted 1∶300, during 4 hours at 4°C under agitation. The samples were washed five times and mounted in 90% glycerol and 10% TRIS, pH 9.0. The parasites were visualized in a Zeiss 510 Meta confocal microscope using an immersion objective. All the parameters and microscope settings used were maintained throughout the process. Tegument purification Cercariae obtained from Biomphalaria glabrata snails exposed to light for two hours were transformed mechanically in skin-stage schistosomula according to Ramalho-Pinto et al [13]. First, cercariae were incubated on ice for 30 minutes and centrifuged for 3 minutes, 1000 rpm, 4°C. The cercariae were resuspended in 1ml of cold ELAC (Earle's salts plus lactalbumin hydrolysate) contain 0.5% lacto albumin, 1% penicillin/streptomycin and 0.17% glucose. The tails were broken by vortex in high speed during 2 minutes. After this, the tails were removed through six washes with ELAC. The schistosomula were incubated for 1 hour and 30 minutes at 37°C in ELAC and washed with apyrogenic physiologic saline. For the tegument removal, the schistosomula were submitted to vortex using high speed, two times of eight minutes each, in a CaCl2 0.3 M solution. The sample was centrifuged at 1000 rpm for 1 minute. The supernatant was collected and centrifuged at 26500 rpm for 1 hour at 4°C. The pellet was dialyzed against physiologic saline 1.7%. Western blot analysis Two micrograms of purified skin-stage schistosomula tegument were submitted to SDS- PAGE 12%, transferred to a nitrocellulose membrane (Millipore) and blocked 16 hours at room temperature with TBST (0.5M NaCl, 0.02M Tris pH 7.5, 0.05% tween, 5% skim milk). The membranes were washed tree times with TBST and probed with anti-Sm29 or naive mice serum diluted 1∶500 in TBST for 4 hours at room temperature. The membranes were washed six times and probed one hour with anti-mouse IgG conjugated to alkaline phosphatase (Invitrogen) diluted 1∶10000 in TBST. After six washes, the membranes were developed using ECF subtract (GE Healthcare) and visualized in a Storm apparatus (GE Healthcare). Immunization of mice Six to eight week old female C57BL/6 mice were divided into two groups of ten mice each. Mice were subcutaneously injected in the nape of the neck with 25 μg rSm29 on days 0, 15 and 30. Sm29 protein concentration for vaccination was determined as previously described [14]. The recombinant protein was formulated with Freund's adjuvant (complete Freund's adjuvant/CFA for the first immunization and incomplete Freund's adjuvant/IFA for the boosters). In the control group, PBS with Freund's adjuvant was administered using the same immunization protocol. Challenge infection and worm burden recovery Fifteen days after the last boost, mice were challenged through percutaneous exposure of abdominal skin for 1 h in water containing 100 cercariae (LE strain). Forty-five days after challenge, adult worms were perfused from the portal veins. Two independent experiments were performed to determine protection levels. The protection was calculated by comparing the number of worm recovered from each vaccinated group with its respective control group, using the formula: where PL = protection level, WRCG = worms recovered from control group, and WREG = worms recovered from experimental group. Oogram and histophatological analysis Following perfusion for the recovery of the schistosomes, fragments of the intestine (terminal ileum) from each animal were separated and transferred to the Petri dishes containing saline as previously demonstrated [15]. The intestines were opened lengthwise and the excess mucus was removed. One-centimeter fragments were weighed and placed between a glass slide and a plastic cover. The preparation was inverted and pressed on a rubber surface padded with filter paper. Then, the slices were examined with a microscope (100 x) and all the eggs were counted. Quantitative oograms were obtained, using the formula: number of eggs/grams of tissue = number of eggs in intestinal fragment/weight of fragment in mg. A qualitative oogram evaluation was performed, in which the developmental stages of the eggs were classified as described previously. The liver fragments were collected from the same animals analyzed in oogram studies and fixed in 10% paraformaldehyde. The fragments were processed for paraffin embedding and histopathological sections performed using microtome at 6–7 μm and stained in a slide with hematoxilin-eosin (HE). The number of granulomas was obtained from the liver sections using 10x objective in a microscope. The area from each liver section was calculated using capture in scanner followed by analysis in the KS300 software connected to a Carl Zeiss image analyzer. Measurement of specific anti-Sm29 antibodies Following immunization, sera of ten mice from each experimental group were collected at two-week intervals. Measurement of specific anti-Sm29 antibodies was performed using indirect ELISA. Maxisorp 96-well microtiter plates (Nunc, Denmark) were coated with 5 μg/ml rSm29 in carbonate-bicarbonate buffer, pH 9.6 for 16 h at 4°C, then blocked for 2 at room temperature with 200 μl/well PBST (phosphate buffer saline, pH 7.2 with 0.05% Tween-20) plus 10% FBS (fetal bovine sera). One hundred microliters of each serum diluted 1∶100 in PBST was added per well and incubated for 1 h at room temperature. Plate-bound antibody was detected by peroxidase-conjugated anti-mouse IgG, IgG1 and IgG2a (Southern Biotechnology, CA, USA) diluted in PBST 1∶10000, 1∶5000 and 1∶2000, respectively. Color reaction was developed by addition of 100 μl per well of 200 pmol OPD (o-phenylenediamine, Sigma) in citrate buffer, pH 5.0 plus 0.04% H2O2 for 10 min and stopped with 50 μl of 5% sulfuric acid per well. The plates were read at 495 nm in an ELISA plate reader (BioRad, Hercules, CA). Cytokine analysis Cytokine experiments were performed using splenocyte cultures from individual mice immunized with rSm29 plus CFA/IFA (n = 5 for each group). Splenocytes were isolated from macerated spleens of individual mice 10 days after the third immunization and washed twice with sterile PBS. After washing, the cells were adjusted to 1×106 cells per well for IL-4, IL-10, IFN-γ and TNF-α assays in RPMI 1640 medium (Gibco, CA, USA) supplemented with 10% FBS, 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 250 ng/ml amphotericin B. Splenocytes were maintained in culture with medium alone or stimulated with rSm29 (25 μg/ml) or concanavalin A (ConA) (5 μg/ml) as previously described [14],[16]. The 96-well plates (Nunc) were maintained in an incubator at 37°C with 5% CO2. For cytokine assays polymyxin B (30 μg/ml) was added to the cultures and this treatment completely abrogate the cytokine response to LPS as previously described [17]. Culture supernatants were collected after 24 h of ConA stimulation, 48 h of rSm29 stimulation for IL-4 and TNF-α analysis and 72 h of rSm29 stimulation for IL-10 and IFN-γ. The assays for measurement of IL-4, IL-10, IFN-γ and TNF-α were performed using the Duoset ELISA kit (R&D Diagnostic) according to the manufacturer's directions. Innate immune responses were assessed using peritoneal macrophages culture obtained from C57BL/6 and TLR4 KO mice that had received an intra-peritoneal injection of thioglycolate four days earlier. The macrophages were recovered through peritoneal washes with PBS and adjusted to 1×106 cells per well for IL-12p40 assays in RPMI 1640 medium (Gibco) supplemented with 5% FBS, 100U/ml penicillin G sodium and 100 μg/ml streptomycin sulfate. Macrophages were maintained in culture with medium alone or stimulated with rSm29 (25 μg/ml) or LPS purified from E. coli (1 μg/ml). Culture supernatants were collected after 24 hrs of stimulation. The assays for measurement of IL-12p40 were performed using the Duoset ELISA kit (R&D Diagnostic) according to the manufacturer's directions. Microarray analysis of gene expression in S. mansoni worms Total RNA was obtained using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA was quantified using the Nanodrop ND-1000 UV/Vis spectrophotometer and RNA integrity was checked by electrophoresis with an Agilent 2100 Bioanalyzer. One microgram of total RNA from each sample was amplified using the T7-RNA polymerase-based linear amplification protocol. Three micrograms of aminoallyl-modified amplified RNA was labeled with Cy3 or Cy5 by indirect labeling (GE Healthcare). The Cy3- and Cy5-labeled RNA samples were combined and hybridized overnight to cDNA microarray slides using ASP hybridization chambers (GE Healthcare) at 42°C and the protocols were followed as recommended by manufacturer [18]. Slides were scanned at 5 μm resolution, 100% laser power (GenePix 4000B, Molecular Devices, USA) and PMT levels were adjusted in order to obtain similar average intensities of red and green signals. Each array contained 4,000 different elements, spotted in duplicate on the left and the right sides of a glass slide (GEO accession: GPL3929). The cDNA clones spotted on the arrays were selected to represent 4,000 unique transcripts that were identified in the large-scale S. mansoni transcriptome project [19]. RNA from the pool of adult worms recovered from mice that had been immunized with the rSm29 vaccine (test sample) was hybridized against RNA from control adult worms recovered from mice treated only with adjuvant (control sample). Each assay consisted of two separate hybridizations, the first with Cy5-labeled test sample and Cy3-labeled control, and the second with dye-swap in order to account for dye bias; for each probe, the intensity of the test sample was computed as the average intensity of the Cy5 and Cy3 measurements; the same was computed for the control sample. Each assay provided two average values for the test and two for the control, arising from the duplicated copies of each gene that are spotted on the left and right halves of the glass slides [18]. A total of four hybridizations were performed, corresponding to two sets of technical replicate assays. Thus, a total of four different average intensity values for the test and four for the control sample were obtained for each probe on the array. Data was extracted from images using the Array Vision 8.0 program. We used a locally weighted linear regression (LOWESS) algorithm to correct for systematic bias due to small differences in the labeling and/or detection efficiencies between the fluorescent dyes [20]. Normalized log2 ratios of the intensities of test/control were used for the statistical analysis with the Significance Analysis of Microarrays tool (SAM) using a 0.14% false discovery rate (FDR), which corresponds to a q-value (analogous to the t-test p-value) of q<0.01, to find significant differentially expressed genes [21]. Genes determined in the previous step were filtered using a median fold-change ≥1.5 as cutoff, where fold-change = 2ˆ|log2 (test/control)|. The fold-change filter was applied after identification of significant differentially expressed genes with the use of SAM, therefore avoiding the bias caused by the use of arbitrary thresholds to trim datasets before significance testing [22]. Putative identity of regulated genes Regulated genes were manually annotated by similarity using BLASTx and the GenBank nr database with a cut-off e-value = 10−10. Sequences were placed into four categories according to the similarity matches, as follows: tegument proteins, membrane proteins, receptor interacting proteins, lipid metabolism and other functions. Real-time RT-PCR Total RNA was extracted with Trizol reagent (Invitrogen), according to the manufacturer's instructions. RNA samples were treated with DNAse, purified and concentrated using RNeasy Micro Kit (QIAGEN), following the manufacturer instructions and 1.5 μg of each sample was reverse transcribed using the SuperScript® III First-Strand Synthesis SuperMix (Invitrogen). Specific primer pairs (Table S2) were designed by Primer Express Program using default parameters (Applied Biosystem) and arbitrarily named primers 1 and 2. Real-time RT-PCRs were run in triplicates in a volume of 20 μl containing 10 μl of Sybr Green PCR Master Mix (Applied Biosystem), 160 nmol of each primer (primers 1 and 2), 0.30 μl cDNA from reverse transcription. Real-time RT-PCR was performed with the 7300 Real-Time PCR System (Applied Biosystem) using the following cycling parameters: 60°C for 10 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec and 60°C for 1 min, and a dissociation stage of 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec, 60°C for 15 sec. Real time data was normalized in relation to the level of expression of actin. p-values were determined for the triplicates with Student's t-test, using one tail distribution and heteroscedastic variance. Microarray data public deposition information All microarray data were deposited in the GEO public database under Accession No. GSE10777. Statistical analysis Statistical analysis was performed with Student's t-test for comparison between two experimental groups using the software package GraphPad Prism. Results Sm29 is a tegument protein of S. mansoni In the present study, we demonstrated the localization of Sm29 in the male and female adult worms and also in the lung-stage schistosomula of S. mansoni using specific antibodies to rSm29 produced in E. coli. The native Sm29 was located exclusively on the surface of lung-stage schistosomula and male adult worms of S. mansoni (Fig. 1A, B, C, D, and Video S1). The female adult worm showed Sm29 located on the surface and also in internal tissues (Fig. 1E). Confocal microscopy analysis also revealed that Sm29 is not present in the cercariae stage of S. mansoni (Figure S1), as expected from the absence of mRNA coding for this antigen in cercariae cDNA library [8]. Additionally, we detected Sm29 in the purified tegument fraction of the skin-stage schistosomula by western blot analysis using specific antibodies to rSm29 (Fig. 2). 10.1371/journal.pntd.0000308.g001 Figure 1 Immunolocalization of Sm29 antigen on male and female adult worm and lung-stage schistosomula of S. mansoni. Polyclonal anti-Sm29 antibodies, serum from mice that received Freund́s adjuvant as negative control, and Cy5-conjugated anti-mice IgG were used. Actin was visualized by falloidin-Alexa fluor 488. The parasites were fixed in Omnifix II and used to whole-mount or section immunolocalization. (A and B) Whole-mount immunolocalization of Sm29 antigen on the surface (outer tegument) of male adult worm and (C) lung-stage schistosomula of S. mansoni. (D) Immunolocalization of Sm29 on the surface (outer tegument) of male adult worm, and (E) on the surface (outer tegument) and in some internal tissues on the female adult worm using deparaffinized sections of the parasites. Localization of Sm29 is identified by the orange color and actin filaments by the green color. 10.1371/journal.pntd.0000308.g002 Figure 2 Identification of Sm29 on skin-stage schistosomula tegument by Western blot. Two micrograms of purified skin-stage schistosomula tegument was applied onto 12% SDS-PAGE and transferred to a nitrocellulose membrane by western blot. After that, the membrane was probed with serum from rSm29 vaccinated mice or naïve animals diluted 1∶500 in TBST. Arrow indicates the native Sm29. The molecular mass markers, from top to bottom, are: 97, 66, 45, 30, 20.1, and 14.4 kDa. Antibody profile following mice immunization To evaluate the level of specific IgG, IgG1 and IgG2a antibodies to Sm29 sera from ten vaccinated animals of each group were tested by ELISA. Significant titers of specific anti-Sm29 IgG antibodies were detected at all time points studied after the first immunization (Figure 3). In order to determine the IgG isotype profile induced by vaccination, specific anti-Sm29 IgG1 and IgG2a antibodies levels were also evaluated. The levels of specific IgG1 and IgG2a increased at 15, 30 and 45 days after the first immunization (Table 1). Furthermore, IgG1/IgG2a ratio was reduced at days 30 and 45 that parallels with elevated anti-Sm29 IgG2a production. The IgG1/IgG2a ratio observed in mice immunized with rSm29 can lead us to speculate that a Th1 type of immune response was induced following vaccination. 10.1371/journal.pntd.0000308.g003 Figure 3 Kinetics of specific anti-Sm29 IgG induced in mice immunized with rSm29. Sera of ten immunized mice per group were collected at days 15, 30, 45, and 90 after the first immunization and assayed by ELISA. Arrows indicate the timing of vaccination. Results are presented as the mean absorbance measured at 492nm for each group. Results represent the mean of two independent experiments. Statistically significant differences of vaccinated mice compared to PBS+adjuvant control group is denoted by one asterisk for (p<0.05). 10.1371/journal.pntd.0000308.t001 Table 1 IgG1 and IgG2a immune profile induced by vaccination with recombinant Sm29. Daysa Groups IgG1 IgG2a IgG1/IgG2a ratio Sm29 PBS Sm29 PBS Sm29 15 0.821±0.559* 0.034±0.022 0.220±0.102* 0.011±0.081 3.72 30 1.290±0.326* 0.006±0.068 0.637±0.228* 0.049±0.070 2.02 45 1.623±0.159* 0.006±0.007 0.812±0.267* 0.031±0.017 1.99 a Days after the first immunization * Statistically significant compared to group of animals immunized with PBS with p<0.05 Vaccination induces a Th1-type of immune response in mice Mice vaccinated with rSm29 showed elevated production of IFN-γ, TNF-α and IL-10 and absence of IL-4 (Table 2). Cultures used for measuring the cytokines were treated with polymyxin B to avoid non-specific stimulation due to eventual LPS contamination in the purified recombinant protein [17]. Additionally, rSm29 has stimulated peritoneal macrophages from C57BL/6 (2016±423 pg/ml) or TLR4 KO (1108±197 pg/ml) mice to produce IL-12 (Fig. 4). It is worth to emphasize that rSm29 has activated macrophages to secrete IL-12 which drives Th1 cell development. Protective immunity in mice against S. mansoni infection, before the onset of egg production, is thought to be the result of a Th1 pattern of immune response [23]. 10.1371/journal.pntd.0000308.g004 Figure 4 Sm29 induces IL-12 and TNF-α production by macrophages from TLR4 knockout mice. Levels of IL-12 (p40) (A) or TNF-α (B) were measured in the supernatants of inflammatory macrophages from TLR4 KO or wild-type mice stimulated for 24 hrs with rSm29 (25 μg/ml), E. coli LPS (1 μg/ml) or medium alone. Significant differences from stimulated TLR4 KO or C57BL/6 macrophages in relation to non-stimulated cells are denoted by an asterisk (*) for p<0.05. 10.1371/journal.pntd.0000308.t002 Table 2 Parasitologic data, cytokine profile, intestinal egg counts and liver granuloma analysis of C57BL/6 mice vaccinated with rSm29 and challenged with 100 Schistosoma mansoni cercariae. Groups a Cytokine profile in splenocytes (pg/ml) Male worms Mean±SD Female worms Mean±SD Total worms Mean±SD (% protection) Intestinal eggs Mean±SD (% reduction) Number of granulomas/liver tissue μm2 10−7 Mean±SD (% reduction) PBS+CFA/IFA IL-4 7.8±3.0 28.2±8.1 29.4±6.4 57.7±12.2 16110.3±5755.2 6.74±1.3 IFN-γ 31.2±5.1 TNF-α 15.6±4.2 IL-10 31.3±3.1 rSm29+CFA/IFA IL-4 7.8±4.6 14.6±1.7 15.1±4.7 29.7±5.3* (51%) 6440.4±3644.2* (60%) 3.3±0.6* (50%) IFN-γ 2538.3±44.6 * TNF-α 426.3±231.5* IL-10 425.2±92.2* a This Table shows one experiment representative of two independent trials with similar results * Statistically significant compared to the control group (p<0.05) rSm29 engenders protective immunity To test the vaccine potential of rSm29 against schistosomiasis, we investigated the protection induced by this recombinant antigen in the murine model of S. mansoni infection. C57BL/6 mice were immunized three times with adjuvant-formulated rSm29, and then challenged with 100 S. mansoni cercariae. Control groups received adjuvanted phosphate-buffered saline. Mice vaccinated with rSm29 showed 51% reduction in adult worm burden, 60% reduction in intestinal eggs and 50% reduction in liver granuloma counts compared to control mice (Table 2). The most critical points to be considered about an efficient anti-schistosomiasis vaccine are the reduction in pathology and transmission [7], aspects that are present in the protection profile induced by rSm29 vaccine. Gene expression profile in worms from vaccinated mice In order to investigate the impact of rSm29 vaccination on S. mansoni, worms that were recovered from vaccinated mice had their gene expression profile analyzed using microarrays. This analysis revealed significant (q<0.01) regulation of 517 genes (Table S1) in worms recovered from mice vaccinated with adjuvanted rSm29 when compared to control mice injected with adjuvanted phosphate-buffered saline; of these, 495 genes (96%) were down-regulated and only 22 genes (4%) were up-regulated. Among down-regulated genes, several of them caught our attention and they fell into four major categories: tegument proteins (Sm23 and CD36-like class B scavenger receptor), membrane proteins (tetraspanin TE736), lipid metabolism (Sm14), receptor interacting proteins (B-cell receptor-associated protein and TGF-beta receptor interacting protein) and others (superoxide dismutase and Sm65) (Table 3). To validate the microarray data, we selected six genes (Sm23, Sm14, superoxide dismutase, CD36-like class B scavenger receptor, B-cell receptor-associated protein and TGF-β receptor interacting protein) and performed real-time RT-PCR. As demonstrated in Figure 5, real-time RT-PCR of all six tested genes confirmed the reduced expression previously determined by microarray analysis. Based on the data shown in Table 3 and Figure 5, we hypothesize that in rSm29 protected mice a reduced expression of these critical surface antigens enables the parasites to adapt to the host in the face of an ongoing antiparasite immune response developed in vaccinated animals. 10.1371/journal.pntd.0000308.g005 Figure 5 Transcript level changes in selected genes of S. mansoni worms recovered from mice vaccinated with rSm29. The figure shows real-time RT-PCR validation experiment for six genes down-regulated in S. mansoni worms recovered from rSm29+adjuvant immunized mice (black bars) compared to worms from PBS+adjuvant injected control mice (white bars). The selected genes were: CD36-CD36-like class B scavenger receptor [S. mansoni]; Sm23-Sm23 kDa integral membrane protein [S. mansoni]; Sm14-Fatty acid-binding protein Sm14 [S. mansoni]; Superox-Superoxide dismutase [S. mansoni]; TGF-β RIP-TGF-β receptor interacting protein 1 [C. sinensis]; B-cell RAP-B-cell receptor-associated protein-like protein [S. mansoni]. Statistically significant differences of vaccinated mice compared to PBS+adjuvant control group is denoted by one asterisk (p<0.03). 10.1371/journal.pntd.0000308.t003 Table 3 Putative identity, categories of biological function and fold change of a subset of down-regulated genes identified in worms recovered from animals vaccinated with rSm29 associated to adjuvant. Contig Putative identity Fold Changea Down-regulation with rSm29 plus adjuvant Tegument proteins C600716.1 CD36-like class B scavenger receptor [S. mansoni] 1.7 C605987.1 Integral membrane protein/sugar transporter family [B. vulgaris] 2.1 C601295.1 Sm23 kDa integral membrane protein [S. mansoni] 2.0 C607243.1 Alkaline phosphatase [M. musculus] 3.1 C610121.1 Annexin [S. mansoni] 2.0 Membrane proteins C604563.1 Tetraspanin TE736 [S. japonicum] 2.1 C609525.1 Transmembrane protein 49 [X. tropicalis] 2.6 C605890.1 ATPase, aminophospholipid transporter [B. Taurus] 1.9 C606907.1 Putative insulin receptor [E. multilocularis] 1.8 C609637.1 vacuolar proton pump [Danio rerio] 1.8 C609954.1 Autocrine motility factor receptor [T. castaneum] 1.5 C608609.1 Voltage-dependent anion channel 1 [X. laevis] 1.6 C601007.1 UDP dual transporter [C. elegans] 2.0 C609782.1 DAD-1-like protein/integral membrane [S. japonicum] 1.9 Receptors interacting proteins C610202.1 Serine/threonine kinase receptor associated protein [P. troglodytes] 1.6 C607204.1 Thyroid receptor interacting protein 13 [H. sapiens] 1.7 C603157.1 syndecan binding protein (syntenin) [D. rerio] 1.5 C606116.1 G protein beta subunit [P. fucata] 2.1 C600861.1 B-cell receptor-associated protein-like protein [S. mansoni] 1.5 C608810.1 rhoGAP protein [M. domestica] 2.7 C714516.1 TGF-beta receptor interacting protein 1 [C. sinensis] 1.8 Lipid metabolism C601385.1 SmINSIG [S. mansoni] 2.3 C601665.1 Fatty acid-binding protein Sm14 [S. mansoni] 1.5 C608771.1 Very low density lipoprotein binding protein [S. japonicum] 1.8 C711918.1 Fatty acid coenzyme A ligase 5 [H. sapiens] 1.7 Other C602958.1 Apoferritin-2 [S. japonicum] 1.7 C610848.1 Ferritin heavy chain 2 [S. mansoni] 2.1 C601146.1 IB1 protein [S. japonicum] 2.9 C608351.1 Tropomyosin [S. mansoni] 1.7 C600304.1 src tyrosine kinase [S. mansoni] 1.7 C603876.1 PrA2 protein [S. mansoni] 1.7 C607402.1 Superoxide dismutase [S. mansoni] 1.5 C604746.1 Sm65 antigen [S. mansoni] 1.5 a statistically significant, q<0.01 Discussion Schistosomiasis is one of the most important neglected tropical diseases (NTDs), and an effective control is unlikely in the absence of improved sanitation and a vaccine. Recently, the tegument proteome of S. mansoni was characterized, and one study in particular revealed the major proteins that are exposed on live adult parasites such as SmTSP-2 and Sm29 [11]. In this study, we investigated murine humoral and cellular immune responses to rSm29 and tested this molecule as a vaccine candidate. The strategic localization of Sm29 in the mammalian skin-stage and lung-stage schistosomula and its absence in cercariae suggests that this antigen has an important role in the adaptation of the parasite to the new environment when S. mansoni enters the mammalian host. Previous studies using mice vaccinated with irradiated cercariae showed that the lung is the key site of elimination of this parasite [24]. Sm29 localization on the surface of schistosomula is an important aspect of this antigen regarding its protective properties. In the mouse model, rSm29 induced high levels of anti-Sm29 IgG after the second immunization and showed reduced IgG1/IgG2a ratio at 45 days after the first immunization (Table 1). This finding suggests a tendency of a Th1 type of immune response induced by rSm29 vaccination. Further, we confirmed by cytokine analysis that rSm29 immunization elicited a Th1-type of immune response characterized by high levels of IFN-γ and no IL-4 and 51% of worm burden reduction. The involvement of IFN-γ in protective immunity to schistosomiasis is well documented in the murine model [25]. In the irradiated cercariae vaccination model, which induces high levels of protection, treatment with monoclonal anti-IFN-γ antibody totally abrogated the protective immunity achieved [26]. Similar results were obtained using IFN-γ knockout mice exposed to the radiation-attenuated vaccine confirming the essential role of IFN-γ in protective immunity against murine schistosomiasis [27]. Further, we tested the ability of rSm29 to induce IL-12 in peritoneal macrophages of TLR4 KO mice. TLR4 is the major receptor for macrophage activation by bacterial LPS [28]. Therefore, using TLR4 KO we obviated any possible effect of LPS contamination in IL-12 synthesis. In TLR4 KO mice, rSm29 has induced the production of large amounts of IL-12 that is the key cytokine involved in Th1 cell development. Pathology which results from granuloma formation around the eggs in murine schistosomiasis is characterized by Th2-type of immune response and the granuloma size can be reduced by neutralization of IL-4 [29]. Thus, morbidity and mortality in murine schistosomiasis were hypothesized to be developed as a direct consequence of the egg-induced Th2 type of immune response. Herein, the intestinal egg numbers was reduced (60%) following rSm29 vaccination compared to control group. Additionally, the immune response elicited by Sm29 was associated with 50% reduction in granuloma counts. Since we detected IL-10 production following splenocyte activation with rSm19, we hypothesize here that this cytokine might be regulating Th2 responses and/or preventing the development of highly polarized Th1 responses and therefore, reducing inflammation and liver pathology [30],[31]. In order to investigate the impact of rSm29 vaccination on S. mansoni, worms that were recovered from vaccinated mice had their gene expression profile analyzed using microarrays. In worms recovered from mice vaccinated with adjuvanted rSm29 when compared to a control group injected with adjuvanted phosphate-buffered saline, 495 genes (96%) were down-regulated and only 22 genes (4%) were up-regulated. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. We propose here that down-regulation of expression of important surface antigens is an important strategy of the parasite resulting in escape from host immune surveillance. As observed in Table 3, Sm23, tetraspanin TE736 (a paralog of Sm-TSP-2), Sm14, Sm65 and superoxide dismutase are significantly (q<0.01) down-regulated in adult-worms recovered from rSm29 protected mice; it is noteworthy that these genes encode important schistosome antigens that are recognized by immune mice and humans. Sm14 is able to induce cellular immune responses in individuals from endemic areas for schistosomiasis [32]. Sm23 and Sm65 are antigens recognized by sera from schistosome infected patients [33],[34]. Moreover, Sm14 and Sm23 induce partial protection in the murine model for schistosomiasis [35],[36]. Regarding tetraspanins, they belong to a family of membrane proteins, such as Sm-TSP-2, that is currently one of the most important vaccine candidates for schistosomiasis [37]. Further, vaccination of mice with naked DNA construct containing Cu/Zn cytosolic superoxide dismutase showed significant levels of protection compared to a control group [38]. It is noteworthy that no significant up-regulation of heat shock or chaperone genes was detected in schistosomes recovered from rSm29 vaccinated mice (Table S1), therefore excluding a common stress response. Rather, lowering the expression of critical surface antigens seems to be an important and specific strategy developed by schistosomes to reduce the damage caused by the host immune system. Therefore, a combined antigen vaccination with Sm29 and one or more of the proteins encoded by these down-regulated critical surface antigens might be the next logical step, thus circumventing the adaptive response of the parasite and eventually boosting the protective effect of vaccination. Among the down-regulated genes (Table 3) were those encoding protein domains from TGF-β receptor interacting protein, B-cell receptor-associated protein, CD36-like class B scavenger receptor, all of which are associated with host immune response. These parasite gene products could interact with or modulate the host immune response. For example, the scavenger receptors are present on different cells such as macrophages, platelets and neutrophils where they are involved with innate immunity by facilitating phagocytosis, cell adhesion and pathogen recognition. Recently, it was demonstrated that schistosome CD36-like class B scavenger receptor binds to host low density lipoproteins [39]. From the parasite perspective, it would not be difficult to envisage that a reduced expression of S. mansoni scavenger receptor-like molecules would decrease pathogen recognition and cell adhesion by host cells. Additionally, we found down-regulated genes involved with parasite carbohydrate and lipid metabolism, protein biosynthesis, intracellular signaling cascade, among others (Table S1). Down-regulated genes include SmINSIG (an ortholog of the mammal Insulin Induced Gene), a recently described gene in S. mansoni [40] that has a key role in HMG-CoA reductase degradation. HMG-CoA reductase is vital for egg production by S. mansoni [41]. An alternative hypothesis to our findings is that down-regulation of these genes may affect the development or survival of parasites in vaccinated animals, thus reducing worm burden, although the worms recovered from mice vaccinated with adjuvanted rSm29 are morphologically similar to those recovered from control mice. Additionally, haemoglobinases and Sm29 gene expression were unaltered in worms recovered from rSm29 immunized animals. Recently, Dillon et al [42] in an attempt to identify antigens responsible for protection induced by irradiated-cercariae vaccination performed microarray expression analysis of irradiated and normal worms. These investigators detected down-regulation of genes involved in neuromuscular activity and cell cycle. Their hypothesis is that attenuated parasite might have an extended stay in the host enhancing immune priming against exposed antigens [42]. Their findings may explain why irradiated-cercariae can elicit protective immunity when normal cercariae do not. Recent advances in antigen discovery with preclinical studies showing promising efficacy have reinvigorated the case for a schistosomiasis vaccine. The irradiated-cercariae vaccine and the use of recombinant antigens, such as Sm-TSP-2 and Sm29, that induce approximately 50% worm burden reduction, have demonstrated that a vaccine to schistosomes is achievable [37]. Furthermore, we identified an ortholog of Sm29 in the ESTs of the Asian schistosome S. japonicum that shares more than 50% identity in the amino acid sequence, indicating that a vaccine based on Sm29 might also be effective against S. japonicum [43]. Another important issue is the selection of a suitable adjuvant and/or delivery system to induce the appropriate immune responses. Herein, we used rSm29 with Freund́s adjuvant, however, it is not suitable for human application. Experiments are underway testing rSm29 with CpG or CpG plus alum as suggested by McManus & Loukas [44]. As a final conclusion of this work, we believe that Sm29 is an efficient vaccine candidate against schistosomiasis in light of the data obtained from murine studies. The best long-term strategy to control schistosomiasis might be immunization with anti-Sm29 vaccine associated with other protective surface antigens and possibly combined with drug treatment. Supporting Information Figure S1 Immunolocaliztion of Sm29 on cercariae by confocal microscopy (0.02 MB PDF) Click here for additional data file. Table S1 Complete list of all genes and fold-changes determined by microarray analysis (0.15 MB PDF) Click here for additional data file. Table S2 List of primers used for validation by real-time RT-PCR (0.04 MB PDF) Click here for additional data file. Video S1 Movie of confocal microscopy images. Immunolocalization of Sm29 on the tegument of male adult worm. File is in AVI format. (7.04 MB ZIP) Click here for additional data file.

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Making sense of the schistosome surface.

Patrick J. Skelly, Greame Wilson (2005)

The syncytial cytoplasmic layer, termed the tegument, which covers the entire surface of adult schistosomes, is a major interface between the parasite and its host. Since schistosomes can survive for decades within the host bloodstream, they are clearly able to evade host immune responses, and their ability is dependent on the properties of the tegument surface. We review here the molecular organization and biochemical functions of the tegument, combining the extensive literature over the last three decades with recent proteomic studies. We have interpreted the organization of the tegument surface as bounded by a conventional plasma membrane overlain by a membrane-like secretion, the membranocalyx, with which host molecules can associate. The range of parasite proteins, glycans and lipids found in the surface complex is evaluated, together with the host molecules detected. We consider the way in which the tegument surface is formed after cercarial penetration into the skin, and changes that occur as parasites develop to maturity. Lastly, we review the evidence on surface dynamics and turnover.

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Enzymatic Shaving of the Tegument Surface of Live Schistosomes for Proteomic Analysis: A Rational Approach to Select Vaccine Candidates

William Castro-Borges, Adam Dowle, Rachel Curwen … (2011)

Introduction The persistence of adult schistosomes in the bloodstream for decades means they must deploy unique and effective immune evasion strategies at their interface with the host. The 1 cm-long worms are covered by a naked syncytial layer of cytoplasm, the tegument, connected by cytoplasmic tubules to underlying cell bodies that contain the machinery for protein synthesis, packaging and export. The tegument surface has a multilaminate appearance, interpreted as a plasma membrane overlain by a lamellate secretion, the membranocalyx [1]. This complex molecular architecture is maintained by export of the contents of multilaminate vesicles, which originate in the cell bodies of the syncytium. There is experimental evidence for slow turnover of the membranocalyx to the external environment [2] whilst recycling of the plasma membrane by internalisation has been anticipated but not conclusively demonstrated [3]. Initial observations suggested the membranocalyx was an amphipathic bilayer, probably composed of phospholipids, which served as a physical barrier to prevent antibody binding or host leukocyte attachment to the underlying plasma membrane. In addition, its supposed hydrophobic properties coincided with a demonstrable ability of worms in the bloodstream to acquire host molecules, particularly erythrocyte glycolipids (the so-called host antigens) [4]. Whether this acquisition is a deliberate process that benefits the parasite or an accidental consequence of the membranocalyx properties, and any relevance it has to immune evasion, remain unclear. Building on techniques developed in 1980s to detach the tegument by freeze-thaw and enrich the surface membrane complex by differential centrifugation [5], we have characterized its composition using proteomic techniques [6], [7]. We developed a differential extraction scheme for the membrane preparation, with chaotropic agents of increasing strength, which enabled us to identify both membrane-associated and membrane-spanning constituents. These compositional findings demonstrated the importance of the tegument for nutrient uptake and maintenance of solute balance, as well as the presence of several hydrolases in the surface layers [6]. In a second study we incubated live worms with membrane-impermeant probes to biotinylate the most externally-accessible proteins and then recovered the tagged molecules by affinity chromatography for MS/MS identification [7]. This approach revealed that only a small subset of transporters, membrane structural proteins, enzymes, and schistosome-unique proteins were labelled, together with host immunoglobulins and complement C3. We concluded that these represented the most “exposed” surface constituents but we could not place them within the multilayered architecture of the surface with any certainty. To add another dimension to our understanding of tegument surface organization we have now used an enzymatic shaving approach on live worms to release the components most accessible to the selected enzymes, for MS/MS analysis. By analogy with techniques for stripping adherent cells from culture flasks, we used trypsin to cleave exposed protein loops or domains without impairing membrane integrity. We also incubated worms with phosphatidylinositol-specific phospholipase C (PiPLC) to release any externally accessible GPI-anchored proteins. As a control for proteins released by the vomiting of gut contents during the incubation period, or damage due to handling, we compared protein release +/− PiPLC, using the iTRAQ technique. Finally we used a phospholipase A2 (PLA2) preparation purified from snake venom to erode the lipid bilayer complex to determine if proteins could be selectively detached. We report that both trypsin and PiPLC removed a small subset of proteins whilst inflicting minimal damage on the worms whereas PLA2 was more destructive. We show that the iTRAQ technique identified both parasite and host proteins enriched by PiPLC treatment whereas trypsin released a different subset, with only Sm200 common to both. The proteins we have identified in the adult are also present as transcripts in the lung schistosomulum. We suggest that collectively they may be candidates for a schistosome vaccine, especially if responses can be targeted to the lungs to interfere with intravascular migration of incoming larvae. Methods Ethics statement The procedures involving animals were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986, and authorised on personal and project licences issued by the UK Home Office. The study protocol was approved by the Biology Department Ethical Review Committee at the University of York. Parasite maintenance and worm recovery A Puerto Rican isolate of S. mansoni was maintained using albino Biomphalaria glabrata snails and NMRI strain mice as laboratory hosts. All animal experiments were approved by the Ethical Review Process Committee of the Department of Biology, University of York. Adult parasites were obtained by portal perfusion of mice seven weeks after exposure to 200 cercariae, using RPMI1640 medium (minus phenol red) buffered with 10 mM HEPES (both from Invitrogen, Paisley, UK). Parasites were extensively washed in the same medium and tissue debris and any damaged individuals removed under a dissecting microscope. No attempt was made to separate males from females. Trypsin treatment of live parasites and recovery of peptides Approximately 800 freshly perfused parasites from 20 mice were used in each of four replicate experiments with trypsin as the shaving enzyme. They were incubated in a 30 mL Corning flask (Corning, NY, USA) containing 5 mL of buffered RPMI, with trypsin MS (Promega, Southampton, UK) added at 10 µg/mL, for 30 min at room temperature (RT). The supernatant was recovered, transferred to a 15 mL Falcon tube and centrifuged at 500×g for 30 min to remove any insoluble material such as the haematin particles in gut vomitus. Streptomycin and penicillin were then added to a final concentration of 100 µg/mL to prevent microbial growth and tryptic digestion was continued overnight at 37°C, after which peptides were reduced and alkylated. Reduction was performed in the presence of 20 mM DTT for 30 min at 65°C in a water bath and, after cooling, alkylation was performed in the presence of 80 mM iodoacetamide for 1 h at RT in the dark. Trifluoroacetic acid (TFA) was then added to a final concentration of 0.1% before recovery of peptides by passage through a solid phase Strata C18-E extraction cartridge (55 µm, Phenomenex, Macclesfield, UK), followed by several column washes in 0.1% TFA and final elution in 750 µL of 50% acetonitrile/0.1% TFA. The eluted fraction was concentrated under vacuum to dryness and peptides resuspended in 20 µL 0.1% TFA. LC-MS/MS A 3 µL aliquot of the tryptic peptide preparation was injected onto a reversed-phase PS-DVB monolith column (200 µm i.d.×5 cm, LC Packings, Amsterdam, Netherlands). Peptides were separated using a two-step linear gradient of 2–31.4% (v/v) acetonitrile in 0.1% aqueous heptafluorobutyric acid over 60 min, followed by 31.4–51% (v/v) in the same solvent over 5 min, at a flow rate of 3 µL/min; UV absorbance at 214 nm was monitored. Fractions were collected onto a MALDI target plate using a Probot (Dionex, Bannockburn, USA) with simultaneous addition of matrix solution (6 mg/mL α-cyano-4-hydroxycinamic acid (CHCA, Sigma, Poole, UK) in 60% (v/v) acetonitrile). Positive-ion MALDI mass spectra (MS) were obtained using a 4700 Proteomics Analyzer with TOF-TOF Optics (Applied Biosystems, Framingham, USA) in reflector mode, over the m/z range 800–4000 and monoisotopic masses obtained from centroids of raw, unsmoothed data. The precursor mass window was set to a relative resolution of 50, and the metastable suppressor was enabled. The default calibration was used for MS/MS spectra, which were baseline-subtracted (peak width 50) and smoothed (Savitsky-Golay with three points across a peak and polynomial order 4); peak detection used a minimum S/N of 5, local noise window of 50 m/z, and minimum peak width of 2.9 bins. The twenty strongest peaks from each fraction, having a signal to noise (S/N) greater than 50 and a fraction-to-fraction precursor exclusion of ±0.2 Da, were selected for CID-MS/MS analysis. Singly-charged peptides were fragmented with Source 1 collision energy of 1 keV, and air as the collision gas. Peak lists from the MS/MS data, containing all m/z values from m/z 20 to the precursor m/z - 60, with a minimum S/N of 10, were provided by TS2 software (version 1.0.0, Matrix Science Ltd., London, UK). Each list, corresponding to one MALDI plate, was then submitted to a local copy of the Mascot program (version 2.1, Matrix Science) and searched against the SmGenesPlusESTs (260448 sequences; 71029272 residues), an in-house database derived from the publically available data in http://www.genedb.org/genedb/smansoni/), and the NCBInr Mus musculus database (139457 sequences) for host proteins. Search parameters specified only tryptic cleavages and allowed for up to one missed site, the variable carbamidomethylation of cysteines, and oxidation of methionines; precursor and product ion mass error tolerance was set to ±0.3 Da. A decoy database, generated by Mascot, was used with the significance threshold for protein identification set to achieve a false positive rate of 1 to 2% and peptide threshold set to ‘least identity’. A protein was considered positively identified if the ion score for a particular peptide had an expect value less than 0.05. Phosphatidylinositol-phospholipase C (PiPLC) treatment of live parasites GPI-anchored proteins were recovered from live worms by in vitro incubation with PiPLC as the shaving enzyme, the experiment being performed twice to provide biological replicates. Downstream analysis required the worms from 40 mice, which were perfused and treated in two separate batches to minimise the time ex vivo; supernatants were then combined. Each batch was incubated at 37°C for 1 h in the presence of PiPLC (from Bacillus cereus, Sigma) at 1.25 Units/mL, with conditions as for trypsin. The supernatant was removed and concentrated at 4°C using a 5000 Da cut-off centrifugation device (Vivaspin 6, West Sussex, UK). The control for secretion, vomitus production and parasite damage due to handling comprised an identical experiment, minus PiPLC. This also served as a ‘background’ control for the other enzyme treatments. For one experiment, PiPLC-released proteins were obtained using PiPLC from a different source (from Bacillus thuringiensis, Europa Bioproducts, Wicken, Cambridigeshire, UK) employing the same conditions as above. On that occasion the GPI-released fraction was used for a 2-DE separation. Fractionation of the PiPLC-released material The composition of released material was evaluated by 1-DE using a pre-cast NUPAGE 4–12% Bis-Tris gel (Invitrogen) after a 45 min run at 200 V. The gel was then fixed in 40% methanol, 10% acetic acid for 30 min, stained with SYPRO Ruby (Invitrogen) for 2 h in the dark and imaged using a Molecular Imager FX (Bio-Rad, Bath, UK). Protein content in each lane of the gel was then estimated by densitometric analysis using Quantity One software (Bio-Rad). Fifty µg of the PiPLC-treated sample was also evaluated by mini 2-DE essentially as previously described [8], [9]. After electrophoresis the gel was first stained with SYPRO Ruby, imaged as above, and restained with Bio-Safe Coomassie (BioRad); all visible spots were selected for “in gel” digestion [8]. An aliquot of 1–2 µL of the digestion supernatant containing the peptides was spotted on a MALDI plate and dried before the addition of 0.6 µL of a saturated solution of CHCA matrix (in 50% acetonitrile/0.1% TFA). Peptide fragmentation data from each gel spot was processed by GPS Explorer Software (Applied Biosystems) underpinned by Mascot (settings as above), to provide a putative identity for the protein. iTRAQ labelling of peptides The relative composition of PiPLC-treated and control samples was characterized using isobaric tagging (the iTRAQ labelling technique) following the protocol provided by the manufacturer (Applied Biosystems). Prior to labelling, two aliquots of treated and control samples containing 10 µg protein were taken to provide technical replicates. Briefly, 10 µg of both control and PiPLC-treated samples were individually denatured, reduced, and alkylated with reagents supplied in the iTRAQ kit. Peptides were generated by trypsin digestion using a 1∶20 enzyme/protein ratio, at 37°C for 24 h, and labelled with iTRAQ reagents at lysine, terminal amine groups and partially at tyrosine residues. Test samples were labelled with tags 116 or 117 and control samples with tags 114 or 115, respectively. A peptide mixture was made by combining the four tagged samples and cleaned up using a strong cation-exchange cartridge to remove the detergents and excess iTRAQ reagents. The peptides were then affinity-purified in a Strata C18-E cartridge, eluted as for tryptic peptides, dried using a vacuum concentrator and resuspended in 10–20 µL of 0.1% TFA. LC-MS/MS was performed as described above. Protein identification and peptide quantification was achieved by submitting the TS2-generated MS/MS raw data files to Mascot, searching against SmGenesPlusESTs and NCBInr databases. Search parameters were tryptic peptides, with 0–1 missed cleavage; fixed modifications, β-methylthiolation of cysteines, iTRAQ tagging of lysines and N-terminal amine groups; variable modifications were oxidation of methionines and iTRAQ tagging of tyrosines. Precursor and product ion mass error tolerance was set to ±0.3 Da. The 114-tagged sample (C1) was taken as the reference for calculating ratios. The Mascot software displays the median normalised geometric mean ratios and a factor from which the geometric standard deviation can be derived. Automatic outlier removal was performed by the Mascot software. A protein was considered enriched if the mean T1/C1 and/or T2/C1 ratios minus the 95% confidence limit exceeded the corresponding C2/C1 ratio plus its 95% confidence limit. For single peptide identifications, a protein was considered enriched if it appeared in both biological replicate experiments, and had a mean treated/control ratio >2. Phospholipase A2 (PLA2) treatment of live parasites The final approach for recovery of parasite surface molecules using enzymatic shaving, involved the treatment of live parasites with PLA2. This enzyme isolated from the venom of Crotalus durissus terrificus (deposited under accession number P24027 at NCBInr) was kindly provided by Prof. Andreimar Soares (Faculty of Pharmaceutical Sciences, University of Sao Paulo, Brazil). During the shaving experiment, approximately 800 freshly perfused parasites were incubated in a 30 mL Corning flask, containing 6 mL of buffered RPMI1640, with PLA2 added at 16 µg/mL for 1 h, at RT. Another batch of parasites was used in a control and parallel experiment in which no enzyme was added to the culture medium. The supernatants from control and treated samples were concentrated using a Vivaspin 6 filtration device (5000 Da cut off) at 500×g, at 4°C until the volume reached 500 µL. The samples were then transferred to 1.5 ml eppendorf tubes and centrifuged at 25,000×g for 20 min. After this step a membranous pellet, recovered from PLA2-treated parasites only, was extracted in 50 µL of 0.5% Triton-X100, yielding approximately 20 µg of protein. Reduction, alkylation, trypsin digestion and peptide clean-up were performed as described for the iTRAQ protocol (omitting the labelling steps). LC-MS/MS of the peptide mixture was performed essentially as described above. Immunocytochemical localization of host membrane proteins by confocal microscopy Host plasma membrane proteins on the surface of adult parasites were investigated either on live worms, perfused from mice using RPMI medium, or by preparing OTC-embedded cryostat sections. Both were incubated with primary antibodies at 1∶100 dilution in PBS for parasite sections and in RPMI for live worms, containing 5% normal goat serum, for 1 h at RT. Monoclonal antibodies used were rat anti-mouse CD44 (558739), rat anti-mouse CD90 (Thy-1; 553016) and hamster anti-mouse CD48 (553682), all from BD Biosciences Pharmingen, New Jersey, USA. Labelling was detected by the use of goat anti-rat IgG conjugated to Alexa fluor 488 at 1∶500 dilution for 30 min in the same buffer, or goat anti-hamster IgG conjugated to Alexa fluor 568 under the same conditions. Transcript searching A searchable database was created in 2005, comprising all S. mansoni transcripts then available from dbEST (http://www.ncbi.nlm.nih.gov/), the Sao Paulo Schistosoma mansoni EST genome project (http://bioinfo.iq.usp.br/schisto/), and the Wellcome Trust Sanger Institute ftp site (ftp://ftp.sanger.ac.uk/pub/pathogens/Schistosoma/mansoni/ESTs). All proteins of interest identified by the enzymatic shaving approach were searched against the compiled EST data to determine the number of transcripts detected in adults and lung stage schistosomula; this provided a very approximate guide to the relative levels of expression in the two life cycle stages. Results Enzymatic shaving using MS grade trypsin Freshly perfused live parasites were subjected to trypsin digestion under controlled conditions, in order to release from the surface membrane complex of the tegument any proteins, or segments thereof, accessible to the enzyme. The vast majority of worms retained a normal appearance and activity over the 30 min incubation. The culture supernatant was recovered and released proteins/peptides allowed to digest further overnight. Tryptic peptides were then reduced and alkylated before their recovery and separation using reversed-phase chromatography (Figure S1A) for subsequent mass-spectrometric identification. The identities obtained by Mascot searching of the MS/MS data were then categorized according to their molecular function and, by inference, their potential cellular origin (Table 1). 10.1371/journal.pntd.0000993.t001 Table 1 Selected identities from trypsin shaving experiment. Category/Protein identity Accessiona Exp1 Exp2 Exp3 Exp4 NCBInr/Smp (SmGenes) Clustered ESTs Host proteins haemoglobin α chain AAA37783 • haemoglobin β-1 chain P02088 • C3 complement AAC42013 • • C4 complement CAA28936 • • • CD44 CAA46880 • • • Membrane and membrane-associated Annexin type IV Smp_074140 Sm04814 • • Annexin type V Smp_074150* • Annexin type VI Smp_077720 Sm03987 • • • • Calpain Smp_157500 Sm11826 • Sm25 Smp_195180 Sm04760 • • • Sm200 Smp_017730* Sm03865 • • • • Proteoglycan-like Smp_020550 Sm08105 • • Secretory and vesicular pathway Potassium-channel inhibitor SmKK7 Smp_194830 Sm12916 • Endophilin (BAR domain) Smp_003230.2 Sm04606 • • • • Endophilin (BAR domain) Smp_163720 Sm00115* • • • • Gut proteases Hemoglobinase (asparaginyl endopeptidase) Sm00989 • Cathepsin B1 isotype 1 precursor Smp_067060* Sm01772* • a Accession numbers for host proteins can be found at Pubmed http://www.ncbi.nlm.nih.gov/protein/Smp and Sm numbers are found at http://www.genedb.org/Homepage/Smansoni. *Peptide hits common to more than one predicted protein. Exp = Experiment. The host complement proteins C3 and C4 and the leukocyte surface marker CD44 were released in two or more of the four replicate experiments. Host haemoglobin alpha and beta chains were also identified in one experiment, presumably an indication that regurgitation from the worm gut was occurring. A number of proteins, known to be associated with the tegument surface, were also released by the treatment. They included three phospholipid-binding annexins and the membrane protease calpain. Of the annexins, Smp_077720 was found in all four experiments and the others on two and one occasions, respectively; calpain was detected in only one experiment. The schistosome-unique proteins of unknown function, Sm200 and Sm25, were found on three and four occasions, respectively. The final membrane protein, not previously associated with the tegument, shows homology with cell surface proteoglycans. A pair of BAR-domain-containing endophilins was also released by the treatment in all four experiments while the putative potassium-channel inhibitor, SmKK7, was found on one occasion. Although worm incubations were short-term, two known gut-derived proteases, asparaginyl endopeptidase and cathepsin B1 were detected. In addition, a total of 12 proteins with unknown function and localization were found (Tables S1 and S2), some containing domains, e.g. Ig-like and EGF-like, which may indicate a surface position. In spite of strenuous efforts to maintain worm viability, the trypsin treatment affected the integrity of the surface membranes to some extent, as evidenced by the appearance of seven cytoskeletal and 17 cytosolic proteins in the medium (Tables S1 and S2). Among the former, actin, fimbrin and severin have been proposed as constituents of the tegumental spines that reside immediately beneath the surface membrane complex. The more numerous cytosolic proteins, comprising glycolytic enzymes, chaperones, and antioxidants indicate the leakage of internal components in at least some of the parasites. Known tegumental surface transporters, ion channels, and enzymes (other than calpain) were conspicuous by their absence in the trypsin preparation. Enzymatic shaving using PiPLC The purpose of treatment with PiPLC was to release GPI-anchored proteins accessible to the externally applied enzyme in live worms; incubation with the enzyme for 1 h appeared to have no morphologically obvious deleterious effect. A 1-DE gel separation of material released by treated and control worms revealed a complex pattern of protein bands with Mr ranging from 10 to >250 kDa (Figure 1A). The two preparations displayed a strong similarity, with only three bands (arrowed) visibly enriched by the PiPLC treatment versus the control; two were of high molecular mass (approx. 200 kDa) while the other was located at the bottom of the gel (approx. 12 kDa). The PiPLC treatment released sufficient protein to permit a mini 2-DE separation (Figure 1B) for subsequent MS/MS analysis of gel spots. The analysis revealed the presence of proteins known to be GPI-anchored, such as ‘Surface protein’ (Sm200) and alkaline phosphatase. Protein orthologues of CD59 and carbonic anhydrase IV were novel features of this 2D map. The absence of gut proteases Sm31 and Sm32 was notable. The identities of other spots revealed the presence of cytosolic and cytoskeletal contaminants, including thioredoxin, fatty acid binding protein (Sm14), Sm22.6, enolase and triose phosphate isomerase (Table S3). The remaining spots on the SYPRO-stained gel (Figure 1B) were not detected by Coomassie staining so no identification could be assigned using single spot tryptic digestion. 10.1371/journal.pntd.0000993.g001 Figure 1 PiPLC-released proteins analysed by 1-DE and 2-DE. (A) Ten µg of proteins present in the culture supernatant +/− PiPLC were separated by 1-DE and the gel stained with SYPRO Ruby. The majority of the proteins were shared between the PiPLC-treated and non-treated samples. Only three bands (arrowed) were visibly enriched by the treatment, two of high molecular mass at approx. 200 kDa and a third at approx. 12 kDa. (B) Fifty µg of proteins present on the PiPLC- treated supernatant were separated by 2-DE and the gel stained in SYPRO Ruby. Labelled spots are those identified by mass spectrometry after Coomassie staining of the gel. The PiPLC protein cluster on the 2-DE map does not correlate with the same levels of PiPLC used for preparation of 1-DE gels as they were from different sources and exhibited differing specific activities. *Less acidic CD59 isoform, see Table S3 for peptide identification data. As proteins were released into the culture medium during the 1 h incubation irrespective of whether PiPLC was present or not, we used the iTRAQ technique to determine the degree of enrichment due solely to the enzymatic shaving. Within an individual labelling protocol, splitting both the control and treatment samples provided technical replicates. After LC separation of the tagged peptide mixture (Figure S1B), fragmentation spectra of the most abundant peptides were generated, each containing signature peaks for the four reporter tags (Figure 2 and Figure S2). In most instances the fragmentation spectra yielded four peaks of approximately equal area (Figure 2A). The 115/114 (C2/C1) ratios approximated to unity, indicating approximately equal protein contributions of the two control samples to the iTRAQ mixture. However, the majority of 116/114 (T1/C1) and 117/114 (T2/C1) ratios were almost invariably less than one, sometimes significantly so (Figure 2A and Figure S2). We have no reason to believe that the control and treatment incubations differed except in the addition of enzyme, and so we must attribute the ratios of less than unity to a dilution effect on the background proteins in the treated samples. This dilution resulted from the addition of PiPLC plus the extra proteins released from the surface by its action. In a minority of fragmentation spectra the four peaks representing the reporter tags were not of approximately uniform area (Figure 2B and Figure S2). The reporter ion peaks from the treated samples were from 2 to 10 fold greater intensity indicating enrichment of the parent peptide by the PiPLC treatment. A total of 52 identities was obtained from fragmentation of the tagged peptides (Figure S2 and Tables S4 and S5) ranging from 1 to 15 per identity, primarily of cytosolic or cytoskeletal origin. The PiPLC-enriched proteins of parasite origin were, in descending order of abundance, Sm29, CD59a, Sm200, carbonic anhydrase, CD59b, alkaline phosphatase and ADP-ribosyl cyclase (Figure 3). In addition two host proteins, CD48 and CD90 (Thy1.2), were also enriched by PiPLC treatment. Proteins of known gut origin, α2-macroglobulin and saposin B, were present in control and treated samples in equivalent amounts indicating similar regurgitation of gut contents over the one hour incubation. Equivalent amounts of cytosolic (e.g, enolase, 14-3-3) and cytoskeletal proteins (e.g, actin, Sm20.8) in control and treated samples (Figure 3 and Table S4) indicated a certain degree of surface damage, but not inflicted by the PiPLC treatment. Although not enriched by PiPLC hydrolysis, because they lack the GPI-anchor, three other proteins are worth noting. Two of these, CD63/tetraspanin (TSP-2) and annexin IV (Smp_074140) were already known to be associated with the tegument surface and the third is an 8 kDa low molecular weight protein (LMWP). This last protein, present in a range of trematodes [10], has a signal peptide and so may be a true secreted protein released along with the membranocalyx; its status as a tegument surface protein needs to be confirmed. 10.1371/journal.pntd.0000993.g002 Figure 2 Discrimination between enzymatic enrichment and parasite leakage/secretion using iTRAQ tags. (A) Low m/z spectral region displaying the iTRAQ reporter ion peaks whose ratios revealed a non-GPI anchored protein. (B) Low m/z spectral region displaying the iTRAQ reporter ion peaks whose ratios revealed a GPI-anchored protein. C1 and C2 represent control samples labelled with the iTRAQ m/z 114 and 115 tags, respectively. T1 and T2 represent PiPLC-treated samples labelled with iTRAQ m/z 116 and 117 tags, respectively. 10.1371/journal.pntd.0000993.g003 Figure 3 iTRAQ consolidated results. Sixteen out of 52 proteins identified in the iTRAQ experiment selected to provide a snapshot of the major findings provided by the PiPLC shaving approach. Fold enrichment on the y axis is plotted relative to the intensity of the m/z 114 C1 tag. The seven GPI-anchored proteins of parasite and the two of host origin are included. Note the equivalent levels of proteins representing cytosolic, cytoskeletal, secretory and gut contaminants present in both PiPLC-treated and control samples. C1, C2, T1 and T2 key as in Fig 2. Bars represent the standard geometric deviation for iTRAQ ratios associated to protein identities obtained with at least 3 peptide fragmentations. iTRAQ ratios and protein identities obtained from fragmentations of 1 and 2 peptides are reported based on their significant Mascot expect score ( 0.6) but no O-glycosylation sites on the amino acid sequence coding for Sm200. It is notable that four of the five potential N-linked sites are located furthest from the GPI anchor site in the N-terminal region where few peptides were identified following trypsin treatment of the live worms. Thus it is plausible that attached N-glycans protect the native Sm200 polypeptide chain in situ on the worm surface from trypsin attack. Discussion Our previous biotinylation studies have provided insights into the disposition of proteins in the complex molecular structure of the schistosome tegument surface. In the present study we have taken an alternative approach to obtain information about the location of tegument components. This involved shaving the surface of live adult worms with selected enzymes to release accessible proteins, and their identification by proteomics. It is axiomatic in this approach that the integrity of the worm surface is not compromised by the treatment. Our results indicate that trypsin and PiPLC largely fulfilled this criterion but PLA2 did not. We must assume that this last enzyme, via its attack on the lipid bilayers, rapidly caused generalized erosion of the surface membranes and loss of integrity. We shall therefore consider only the results of the trypsin and PiPLC treatments to make inferences about tegument surface organization. In addition, the male parasite has at least two times the surface area of the female, assuming all surfaces are accessible to the enzymes. If the gynaecophoric canal is effectively sealed, then the male dorsal surface would have contributed the bulk of released protein. It was inevitable that the in vitro culture of live worms resulted in the contamination of the enzyme-released proteins by gut content. Thus the detection of proteins such as α2-macroglobulin, saposin B, and hemoglobinase (asparaginyl endopeptidase) in the two enzymatic shaving experiments was to be expected. Conversely, even with the most careful handling of parasites including their recovery from mice by perfusion with culture medium, the presence of cytosolic and cytoskeletal proteins must be attributed to some degree of worm damage. In the present study, we used the iTRAQ labelling technique on test and control samples to discriminate enzymatic enrichment from normal secretion or protein leakage due to invisible damage. This approach successfully overcame the dominance of abundant peptides from contaminating cytosolic and cytoskeletal proteins to highlight the few proteins released by PiPLC treatment. Nevertheless, one must bear in mind that as enzyme accessibility has not been addressed in this investigation, caution should be exercised when considering the fold enrichment found for GPI-anchored molecules. In this regard, it is possible that a higher fold enrichment for a given protein may only indicate a more exposed/accessible location at the parasite surface rather than imply protein abundance. A major finding of our study is that seven parasite proteins can be detached from the surface by treatment with PiPLC and enriched compared to control incubations. From this we infer that each is inserted into the parasite surface by a GPI-anchor. As these anchors are invariably located at the C-terminus of such proteins we conclude that the extraneous 30 kDa PiPLC enzyme is able to access the anchor in proximity to a lipid bilayer. Whether this bilayer is the secreted membranocalyx or the underlying plasma membrane is problematic. Access to the latter would mean that the digesting enzyme had to pass through the protective membranocalyx. One possible inference is that the seven GPI-anchored proteins are inserted into the membranocalyx. For carbonic anhydrase this seems unlikely because of its assumed function in regulating acid-base balance at the parasite surface. It would be best placed to do this if located between the two lipid bilayers so that, by analogy with the erythrocyte, CO2 generated inside the worm could be converted to HCO3 − for diffusion into the bloodstream. Indeed, the reverse diffusion of a chloride ion through the membranocalyx would be required to balance the charge, bringing it into close proximity with the anion transporters, which our previous studies have shown are present in the tegument plasma membrane [6]. The gene model for carbonic anhydrase is incomplete, lacking both the N-terminal exon encoding the signal peptide and the C-terminal exon(s) encoding the site for the attachment of a GPI-anchor. However, its enrichment by PiPLC treatment of live worms attests to the presence of a GPI-anchor. Two isoforms of CD59 orthologues, identified by possession of the CCxxDxCN motif, were also highly enriched by the PiPLC treatment, implying their outer location at the surface. These proteins are potentially significant because the human CD59 protein protects self-cells against complement fixation by blocking formation of the C5 to C9 membrane attack complex [11]. A similar role for the two schistosome molecules is very attractive, as a component of the parasite's mechanisms of immune evasion. It is of note that in the S. mansoni genome there are four additional gene models encoding CD59 orthologues, which could also be tegument-associated [12]. These CD59 orthologues in the tegument surface are better candidates for inhibition of complement fixation than the SCIP-1 protein [13], subsequently identified as paramyosin [14], [15]. The protein most highly enriched by PiPLC treatment was Sm29 that has no known function. It was originally selected by a bioinformatic search as a protein with a single membrane spanning domain [16]. It was identified in the final pellet after differential extraction in a compositional analysis of the tegument membranes [6] and was accessible to biotinylation in live worms [7]. Subsequent confocal microscopy revealed its association with the tegument although technical issues do not allow a firm conclusion about its precise location [17]. Our PiPLC shaving results demonstrate both its peripheral location and the fact that it is GPI-anchored, the latter prediction also made by big-PI Prediction server (http://mendel.imp.ac.at/gpi/gpi_server.html). The peripheral location of Sm200, again with no known function, was also revealed by PiPLC shaving. This protein first cloned by Hall et al. [18] was already known to be GPI-anchored and located in the tegument surface [19], [20]. Surprisingly therefore it was not found in compositional analysis of the tegument membrane [6], but was accessible to biotinylation in live worms [7]. These observations suggest that whilst definitely surface-attached it is readily lost during the processing of tegument membranes by differential extraction for MS analysis, a feature that may be linked both to its size and GPI-anchor. Moreover, Sm200 has recently been identified in circulating lipoprotein particles from the blood of schistosome-infected humans, confirming its turnover into the vascular environment [21]. The two remaining GPI-anchored proteins shown to be enriched by iTRAQ labelling after PiPLC treatment were alkaline phosphatase and ADP-ribosyl cyclase. The former is well characterized and was previously shown to be GPI-anchored in schistosomula [22]. It has been used as a membrane marker in the development of methods for tegument surface isolation [5]. It was identified by compositional analysis of the tegument surface membranes in the urea/thiourea/CHAPS/sulfobetaine (UTCS) fraction and insoluble pellet [6], and is accessible to biotinylation in live worms [7]. Its presence in the UTCS fraction indicates that it is, at least partially, loosely associated with the surface membranes i.e not membrane spanning. The recently characterised ADP-ribosyl cyclase was also shown to be GPI-anchored and localized to the outer tegument of the adult schistosome [23]. Both schistosome enzymes must be able to access their substrates in live worms but their precise function at the tegument surface remains to be established. In the case of ADP-ribosyl cyclase, a role in calcium mobilization has been proposed [23] but alternatively it could function in immune evasion by regulating ecto-NAD+ levels, thereby reducing substrate availability for CD38- and CD157-mediated effector functions of lymphocytes. Corroborating the identification of GPI-anchored molecules using the iTRAQ technique, four out of the seven proteins assigned as GPI-anchored were also detected by our 2-DE analysis. These were Sm200, alkaline phosphatase, two isoforms of CD59 orthologues and carbonic anhydrase. A definite proof of their enrichment due to PiPLC's activity is the fact that these molecules are underrepresented and are not easily identifiable in 2-DE maps produced using soluble worm preparations [8] or membrane-extracted proteins from crude or differentially-extracted S. mansoni tegument [6]. Use of the iTRAQ technique to identify proteins enriched by trypsin treatment was not possible because the released material was already partially digested. This prevents accurate quantification of protein for labelling and therefore the trypsin shaving fraction was subsequently processed as a peptide digest. We have taken as a reference of cytosolic/cytoskeletal contaminants in the trypsin shaving, the number and diversity of those proteins released during 1 h incubation at 37°C in the absence of added enzyme. When we compared the number of proteins that could indicate leakage or vomitus with the number of proteins in the trypsin-treated parasites, we observed we had less contamination, most likely due to a shorter incubation time, 30 min. In addition, we are assuming that as trypsin should not be able to permeate the parasite tissues, the peptides originating from membrane proteins are likely to represent the most exposed/accessible domains at the parasite surface. We therefore focus only on (1) membrane and membrane-associated, (2) vesicular pathway and secreted, and (3) host proteins. The peripheral location of Sm200 was again confirmed but it was the only one of the seven PiPLC-released proteins that was also released by trypsin. This suggests that the rest may be protected from proteolysis, potentially by their N and/or O-linked glycans or by a sequestered location. The release of three annexins by the trypsin treatment indicates their superficial location. One (Smp_077720) was previously detected by both biotinylation and compositional analysis and two are new to this study (Smp_074140 and Smp_074150). The known phospholipid-binding properties of this group of proteins means that they could have a role in promoting adhesion of the membranocalyx to the plasma membrane, acting like a molecular ‘velcro’ via the four binding domains that each possesses. More recently, certain annexin isoforms have been implicated in immunomodulatory functions such as the resolution of inflammation [24]. They are able to interact with receptors on the surface of leukocytes to control apoptosis and their clearance by macrophages. The existence of such a process at the surface of the tegument, mediated by schistosome annexins, accords with the absence of leukocyte binding to adult worms in vivo [25]. However, a comparison of the orthologies of the schistosome and human annexins is difficult because of the evolutionary distance. Thus, BLAST searching of the schistosome sequences against the NCBInr database reveals the closest homologues of Smps 077720, 075150 and 075140 are human annexins A13, A7 and A8 respectively, not the A1 isoform that has been most implicated as an anti-inflammatory agent. The conjecture will only be resolved by expression of the schistosome annexins for assays of function. The presence of Sm25 as a tegument surface protein has a chequered history. It was proposed as a vaccine candidate because anti-Sm25 antibody levels correlated with protection in mice vaccinated with a crude tegument membrane preparation [26]. It was then cloned and designated as an N-glycosylated integral membrane protein [27] but later characterized as a palmitoylated protein with the implication that it was on the cytosolic leaflet of the plasma membrane [28]. Further immunocytochemical studies suggested it was distributed throughout the tegument syncytium but not associated with the surface membranes [29] and it could only be biotinylated when parasites were permeabilized by Triton X-100 [30]. It was not found in either our compositional or biotinylation studies on the tegument surface. The removal of Sm25 from live worms by trypsin, when so few other membrane proteins are released, suggests a unique accessibility. The proteolytic enzyme calpain was previously identified at the tegument surface by both compositional analysis [6] and biotinylation [7]; its potential as a vaccine candidate has already been exploited [31], [32]. The final member of this group is a heparin sulphate-like proteoglycan which has not been identified in any previous studies and merits further investigation. In the secreted protein category, this is the first report of SmKK7 release from the tegument surface; it has previously only been reported from cercarial secretions [33]. The detection of a pair of BAR domain proteins in all four experiments after trypsin treatment is intriguing since these banana-shaped molecules located on the cytosolic surface of plasma membranes function to promote membrane curvature in exo- and endocytosis [34]. This suggests that the trypsin may be entering the fine tubules located at the base of tegument pits where multilaminate vesicle fusion with the tegument plasma membrane occurs [35], [36]. That conclusion would place Sm25 and the proteoglycan in a similar location where constituent proteins are poorly protected by overlying membranocalyx, perhaps loosely analogous to the relationship of the flagellar pocket of trypanosomes [37] to variant surface glycoprotein export. The release of complement C3 from the parasite surface by trypsin treatment is consistent with our previous biotinylation study [7]. We can now add C4, its precursor in the complement cascade, but not the C5 to C9 components of the membrane attack complex, suggesting complement fixation but then inhibition of the cascade. The failure to detect any immunoglobulins, when murine IgM, IgG1 and IgG3 heavy chains were biotinylated [7], is puzzling as they would be expected to initiate complement fixation. However, there is evidence that these proteins are resistant to trypsin degradation, particularly under the low concentration conditions employed in our experiments [38]. In fact, aiming to preserve worm viability, trypsin was used at a concentration approximately 100 times lower than that typically employed for stripping adherent cells from culture flasks. A positive consequence of such a gentle shaving treatment on live worms is justified by the reasonable number of genuine membrane proteins that were identified. In contrast, a previous study on S. bovis with trypsin treatment, used methanol-fixed parasites [39] and found an overall higher number of protein identifications. However, membrane-associated and/or integral proteins were poorly represented, and the results are not comparable with our approach using live worms. Our enzyme shaving experiments extend the list of host proteins known to be firmly associated to the parasite surface. The release of CD48 and CD90 (Thy1.2) by PiPLC confirms their possession of GPI-anchors and most likely accounts for their transference from the host (leukocytes) in the same manner as host glycolipids transfer from erythrocytes through a process currently termed cell-painting [40]. Supporting this finding is the demonstrable ability of purified Thy-1to reincorporate into the plasma membrane of murine Thy-1- cells directly from aqueous suspension and without the use of detergents [41]. However, failure to detect the presence of both CD48 and CD90 by immunocytochemistry indicates their relative paucity. The detection of host CD44 is more surprising since it is an extracellular protein anchored in the membrane of a range of cell types by a single transmembrane domain [42]. However, the intense staining of the tips of spines on the dorsal tubercles, the point of contact between the worm and the vascular endothelium during peregrination around the portal vasculature, suggests that the transfer occurs when the apposing membranes of tegument and endothelium are pressed together. The observations on CD44 staining are a testament to the acquisitive properties of the schistosome surface. Our studies on the surface accessibility of tegument proteins are relevant to the development of schistosome vaccines. In mice immunized with attenuated cercariae challenge schistosomula are eliminated in the lungs by inflammatory foci that block their onward migration [43]. Both priming of the immune response [44] in skin-draining lymph nodes and the pulmonary effector responses appear to be mediated by proteins on the schistosomular tegument surface [45]. Although we have characterised surface proteins on the adult worm, the presence of the encoding mRNAs in the lung schistosomulum provides support for the suggestion that those same proteins are exposed on the larval tegument surface. In this context Sm29 has already been used successfully as a vaccine candidate [17]. We therefore suggest that the other six PiPLC-anchored proteins would also repay investigation as putative vaccine candidates, especially Sm200 because it was also removed by trypsin. Indeed, the presence of a GPI-anchor may make proteins in this group more prone to detachment from the surface for transcytosis across the pulmonary capillary endothelium, processing by accessory cells and presentation to reactive CD4+ Th1 cells in the lung parenchyma. To this list we can add the three annexins released by trypsin and LMWP that may be a tegument secretion, noting also that calpain is a proposed vaccine candidate [31]. It is conceivable that a cocktail of these proteins would elicit strong protection against intravascular migrating schistosomula, especially if targeted to the lungs, whereas immunisation with a single protein has only modest success [46]. Supporting Information Figure S1 Trypsin and PiPLC shaving experiments. Peptide Chromatograms at 214 nm. (0.13 MB PDF) Click here for additional data file. Figure S2 Protein identities and iTRAQ ratios from PiPLC shaving approach. Bars represent the standard geometric deviation for iTRAQ ratios associated to protein identities obtained with at least 3 peptide fragmentations. iTRAQ ratios and protein identities obtained from fragmentations of 1 and 2 peptides are reported based on their significant Mascot expect score (<0.05) in three independent iTRAQ experiments. (0.53 MB PDF) Click here for additional data file. Figure S3 Sm200 tryptic peptides identified using trypsin and PiPLC shaving of live parasites. Putative N-linked glycosylation sites are indicated in red. (0.01 MB PDF) Click here for additional data file. Table S1 Other identities from trypsin experiment. (0.03 MB XLS) Click here for additional data file. Table S2 Trypsin shaving peptide data. (0.10 MB XLS) Click here for additional data file. Table S3 Identities from 2-DE gel of PiPLC-released proteins. (0.03 MB XLS) Click here for additional data file. Table S4 PiPLC shaving identities/iTRAQ ratios. (0.04 MB XLS) Click here for additional data file. Table S5 PiPLC shaving peptide data. (0.06 MB XLS) Click here for additional data file. Table S6 PLA2 shaving identities and peptide data. (0.03 MB XLS) Click here for additional data file.

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Journal ID (nlm-ta): PLoS Negl Trop Dis

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Publication date (Electronic): 17 January 2017

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[1 ]Department of Infectious Disease and Global Health, Cummings School of Veterinary Medicine, Tufts University, North Grafton, Massachusetts, United States of America

[2 ]Departamento de Bioquímica e Imunologia do Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

Queen's University Belfast, IRELAND

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The authors have declared that no competing interests exist.

Conceptualization: GKP CBS.
Data curation: CBS.
Funding acquisition: PJS CBS.
Investigation: GKP MD JMT AAD.
Methodology: GKP MD JMT SCO AAD PJS CBS.
Project administration: CBS.
Resources: SCO AAD PJS CBS.
Supervision: CBS.
Visualization: CBS.
Writing – original draft: CBS.
Writing – review & editing: GKP MD JMT SCO AAD PJK CBS.

* E-mail: charles.shoemaker@ 123456tufts.edu

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Charles B. Shoemaker http://orcid.org/0000-0001-9738-1361

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DOI: 10.1371/journal.pntd.0005306

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Date received : 19 October 2016

Date accepted : 6 January 2017

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Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;

Award ID: R01-AI056273

Award Recipient : Patrick J. Skelly

Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;

Award ID: R21-AI061517

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ORCID: http://orcid.org/0000-0001-9738-1361

Charles B. Shoemaker

This work was supported by: National Institute of Health, National Institute of Allergy and Infectious Diseases, R01-AI056273 (PJS) and R21-AI061517 (CBS); NIAID Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, MD); and Biomedical Research Institute provided the 5 day cultured schistosomula used in these studies under NIH-NIAID Contract HHSN272201000005I. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins

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