A Comparative Proteomic Analysis of Praziquantel-Susceptible and Praziquantel-Resistant Schistosoma mansoni Reveals Distinct Response Between Male and Female Animals

Schistosomiasis is a chronic neglected tropical disease saddling millions of people in the world, mainly children living in poor rural areas. Praziquantel (PZQ) is currently the only drug used for the treatment and control of this disease. However, the extensive use of this drug has brought concern about the emergence of PZQ-resistance/tolerance by Schistosoma mansoni. Studies of Schistosoma spp. genome, transcriptome, and proteome are crucial to better understand this situation. In this in vitro study, we compare the proteomes of a S. mansoni variant strain stably resistant to PZQ and isogenic to its fully susceptible parental counterpart, identifying proteins from male and female adult parasites of PZQ-resistant and PZQ-susceptible strains, exposed and not exposed to PZQ. A total of 60 Schistosoma spp. proteins were identified, some of which present or absent in either strain, which may putatively be involved in the PZQ-resistance phenomenon. These proteins were present in adult parasites not exposed to PZQ, but some of them disappeared when these adult parasites were exposed to the drug. Understanding the development of PZQ-resistance in S. mansoni is crucial to prolong the efficacy of the current drug and develop markers for monitoring the potential emergence of drug resistance.


INTRODUCTION
Schistosomiasis is one important intravascular parasitic infection, being endemic in 76 countries with more than 97% of infected people living in Africa and parts of South America. This disease infects an estimative of 290 million people worldwide, especially children, living in poor rural areas of low and middle-income countries (1)(2)(3)(4). Schistosoma mansoni and S. haematobium are the two major endemic parasite species causing Schistosomiasis. S. mansoni cercariae (shredded by snail hosts) penetrate the human skin and migrate into the vascular system, where mature male and female worms mate and produce ≈ 300 eggs per day (5). S. mansoni eggs provoke inflammatory responses and liver damages, which can lead to liver cirrhosis and portal hypertension, as well as an inability to perform a daily activity, headache, fever, syncope, nausea, elevated liver enzymes, malaise, fatigue and nocturnal sweats (4,5).
Despite many efforts to control the transmission of this disease (6)(7)(8), essentially after the introduction of chemotherapeutic treatment in the 1980s, schistosomiasis is still highly prevalent (9). Its control is based on chemotherapy treatment using Praziquantel (PZQ) (10)(11)(12)(13)(14)(15)(16), which is effective, inexpensive, and easy to use (10,16). However, frequent schistosome reinfection occurs, PZQ has minor activity against juvenile parasite forms but has a strong impact on adult worms (10,17,18). Because of its high prevalence, schistosomiasis has earned a Category II disease, ranking next to malaria, for importance as a target tropical disease by the World Health Organization special program for research and training in tropical diseases (1)(2)(3). Although the impact of schistosomiasis could be dramatically reduced by improvement in education and population awareness, basic sanitation conditions, as well as elimination of the intermediate host snails, such methods are not sufficient to control or eradicate this parasitosis. In the absence of vaccines (4), the control of this disease relies on chemotherapy to ease symptoms and reduce transmission. It is possible to induce resistance of S. mansoni and S. japonicum to PZQ in mice under laboratorial conditions, and reduced susceptibility to PZQ in the field isolates of S. mansoni has been found in many foci. Also, there are several schistosomiasis cases caused by S. haematobium infections in which repeated standard treatment fails to clear the infection (10,19,20). Understanding the development of PZQ-resistance in S. mansoni is crucial to prolong the efficacy of the current drug and develop markers for monitoring drug resistance (19,20).
The identification of proteins is important for understanding how schistosomes regulate host immune systems to establish chronic infections and also elucidate other aspects of parasite-host interaction (21). Furthermore, a comprehensive deciphering of the schistosome genome, transcriptome, and proteome has become increasingly central for understanding the complex parasite-host interplay (12,(22)(23)(24). Therefore, such information can be expected to facilitate the discovery of vaccines and new therapeutic drug targets, as well as new diagnostic reagents for schistosomiasis control (21)(22)(23), and may aid the development of protein probes for selective and sensitive diagnosis of schistosomiasis (25). Proteomics approaches encompass the most efficient and powerful set of tools for the identification of protein complexes (26)(27)(28) and have been widely used to decipher the proteome of parasites such as nematodes (29) and trematodes (30)(31)(32)(33). For Schistosoma spp., the proteome has been studied in many developmental life stages (34), including lung-schistosomula (35,36), cercariae (36)(37)(38), egg (36,39) and, adult worms (24,25,36,(40)(41)(42).
Two-dimensional electrophoresis (2-DE) is a technique for the separation and identification of proteins, based on pI values (isoelectric focusing, IEF) in one dimension and molecular mass by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in the other (43). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is one of the most commonly used methods to identify and characterize peptides and proteins in complex samples (44). To our knowledge, the S. mansoni PZQ-resistant strain proteome has not been yet reported thus an examination of global changes in gene expression following drug treatment or between isolates showing differential drug susceptibility may provide an entreé into the identification of genes underlying drug action or resistance.
Based on the need to identify the genes that underly drug action or resistance, this work aimed to compare the proteomes of a S. mansoni variant strain stably resistant to PZQ and isogenic to its fully susceptible parental counterpart, identifying proteins from male and female adult parasites of PZQ-resistant and PZQ-susceptible strains, exposed and not exposed to PZQ. This information represents substantial progress towards deciphering the adult parasite proteome. Furthermore, these data may constitute an informative source for further investigations into PZQ-resistance and increase the possibility of identifying proteins related to this condition, possibly contributing to avoid or decrease the likelihood of development and spread of PZQ-resistance. Improving the understanding of PZQ-resistant and PZQ-susceptible proteome would increase the possibilities of clarification of PZQ mode of action and resistant phenotype.

Ethical Statement
The

Parasite Samples
A resistant strain (RS) of S. mansoni that tolerates up to 1,200 mg PZQ/kg of mouse body weight (this dose compares to a dose of a single-dose treatment regime of 80 mg/kg in humans) was developed recently (45). As previously described in this strain the female and male parasites phenotypically presented different reactions to PZQ both in vivo and in vitro (males being more resistant than female parasites). This S. mansoni variant strain was selected from a fully susceptible parasite strain, by stepwise drug pressure, and is isogenic, except for the genetic determinants of PZQ-resistance phenotypes, and significantly different from the counterpart S. mansoni susceptible strain. As such, this S. mansoni PZQ-resistant strain represents a distinct and valuable model for the study of PZQ-resistance.
Thus, two different parasite isolates were used in this study: S. mansoni BH PZQ-susceptible strain (SS) from Belo Horizonte (BH), Minas Gerais state, Brazil, and a stable PZQ-resistant strain (RS) (IHMT/UNL) obtained from the same BH strain, as described by Pinto-Almeida et al. (45). These two parasite strains are routinely kept in their intermediate host Biomphalaria glabrata snails at the IHMT/UNL (Instituto de Higiene e Medicina Tropical, IHMT; Universidade Nova de Lisboa, UNL, Lisboa, Portugal). Mus musculus CD1 line male mice was chosen as the animal model for S. mansoni infection because it is a good host for this parasite mimicking the S. mansoni human infection (46). Mice infection occurred by natural transdermal penetration of cercariae, by exposing mice tails to about 100 cercariae of S. mansoni each.
Eight-to ten-week old adult parasites were recovered by perfusion of the hepatic portal system and mesenteric veins, according to Lewis (47), and washed twice in RPMI-1640, to remove contaminating hair and blood clots. Males and females of S. mansoni (resistant strain-RS and susceptible strain-SS to PZQ) were analyzed separately. Regarding the groups of adult parasites exposed to PZQ (EPZQ), after collecting, the parasites were transferred to 24-well culture plates containing RPMI-1640 culture medium, 0.2 mol/L L-glutamine, 0.01 mol/L HEPES, 0.024 mol/L NaHCO 3 , 10,000 units penicillin, and 10 mg streptomycin/ml, pH 7 and supplemented with 15% FBS. Five adult parasites were added on each well for each studied group for PZQ treatment: 1) PZQ-susceptible male (SM); 2) PZQsusceptible female (SF); 3) PZQ-resistant male (RM), and 4) PZQ-resistant female (RF). Adult parasites were treated in culture, with 3.0 × 10 −5 mol/L of PZQ during 24 h (this dose was chosen and used because it was optimized in our previous published work (45), it allowed us to differentiate between susceptible and resistant parasites in culture, it has an effect on the parasites but that it does not kill them) and, then washed twice with saline solution to clean any traces of culture medium and stored in Trizol at −80°C, for protein extraction later.
For the groups of adult parasites not exposed to PZQ (NEPZQ), adult parasites were kept in RPMI-1640 medium with no addition of drug during 24 h and then washed twice with saline solution to clean any traces of culture medium and, also stored in Trizol at −80°C, for post protein extraction. There are three experimental variables; (1) exposure (E) to Praziquantel (PZQ) or non-exposure (NE), (2) the strain of S. mansoni, susceptible (S) or resistant (R), and (3) the sex of the parasite (M/F). Accordingly, the experimental set up consisted of eight sample groups, four for parasites not exposed to PZQ (RM-NEPZQ, RF-NEPZQ, SM-NEPZQ, and SF-NEPZQ), and four for parasites exposed to PZQ (RM-EPZQ, RF-EPZQ, SM-EPZQ, and SF-EPZQ), all experimental groups were done in technical triplicate (2-DE gels were run three times on a single drug treated sample). After the treatment period, the proteins were immediately extracted, all worms were alive prior the protein extraction. In each experimental group proteins were extracted in bulk. The effect of male and female separation on parasite development was not tested in vitro in this work.

Preparation of Protein Extracts
S. mansoni adult parasite protein extracts were obtained using Trizol protocol, according to the manufacturer's instructions (48). Briefly, the parasites were lysed and homogenized directly in Trizol reagent at room temperature (≈25°C). The homogenized samples were incubated at room temperature to permit complete dissociation of the nucleoprotein complex. After homogenization, we proceeded to the separation phase, adding chloroform and centrifugation of samples. The aqueous phase was removed and the interphase and organic phenolchloroform phase was used for the protein isolation procedure. Next, isopropyl alcohol precipitation was performed and the pellet was solubilized in SBI buffer (7 mol/L urea, 2 mol/L thiourea, 0.015 mol/L DHPC, 0.5% Triton X-100, 0.02 mol/L DTT, and complete mini protease inhibitor cocktail tablets), according to Babu et al. (49) and stored at −80°C until use. Protein concentration in protein extracts was measured by Bradford assay (50), and the quality of the extract was verified in 12% uniform SDS-PAGE gels (42).

Two-Dimensional Electrophoresis
Two-dimensional electrophoresis (2-DE) gels was performed in triplicate with 240 mg of protein extracts, for each eight sample group (four not exposed and four parasites exposed to PZQ). To prepare samples for 2-DE, protein samples were diluted in rehydration solution containing 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 0.5% IPG buffer, 1% DTT, and 0.002% bromophenol blue. The rehydration was carried out passively overnight for 12 h in a 13 cm pH 3-10 strip. The strips were then applied on an Ettan IPGphor 3 System (GE Healthcare, Piscataway, NJ, USA), for protein separation by isoelectric focusing (IEF), using the following conditions developed for this work: at a constant current 50 mA/strip, the voltage program started with a gradient up to 3.5 kV in 3 h, then a step of 3 h at 3.5 kV, then a total voltage of 64.0 kVh to the end.
After focusing, the strips containing proteins were reduced in an equilibration solution (50 mol/L Tris-HCl, pH 8.8, 6 mol/L urea, 20% glycerol, 2% SDS) containing 2% DTT, and then alkylated in the same solution containing 2.5% IAA. The Immobilized pH gradient (IPG) strips and molecular weight standards were then transferred to the top of 12% uniform SDS-PAGE gels, and sealed with 0.5% agarose. The second dimension was carried out using a Protein Plus Dodeca cell system (Bio-Rad Laboratories, Inc., Hercules, CA) under an initial current of 15 mA/gel for 15 min, followed by increasing the current to 50 mA/ gel until the end of the run.
For 2-DE experiments, three biological sample replicas were done for the two-dimensional polyacrylamide gel electrophoresis for each group, thus making our experimental procedure more reproducible. Gels were fixed in 40% methanol/10% acetic acid solution and stained with coomassie brilliant blue R-350. The spots were visually normalized and evaluated using the ImageMaster ® 2D Platinum 7.0 software (GE Healthcare Bio-Sciences, Uppsala, Sweden), according to the number of spots matching, and the results were exported and evaluated in SIMCA-P software (Umetrics, Umeå, Sweden) as previously described. Here, we built PCA models, from which it was possible to obtain statistically valid spots between groups using t-test and Jackknife. This was done in a way that the software would inform us on the proteins spots of interest (51).

In-Gel Digestion and Peptide Preparation for Mass Spectrometry Analysis
We selected spots that were consistently found in all replicates for each condition, and were large enough to excise and retain sufficient material for downstream processing. The protein spots selected for each group were manually excised, destained, reduced, alkylated, and digested in-gel with trypsin from the corresponding 2-DE gel for mass spectrometry (MS) identification. First, spots were washed in ultrapure water and then destained in a solution containing 50% methanol/2.5% acetic acid, for 2 h at room temperature. This step was repeated until clear of blue stain. The gel fragments were incubated in 100% ACN with occasional vortexing until gel pieces became white and shrank. Then, the solution was removed and spots were completely dried and ready for digestion. The in-gel digestion with trypsin modified sequencing grade reagents was done according to Shevchenko et al. (52). Briefly, protein digestion was conducted at 37°C overnight. After the incubation, the supernatant was transferred to a clean tube, and 30 ml of 5% FA/60% ACN solution was added to gel spots for the extraction of tryptic peptides. This procedure was performed two times for 30 min, under constant agitation. The supernatant was pooled to the respective tube containing the initial peptide solution. This solution was dried in a Speedvac concentrator Thermo Fisher Scientific (Waltham, MA) and the peptides were re-suspended in 8 ml of 0.1% FA solution. The peptides were desalted in reverse phase micro-columns ZipTip ® C18 (Millipore Corporation, Billerica, MA), according to the manufacturer's instructions (53). Peptides were dried again and resuspended in 50% ACN/0.1% TFA solution.

Nanoflow LC-MS/MS Analysis and Protein Identification
The digested peptides (50 µg) were analyzed in an Easy-nLC II nanoflow liquid chromatography system (nano-LC-MS/MS) (Thermo Fisher Scientific, Waltham, MA) in tandem with an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific), which was equipped with a nanoelectrospray Nano-Flex II (Thermo Fisher Scientific) operating in a positive ion mode (24). Nanoelectrospray voltage was set to 4.5 kV and source temperature at 220°C. The precursor ion was isolated using the data-dependent acquisition mode with a 2 m/z isolation width and sequentially the ten most intense ions were selected for fragmentation event by collision-induced dissociation (CID), at 35% normalized collision energy and 10 ms activation time. Maximum ion injection times were set to 100 ms for MS and 500 ms for MS/MS, with a resolution of 60,000 and a scan within m/z range from 400 to 2,000. Chromatographic separation was carried out a Thermo C18 capillary column (10 cm × 75 µm, 3 µm, 120 Å) protected by a guard Thermo C18 capillary column (2 cm × 100 µm i.d., 5 µm particle, 120 Å pore size). Ultrapure water containing 0.1% FA (solvent A) and ACN containing 0.1% FA (solvent B) was used as mobile phase. The separation was performed at room temperature with a constant flow rate of 0.3 µl/min, with a total acquisition time of 90 min, by employing the elution program as follows: linear gradient of 5% of solvent B during 5 min, ranging to 80% of solvent B over 85 min. Data acquisition was controlled by Xcalibur ® 2.0.7 Software (Thermo Fisher Scientific) and converted in mgf files using MassMatrix ® MS Data file conversion version 3.9 software.

Bioinformatics
The list of peptide and fragment mass values generated by the mass spectrometer for each spot were submitted to an MS/MS ion search using the Mascot ® 2.0 software online search engine (Matrix Science Inc, Boston, MA, USA) to search the Nanoflow LC-MS/MS data against the NCBInr database Schistosoma_mansoni_NCBI_112014, November 2014. Mascot ® software (used at the Mass Spectrometry Laboratory at Brazilian Biosciences National Laboratory, CNPEM, Campinas, Brazil) and also against WormBase ParaSite, was set with two tryptic missed cleavages, a peptide ion mass tolerance of 10 ppm, a fragment ion mass tolerance of 0.02 Da, a peptide charge state of 2+ and 3+, variable modifications of methionine (oxidation), and fixed modifications of cysteine (carbamidomethylation).
During the analysis, our samples were checked against a contaminant database supplied by the Global Proteome Machine [GPM-web-based, an open-source user interface for analyzing and displaying protein identification data by Craig and Beavis (54)]. All validated proteins had at least two independent spectra, with greater than 95.0% probability estimated by the Peptide Prophet algorithm of being present in the S. mansoni database as at least one unique peptide. To avoid random matches, only ions with an individual score above the indicated by the Mascot ® to identity or extensive homology (p <0.05) were considered for protein identification. However, when the Mascot ® score was not significant, but the percentage coverage and root mean squared error (RMSE) were in the same range as those of proteins with a significant match, proteins were deemed identified if additional parameters, such as its calculated pI and Mw, were in agreement with those observed for the actual gel spot and the species matched was S. mansoni.
The molecular function and biological process were assigned for the proteins identified according to information obtained from the Gene Ontology ® (GO) database (55). The exact annotation for each protein was used in most cases. However, the catalytic activity category was used for all proteins with molecular functions associated with (GTPase, hydrolase, isomerase, kinase, ligase, lyase, oxidoreductase, transcription, and transferase activities). Binding category was used for all types of ligand identified (actin, ATP, calcium, GTP, magnesium ion, metal ion, protein domain-specific, and nucleotide bindings). Furthermore, there were other molecular function categories classified such as chaperone, motor, regulation of muscle contraction, structural, and transport. The proteins that had no associated known function were classified as "unknown".
The list of proteins that were identified in all the experiments was analysed using the webserver g:Profiler, to perform functional enrichment analysis. Briefly, the g:GOSt functionality was used, considering a g:SCS threshold <0.05 for statistical significance (56).

2-DE Separation of Proteins From S. mansoni PZQ-Resistant and PZQ-Susceptible Adult Parasites
Eight protein extracts were analyzed: four from parasites not exposed to PZQ (NEPZQ) [resistant males and females (assigned as RM-NEPZQ and RF-NEPZQ, respectively), and susceptible males and females (assigned as SM-NEPZQ and SF-NEPZQ, respectively)] and another four samples from parasites exposed to PZQ (EPZQ) [resistant males and females (assigned as RM-EPZQ and RF-EPZQ, respectively), and susceptible males and females (assigned as SM-EPZQ and SF-EPZQ, respectively)]. All protein extracts presented high purity and good quality for posterior 2-DE ( Figure 1) and nano-LC-MS/MS analysis. The quality/purity of the protein extract was assessed as previously described by Rayna et al. (57), the quality/purity of the extract was verified in 12% uniform SDS-PAGE gels.
Analytical 2-DE gels were produced, to reproducibly resolve protein spots in a broad pH range and molecular weight, and to compare the protein pattern of S. mansoni proteome from the two strains (PZQ-resistant and PZQ-susceptible) not exposed ( Figure 2) and exposed to PZQ (Figure 3). See Figures 2, 3, and Supplementary Data.
Two-DE maps constructed with coomassie blue-stained gels showed reasonably comparable numbers of spots in all the samples. In total 133 ± 14, 265 ± 20, 142 ± 8, and 188 ± 34 spots were detected in proteins from RM-NEPZQ, RF-NEPZQ, SM-NEPZQ, and SF-NEPZQ, respectively ( Table 1). For parasites exposed to PZQ, 203 ± 14, 133 ± 9, 220 ± 34, and 99 ± 19 spots were detected in RM-EPZQ, RF-EPZQ, SM-EPZQ, and SF-EPZQ, respectively ( Table 1). Table 1 includes the data from the replicates. The spots that were validated were the same in each replicate. The only protein spots whose analysis was continued, were those where a protein spot is consistently expressed in the three replicates, if the protein spot is only expressed in one or two replicas then this spot is excluded from the subsequent analysis.

Nanoflow LC-MS/MS Analysis and Protein Identification
Gels were compared between replica and the only spots that were chosen to be excised were the ones that were more intense and that were present in all three replicas. The spots were excised from preparative gels for each sample, digested by trypsin, and  Tables 1, 2. Some proteins were identified in only one individual spot, but on several occasions, more than one spot in a gel corresponded to the same protein, or better, same protein species (Figures 2, 3, and Supplementary Material Tables 1-3).
Sixty individual protein species were identified on samples from parasites not exposed to PZQ, of which 45 were present in RM-NEPZQ, 52 in RF-NEPZQ, 45 in SM-NEPZQ, and 47 proteins in SF-NEPZQ (Supplementary Material). In this group of NEPZQ parasites, 35 proteins were common to all four protein extracts, eight occurred only in resistant adult parasites, eight only in susceptible adult parasites, four in female parasites, and three were only present in resistant females ( Figure 4). Interestingly, two proteins that have shown to be common in RM-NEPZQ, RF-NEPZQ, and SM-NEPZQ preparations, namely serine/threonine phosphatase and troponin I, did not appear in SF-NEPZQ ( Table 2).
The total number of proteins identified was reduced to 55 proteins on the protein extracts from parasites exposed to PZQ.  Tables 1-3). Forty-two proteins appeared to be common to all the four protein extracts of EPZQ parasites. Besides, four proteins were present only in resistant strain, one in susceptible strain, three were exclusive of female parasites, two proteins were present only in male parasites, and three were only in resistant females ( Figure 4). Five proteins that were present on parasites not exposed to PZQ, namely, collagen alpha chain, dopamine transporter, twister (putative), ubiquitin-specific peptidase 30 (C19 family), and Smp_162220, did not appear here ( Table 2).

Molecular Function of Identified Proteins
The proteins identified by nano-LC-MS/MS, summarized in Supplementary Tables 1, 2, were generally categorized by their molecular function, according to information obtained from the GO database (Table 3), we consider that this general classification in large groups of protein functions is fundamental as a basis for future proteomics and transcriptomic tests. When proteins had another function annotation, they were shown in brackets. The biological process and subcellular localization assigned to each protein in that database are also included in Table 3. The results allowed the identification of proteins categorized as binding, catalytic, transport, regulation of muscle contraction, chaperone, motor, structural activities, and proteins of unknown functions. Among the molecules identified as binding proteins, most of them were ATP, nucleotide, protein, and ion binding proteins. The proteins categorized correspond to a variety of biological processes, nevertheless, most of them were glycolytic enzymes and proteins related to the metabolic process.
Regarding the subcellular localization, the proteins identified were classified, as cytoskeletal, cytosolic, nuclear, membrane proteins, and some of them were located on extracellular matrix. Among them, those most abundantly identified were cytosolic proteins. There were fifteen and seven proteins whose biological process and subcellular localization, respectively, were not predicted ( Table 3).

Functional Enrichment Analysis
In all experimental conditions, except in both exposed or not exposed susceptible females, the GO Molecular Processes with the highest p-values were related to muscle contraction (GO:0006936, GO:0003012, GO:0003008), corresponding to UniProt geneIDs P42637, P42638, G4VGQ7, G4LYF7. However, in susceptible females, both exposed and unexposed, the Molecular Processes with the highest p-values were unique and related to ADP metabolic process and ATP generation from ADP (GO:0046031, GO:0006757, GO:0009135, GO:0006096, GO:0009179, GO:0009185, GO:0006165, GO:0046939, GO:0006090, and GO:0009132). These GO terms were associated with gene IDs P53442, P20287, G4VJD5, and G4VQ58 (

DISCUSSION
Nowadays the control of schistosomiasis is based on PZQ, which is the only drug available for its treatment. The WHO strategy for schistosomiasis control focuses on reducing disease through periodic, targeted treatment with PZQ through the large-scale treatment of affected populations. It involves regular treatment of all at-risk groups (6,8,12,16). However, the existence of reports of PZQ-resistance cases by S. mansoni (as well as by S. haematobium) has become a serious problem that needs to be solved. Some studies have suggested that resistance to Schistosoma infection can be acquired naturally or induced by the drug (20,45,(58)(59)(60). For example, Sanchez et al. (20) examined the effect of PZQ in strains of S. mansoni. The authors noted a reduced miracidia susceptibility after exposure to 2 × 50 mg/Kg of PZQ. It has also been reported the susceptibility of worms in vivo (mice infected with schistosomes) after a lethal dose of 3 × 300 mg/kg of PZQ, reducing in only 10% the worm number in infected rodents. Besides experimental evidence, reports of treatment failure in Senegal and Egypt with reduced susceptibility to PZQ were obtained (61,62) and, further in vitro experiments have confirmed the development of PZQ-resistance (9,10,43,(61)(62)(63)(64).
In the present study we identified 60 different proteins on S. mansoni proteome, this corresponds to 0.42% of the full S. mansoni proteome (https://www.uniprot.org/proteomes/ UP000008854). All those proteins were present in adult parasites not exposed to PZQ, but some of them disappeared when these adult parasites were exposed to the drug. This result could indicate the involvement of PZQ exposure on those protein expressions in resistant and susceptible strains. With this, we are not unequivocally stating that these proteins are the only ones or that they are at all responsible for the phenotype of PZQ drug resistance but this work opens the door to new studies using proteomics, transcriptomics, and metabolomics on the importance of these proteins in the phenotype and genotype of resistance to PZQ.
Although previous studies of Schistosoma proteome had been performed using protein extracts and Schistosoma species different from ours, some proteins, such as 14-3-3 protein, HSP-70, GAPDH, glutathione S-transferase 28 kDa, enolase, fructose-bisphosphate aldolase, actin, triosephosphate isomerase, calponin, elongation factor 1-a, phosphoglycerate kinase, phosphoglycerate mutase, myosin, and paramyosin were commonly identified (36,42,(65)(66)(67)(68)(69)(70). In addition, some proteins that have already been tested as vaccine candidates, as glutathione S-transferase 28 kDa (71), triosephosphate isomerase (71,72), and paramyosin (71), were also identified in the present study. Major egg antigen, troponin T, disulphide-isomerase ER-  The only protein spots whose analysis was continued, were those that where a protein spot is consistently expressed in the three replicates, if the protein spot is only expressed in one or two replicas then this spot is excluded from the subsequent analysis.
In bold Mean ± SD.
60, and actin, proteins that we also found, have already been clustered as immunoreactive proteins in serum pools of infected or non-infected individuals from endemic area (42). Looking at the proteomes from both genders, in this survey, four proteins were only present in females from both strains, even under exposure to PZQ, namely, cytosol aminopeptidase, inosine-5′-monophosphate dehydrogenase (IMPDH), ubiquitin protein ligase E3a, and collagen alpha chain. Cytosol/leucine aminopeptidase catalyzes the hydrolysis of amino acid residues from the N-terminus of proteins and peptides (73), and it has already been assessed as a vaccine candidate against the infection of Fasciola hepatica (74). This protein has previously been identified in S. mansoni eggs (75). Regarding IMPDH, this protein is responsible for the rate-limiting step in guanine nucleotide biosynthesis (76), and it has previously been identified in the Schistosoma genome and transcriptome (77). E3 ligase enzyme catalyzes protein ubiquitination, which regulates various biological processes through covalent modification of proteins and transcription factors, and ubiquitin is the most important protein of this process (78,79). Ubiquitination is of interest in S. mansoni because this process could be a potential target for the design of new drugs (80), being ubiquitin protein ligase E3a a good target to be studied. Concerning collagen alpha chain, Yang et al. (81) described that silencing the expression of a type of collagen (type V collagen) significantly affects the spawning and egg hatching of S. japonicum, and it also affects the morphology of the adult parasites (81). Therefore, it would be very interesting to evaluate the role of each of these proteins in PZQ-resistance, especially, collagen alpha chain, since it seems to produce morphological alterations in eggs and adult parasites of S. mansoni PZQ-resistant strain (10,11). Moreover, females of this species, the only gender in which those proteins were found in this work, are more tolerant to PZQ treatment than males (45). FIGURE 4 | Number of shared proteins identified from parasites not exposed and exposed to Praziquantel. TABLE 2 | Summary comparison of the specific proteins identified from parasites not exposed (NEPZQ) and exposed (EPZQ) to Praziquantel.

Proteins
Not exposed to PZQ Exposed to PZQ   Another large difference between the proteome of both genders was the expression of troponin I and serine/threonineprotein phosphatase. These proteins were present in males independently of drug exposure, but in females, they were only present in resistant females not exposed to PZQ. Troponin I belongs to the troponin complex that mediates Ca 2+ regulation that governs the actin-activated myosin motor function in striated muscle contraction (82). On the other side, protein kinases and phosphatases, as is the case of serine/threonine-protein phosphatase, are essential for the normal functioning of signaling pathways since it is well known that reversible phosphorylation of proteins is a ubiquitous mechanism crucial for the regulation of most cellular functions (83). In S. mansoni, a phosphatase 2B (calcineurin) has been described as a heterodimer with a catalytic subunit and a regulatory subunit, which bind to Ca 2+ increased the phosphatase activity (84). Thus, protein phosphatases represent crucial molecules for the parasite and hence potential chemotherapeutic targets (85). Regarding resistant strain parasites, it is notable the finding of eight proteins exclusively present in those S. mansoni adult parasites. From those eight proteins, dopamine transporter, twister (putative), ubiquitin-specific peptidase 30 (C19 family), and uncharacterized protein smp_162220 were not detected in the proportion of the proteome we analysed in S. mansoni PZQexposed adult parasites. However, galactokinase, ornithine aminotransferase, Rab6-interacting protein 2 (ERC protein 1), 4 | Summary comparison of the specific proteins identified from parasites not exposed (NEPZQ) and exposed (EPZQ) to Praziquantel on gene ontology analyses.

Exposed
Non Exposed and transducin beta-like remained after drug exposure. Dopamine/norepinephrine transporter (SmDAT) gene transcript, characterized in S. mansoni, is essential for the survival of the parasite as it causes muscular relaxation and a lengthening in the parasite, controlling movement (86). Galactokinase catalyzes the second step of the Leloir pathway, a metabolic pathway found in most organisms for the catabolism of b-D-galactose to glucose 1-phosphate (87). Galactokinase and hexokinase have a similar enzymatic function on sugar phosphorylation (88), and characterization of schistosome hexokinase has been described as pertinent to understanding the metabolic response of S. mansoni cercariae to an increased glucose availability (89). Ornithine aminotransferase was already identified in S. mansoni (90) and it has been characterized as playing a central role in ornithine biosynthesis (91). It seems responsible for catalyzing the transfer of the delta-amino group of L-ornithine to 2-oxoglutarate, producing L-glutamategamma-semialdehyde, which in turn spontaneously cyclizes to pyrroline-5-carboxylate, and L-glutamate (92). Rab6-interacting protein 2 is a member of a family of RIM-binding proteins, which are presynaptic active zone proteins that regulate neurotransmitter release (93). Ubiquitin-specific peptidase 30 (C19 family) belongs to a metabolic pathway that had previously been associated with the development of artemisinin and artesunate-resistance in Plasmodium chabaudi (94), which is a very interesting result. All those proteins specifically found in the resistant strain should be further studied to better understand if they could have a fundamental role in PZQ-resistance development. Yet, for the resistant strain parasites, there are three proteins, beta 1,3-galactosyl-transferase, cathepsin L, and receptor for activated Protein Kinase C (PKC) that are exclusive to resistant females, even after exposure to PZQ. Beta 1,3-galactosyltransferase has previously been identified in the Schistosoma genome and transcriptome (77). Cathepsin L activity is believed to be involved in hemoglobin digestion by adult schistosomes (95), and these authors suggested the involvement of cathepsin proteinases in several key functions render them as potential targets to novel antiparasitic chemotherapy and immunoprophylaxis. Putative PKC exists in kinomes of the blood flukes S. mansoni (20,96,97), S. japonicum (98), S. haematobium (99), and regulates movement, attachment, pairing, and egg release in S. mansoni, being considered a potential target for chemotherapeutic treatment against schistosomiasis (100). These results need further investigation thus considering that the PZQ mode of action is not completely clarified nor is its genetic process of susceptibility lost, our results might help in the process of clarification of a possible relationship between those proteins and PZQ-resistance in S. mansoni females (20,45). Concerning the PZQ-susceptible strain, we noted eight proteins that only appeared in this strain. Phosphopyruvate hydratase (enolase), an important glycolytic enzyme that has the functions of activating the plasminogen, involving in the processes of infection and migration of parasites, reducing the immune function of the host as well as preventing parasites from the immune attack of the host (101), is the only protein from those eight proteins that continued to be present after PZQ exposure.
Our gene enrichment analyses also provided functional differences when comparing males and females. The results identified in susceptible females, either exposed or not exposed, a significantly higher p-value for GO terms that were related to ATP generation from ADP and other GO terms within the ancestor chart of this term. On the other hand, susceptible males presented significantly higher p-values for GO terms associated with muscle contraction. This give us an indication of a higher fitness and active metabolic status of susceptible females both in the presence or absence of Praziquantel when compared to susceptible males. Also, processes related to monocarboxylic acid metabolic processes were identified only in susceptible males and females, both exposed and unexposed. This could also suggest that loss of susceptibility/resistance to Prazinquantel come at the compromise of the fitness level, with lower metabolic levels both in males and females.
These results altogether represent an important finding for the study of PZQ-resistance/susceptibility in S. mansoni, since they allow comparing directly the proteome under both conditions. We believe that the most promising candidates are proteins that appeared associated only to one of the strains, especially those with functions possibly related to the phenotypic alterations observed by Pinto-Almeida et al. (11), or previously associated with resistance by other parasites to different drugs. These candidates require special attention in more studies, assessing for instance the level of protection induced by these proteins in animal models infected by both S. mansoni strains, as they may have some involvement in the PZQ-resistance phenomenon, although further studies are needed to deepen this knowledge.

CONCLUSIONS
Here, we investigated the proteome of a PZQ-resistant S. mansoni strain and the respective isogenic susceptible strain, an important step towards a full clarification on the genetic process of PZQ-resistance considering that we were able to compare a PZQ-resistant parasite line with the susceptible one from with it was obtained allowing a more accurate comparison between strains that differ solely on PZQ susceptibility phenotype. This study allowed us to identify proteins that in our proteome analyses differ between PZQ-susceptible and PZQresistant parasites, however, their relationship or contribution towards the mechanism of resistance remains unclear and strongly needs further clarification. Since these proteins may be involved with the PZQ-resistance phenomenon, their functional characterization should be pursued in future studies aiming at the discovery of new drug targets for schistosomiasis control and also studies that could clarify the PZQ mode of action.
The identification of the proteins putatively associated with PZQ-resistance in S. mansoni permits also to investigate the possibility of developing a diagnostic test to distinguish patients carrying PZQ-resistant strains from those with PZQ-susceptible S. mansoni. The development of such a test would constitute a major step towards schistosomiasis control as it would render it possible to adjust drug administration to increase treatment efficacy, perhaps even by combining PZQ with efflux pump inhibitors as suggested previously (45). The proteome analysis made it also possible to identify proteins that were present only in females, being them good targets to identify the mechanisms underlying the decreased PZQ-susceptibility of females, when compared to males. Furthermore, some of these proteins may constitute targets, for schistosomiasis control. They should, therefore, be the object of further analysis. In this context, the development and use of other techniques, such as genetic manipulation methods, will be crucial to further unravel the phenomenon/problem of PZQ-resistance/tolerance/loss of susceptibility. Therefore, this work opens doors to other PZQresistance studies, and could represent a basis to find a solution to the PZQ-resistance problem in a disease that affects millions of people worldwide.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are publicly available. This data can be found here: https://datadryad.org/ stash/share/_nzZNotPFuNIsBY1R5x1XjDy3XUFKGAEsGBDF Nl3yWM.

ETHICS STATEMENT
The research project was reviewed and approved by the Ethics

AUTHOR CONTRIBUTIONS
AP-A: Conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writingoriginal draft, writing -review and editing, and visualization. TM: Conceptualization, methodology, investigation, resources, writing -original draft, and writing -review and editing. PF: Writing -original draft, writing -review and editing, and experimental studies. ABA: Writing -review and editing, and gene ontology analysis. SB: Conceptualization, writing -original draft, and writing -review and editing. FA: Writing -original draft, writing -review and editing, and funding acquisition. SA: Resources, writing -original draft, and writing -review and editing. CG: Writing -review and editing. EC: Conceptualization, methodology, software, validation, formal analysis, data curation, writing -original draft, writing -review and editing, visualization, supervision, project administration, and funding acquisition. AA: Conceptualization, methodology, software, validation, formal analysis, investigation, research, data curation, writing -original draft, writing -review and editing, visualization, supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We acknowledge the Mass Spectrometry Laboratory at Brazilian Biosciences National Laboratory, CNPEM, Campinas, Brazil for their support with the mass spectrometry analysis. The authors would like to thank Professor Ana Tomaś from IBMC and GABBA program (Porto, Portugal), for very helpful suggestions that improved greatly this paper.