International Journal for Parasitology 45 (2015) 385–391

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International Journal for Parasitology

journal homepage: www.elsevier .com/locate / i jpara

Revealing praziquantel molecular targets using mass spectrometryimaging: an expeditious approach applied to Schistosoma mansoni

http://dx.doi.org/10.1016/j.ijpara.2014.12.0080020-7519/� 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

⇑ Corresponding authors at: Rua Cinco de Junho, 350 – Barão Geraldo, 13083-877Campinas, SP, Brazil. Tel.: +55 19 3521 9138.

E-mail addresses: sallegre@unicamp.br (S.M. Allegretti), rrc@fcm.unicamp.br(R.R. Catharino).

1 These authors contributed equally to this work.

Mônica Siqueira Ferreira a,1, Rosimeire Nunes de Oliveira b,1, Diogo Noin de Oliveira a,Cibele Zanardi Esteves a, Silmara Marques Allegretti b,⇑, Rodrigo Ramos Catharino a,⇑a Innovare Biomarkers Laboratory, Medicine and Experimental Surgery Nucleus, University of Campinas, Campinas, São Paulo, Brazilb Biology Institute, Animal Biology Department, University of Campinas, Campinas, São Paulo, Brazil

a r t i c l e i n f o

Article history:Received 23 July 2014Received in revised form 21 October 2014Accepted 19 December 2014Available online 23 March 2015

Keywords:Schistosoma mansoniMass spectrometry imagingPraziquantelBiomarkersLipids

a b s t r a c t

Finding specific molecular targets and the mechanism of action of praziquantel in the treatment ofschistosomiasis remains a challenging task. Our efforts were focused on obtaining further informationon worm composition before and after exposure to praziquantel in the treatment of schistosomiasis toelucidate the potential sites of action of this drug. Evidence indicates that the lipid bilayer is changedby treatment with praziquantel. Following this rationale, we employed a mass spectrometry imaging-based approach that helped to characterise lipids in specific locations, which are directly involved inthe biochemical pathways of the BH strain of Schistosoma mansoni, as well as differentiating the molecu-lar response that each worm sex presents in vivo. Our findings demonstrated significant differencesbetween the chemical markers found in adult worms before and after praziquantel exposure, especiallyin phospholipids, which were predominantly identified as chemical markers in all samples. Results alsoindicate that distinct molecular pathways in both male and female worms could be differentially affectedby praziquantel treatment. These data shine new light on the mechanism of action of praziquantel, takinga further step towards its full understanding.

� 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Praziquantel (PZQ) is the most commonly prescribed drug forall forms of schistosomiasis, since it is equally effective againstSchistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi,Schistosoma intercalatum and Schistosoma haematobium (Fenwicket al., 2003; Webbe and James, 1977). However, it does not showfull efficacy against schistosomula and the juveniles stages of S.mansoni, resulting in lower cure rates in areas with high endemic-ity (Gönnert and Andrews, 1977; Pica-Mattoccia and Cioli, 2004;Sabah et al., 1986).

The PZQ mechanism of action is continually being studied(Andrews, 1985; Doenhoff et al., 2008; Hu et al., 2004; LoVerdeet al., 2004); some physiological and morphological aspects havebeen understood for a relatively long time, such as rapid Ca2+ ionuptake (Pax et al., 1978) and vacuolation and blebbing near andon the surface of worms (Becker et al., 1980). In male worms, in

addition to its effects on Ca2+ concentration, PZQ stimulates Na+

influx in a non-ionophore mechanism (Pax et al., 1978).Furthermore, PZQ induces modifications in membrane fluidity aswell as in phospholipid (PL) composition, producing alterationsin its permeability to ions or resulting in indirect effects on mem-brane receptors and channels (Harder et al., 1988; Lima et al.,1994). However, the full mechanism still remains unknown; forexample, the pathway of Ca2+ homeostasis disruption by PZQ inadult schistosomes (Day et al., 1992; Redman et al., 1996) andthe mechanism of PZQ binding to its molecular targets (Tallimaand El Ridi, 2007; Troiani et al., 2007) are yet to be determined.

To better understand some of these mechanisms, modern ana-lytical approaches, e.g. chromatographic techniques combinedwith MS, have been employed for chemical characterisation ofadult schistosomes and PZQ metabolites in the host (Meier andBlaschke, 2000, 2001; van Balkom et al., 2005). More recently,MALDI-MS has been applied as the main analytical tool (Franket al., 2012). Approaches using MS Imaging (MALDI-MSI) (Cornettet al., 2007) were developed to identify the spatial distribution ofcompounds in any physical sample such as tissue sections (Solon,2007), single cell (Ferreira et al., 2014a), drug tablets (Rodrigueset al., 2014) and cosmetic products (de Oliveira et al., 2013).

386 M.S. Ferreira et al. / International Journal for Parasitology 45 (2015) 385–391

Moreover, MALDI-MSI was applied, for the first known time, in S.mansoni adult worms to demonstrate different chemical markersaccording to schistosome sex and strain (Ferreira et al., 2014b).In that study we localised each identified compound in the bodyof the worm. Since schistosomes show stage- and sex-dependentdifferences in susceptibility to PZQ (Pica-Mattoccia and Cioli,2004), information on possible targets and pathways of the drugcould be clarified by chemical marker localisation.

Based on this, the present work has employed the metabolomicplatform to characterise both sexes of S. mansoni adult worms (BHstrain) treated with PZQ. This report identifies the spatial dis-tribution of chemical markers using MALDI-MSI technology, aidingthe search for the understanding of possible targets and pathwaysof this anti-schistosomal drug.

2. Materials and methods

2.1. Mouse infection with S. mansoni

BALB/c albino female mice, 30 days-old, weighing 18–20 g,were individually infected with approximately 70 S. mansoni cer-cariae of the BH strain (from Belo Horizonte, MG, Brazil). At thisstage, the utilised procedure was caudal immersion for 2 h, withlight exposure and controlled temperature at 28 �C (Olivier andStirewalt, 1952). After 45 days p.i., animals were divided into twogroups (n = 5/group): (i) treated with PZQ (Merck, Darmstadt,Germany) and (ii) negative control. The first group received a sin-gle oral dose of 40 mg/kg of PZQ. The control group received 1% PBSsolution. The oral dose of 40 mg/kg of PZQ used in the presentstudy is considered curative in humans, as adopted by theschistosomiasis treatment and control programs in Brazil with acure rate of 80–90%. This study was carried out in strict accordancewith the recommendations in the Guide for the Care and Use ofLaboratory Animals. The protocol was approved by theInternational Ethics Commission for the Use of Animals (CEUA/ICCLAs, protocol n� 2170-1, University of Campinas, Brazil).

2.2. Recovery of S. mansoni worms

Two weeks after treatment, mice were subjected to cervical dis-location. Schistosoma mansoni adult worms (n = 2/mouse) wererecovered by perfusion of the hepatic portal system and mesen-teric veins (Pellegrino et al., 1962). All worms were recovered alive

Fig. 1. Principal Component Analysis of all compounds of Schistosoma mansoni adult wor(n = 5/group). The explained variances (X-expl) are shown below the figure. N, male negattreated with praziquantel.

and washed carefully in a 0.9% saline solution at a temperaturebetween 28 and 30 �C. Samples were sequentially transferred toan Eppendorf tube containing 1 mL of MilliQ H2O at the same tem-perature. The maximum time of washing the worms was 30 min.

2.3. MALDI-MSI analysis

All adult S. mansoni worms were deposited on a TLC plate(Merck). Matrix coating was performed using a commercial air-brush, spraying a-cyano-4-hydroxycinnamic acid (Sigma–Aldrich,Pennsylvania, USA) (10 mg/mL in 1:1 Acetonitrile/Methanol solu-tion). Images and MS were acquired in a MALDI-LTQ-XL instrumentequipped with an imaging feature (Thermo Scientific, California,USA). The instrument uses a nitrogen laser as the ionisation sourceand a quadrupole-ion-trap analysing system. All data wereacquired in the positive ion mode. For image acquisition, a50 lm raster width was selected, with three shots taken per spec-trum. Fragmentation data (MS/MS) were acquired by setting thecollision-induced normalised energy to 40. Helium was used asthe collision gas. Each ion was fragmented in triplicate. All imagingdata were then processed using ImageQuest software v.1.0.1(Thermo Scientific). Both spectral and imaging data were nor-malised according to a signal-to-noise ratio threshold of 3:1.

2.4. Statistical analysis and biomarker identification

Mass and intensity values for each spectrum were included inthe Principal Component Analysis (PCA), which was performedusing Unscrambler v.9.7 (CAMO Software, Trondheim, Norway).After discrimination by PCA, potential biomarkers were selectedand, to identify the analytes, MS/MS reactions were performed togenerate their fragmentation pattern. In addition to the spectrum,MALDI-MSI generated a chemical image allowing us to observe thespatial distribution of the analyte precursors, which were pre-viously characterised as lipids. For lipid identification, MS/MS pat-terns and error values were considered, with the assistance ofonline databases such as Lipid MAPS (University of California,San Diego, CA, USA – www.lipidmaps.org) and METLIN (ScrippsCenter for Metabolomics, La Jolla, CA, USA), in order to guide thechoice for potential lipid markers. Their structures were later pro-posed using Mass Frontier software v.6.0 (Thermo Scientific)(Urayama et al., 2010).

ms. Ion biomarkers for each group were separated by Principal Component Analysisive control; d, female negative control; �, male treated with praziquantel; j, female

M.S. Ferreira et al. / International Journal for Parasitology 45 (2015) 385–391 387

2.5. High resolution electrospray ionisation-MS (ESI-MS) analysis

To confirm the chemical markers identities, male and femaleworms, treated or not with PZQ, were submitted to Bligh–Dyerextraction (Bligh and Dyer, 1959). Lipid extracts were resuspendedin 50 lL of MilliQ H2O and 10 lL was diluted in 990 lL of methanoland 0.1% formic acid. Data acquisition was performed in an LTQ-XLOrbitrap Discovery instrument (30,000 FWHM, Thermo Scientific,Bremen, Germany) in the positive ion mode and at the m/zrange of 600–2000 for complex lipid identification. Structuralpropositions were performed using high resolution as the mainparameter. Mass accuracy was calculated and expressed in termsof parts per million (ppm) chemical shifts, according to Machadoet al. (2013).

3. Results

3.1. Metabolic fingerprints of adult worms

Male and female adult worms, treated or not with PZQ, weresubjected to MALDI-MSI analysis, as described in Section 2.1. All

Table 1Lipid chemical markers identified via MS imaging (MALDI-MSI) of Schistosoma mansoni aduany of the positional isomers. Identification is based on MS/MS data, exact mass of each c

Adult worms Parental ion(m/z)

Product ions(m/z)

Molecule LM ID

FCT/MCT 601 557, 583 (DG(17:2/18:3/0:0)+H)+ LMGL617 558, 457, 413 (PA(12:0/18:2)+H)+ LMGP619 448, 430, 560 (PA(13:0/17:1)+H)+ LMGP620 475, 502, 576 (PE(12:0/15:1)+H)+ LMGP724 680, 606, 579, 706 (PC(12:0/20:5)+H)+ LMGP726 682, 581, 710 (PC(14:0/18:4)+H)+ LMGP

FPZQ 651 584, 462, 480 (PG(12:0/15:1)+H)+ LMGP652 463, 585, 608 (PS(14:0/12:0)+H)+ LMGP649 460, 605, 582 (PE-Cer(14:1/18:0)+H)+ LMSP0

MPZQ 853 809, 708, 664 (PI(12:0/22:4)+Na)+ LMGP855 666, 811, 684 (PI(13:0/20:4)+K)+ LMGP827 768, 682, 783 (PI(12:0/20:3)+Na)+ LMGP

LM ID, Lipid MAPS ID (online database); MID, METLIN ID (online database); ppm, parts ptreated with praziquantel (PZQ); MPZQ, male worm treated with PZQ; DG, diacylglycerphosphoglycerol; PS, phosphatidylserine; PI, phosphoinositol; Cer, ceramide.

Fig. 2. Schistosoma mansoni male control adult worm images generated by MS precursor

of them presented clear differences in their spectra when com-pared with each other (Supplementary Fig. S1).

3.2. Statistical analysis and biomarker identification

Preliminary analyses on MALDI-MSI were performed at the m/zrange of 50 and 2000 in both positive and negative ion modes (datanot shown). Accurate results were bound to the m/z range of 600–2000 in the positive ion mode, since they exhibited clear discrim-ination in PCA.

Statistical analyses were performed by the comparison betweenmale and female worms, treated or not with PZQ. As shown inFig. 1, worm control groups (MCT, male control and FCT, femalecontrol) were clearly separated from worms submitted to PZQtreatment, with an accuracy of 99%. The identified ions composethe final model of optimised PCA.

Table 1 presents chemical markers identified in each adultworm, as well as the precursor ion fragmentation and the masserrors for each signal observed in high resolution Fourier transformmass spectrometry (HR-FTMS), measured in ppm (with all resultspresenting a deviation of less than 2 ppm).

lt worms (positive ion mode). The assigned IDs are for general structures and can beompound and Lipid Maps and METLIN databases.

Theoretical mass Experimental mass Error (ppm) MID

02010051 601,4826 601,4818 �1,330046788 435710010051 617,4177 617,417 �1,133754345 81,21710010072 619,4333 619,433 �0,484313646 81,23802010361 620,4286 620,4291 0,805894506 76,59601011333 724,4912 724,4899 �1,79436272 75,61301010499 726,5068 726,506 �1,101159686 59,32704010048 651,4232 651,424 1,228080302 78,87303010931 652,4184 652,4175 �1,379482859 78,5953020059 649,4915 649,4905 �1,539666031 103,098

06010035 853,4837 853,4833 �0,468667416 80,05706010054 855,442 855,4431 1,285884958 80,07606010029 827,4681 827,4688 0,845954061 80,051

er million; FCT, female worm control; MCT, male control worm; FPZQ, female wormol; PA, phosphatidic acid; PE, phosphoethanolamine; PC, phosphatidylcholine; PG,

s. Pos, posterior portion; Ant, anterior portion; T, tegument; OS, oral sucker; G, gut.

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3.3. Spatial distribution of biomarkers in adult worms

Individual worms were submitted to MS/MS analysis. In Figs. 2–5,images clearly revealed differential spatial distribution of chemicalmarkers, where the darker the scale, the higher the concentrationin the site. It was possible to distinguish some characteristicanatomical structures of the worms, e.g. suckers, gut, reproductivesystem and tegument. The increased intensity in grey-scale indi-cates higher concentrations of these biomarkers in each location.

Fig. 3. Schistosoma mansoni female control adult worm images generated by MS precurstegument.

4. Discussion

In the present report, we applied the same tools to identifymetabolic alterations caused by PZQ treatment in the S. mansoniBH strain (in vivo). The results demonstrate the specificity of themethod for schistosomes, identifying and differentiating each sex(Supplementary Fig. S1A, B). Recently, a study performed in ourlaboratory demonstrated the spatial distribution of lipids in adultworms of two Brazilian strains (BH and SE) of S. mansoni

ors. Pos, posterior portion; Ant, anterior portion; R, reproductive system; G, gut; T,

Fig. 4. MS precursor-generated images of a Schistosoma mansoni male adult wormtreated with praziquantel. Pos, posterior portion; Ant, anterior portion; R,Reproductive system; OS, Oral sucker; G, Gut.

Fig. 5. MS precursor-generated images of a Schistosoma mansoni female adult worm treasystem; T, tegument; G, gut; OS, oral sucker; VS, ventral sucker.

M.S. Ferreira et al. / International Journal for Parasitology 45 (2015) 385–391 389

(in vitro) (Ferreira et al., 2014b) using MALDI-MSI and the lipido-mics approach (Ferreira et al., 2014b). However, as expected dueto biochemical interactions with the host, the in vivo lipid profilediffered from the in vitro profile, as it has been described thatschistosomes use compounds from the host in lipid synthesis(Furlong, 1991).

Furthermore, in addition to tegumental damage, PZQ signifi-cantly changed the metabolic profile of both sexes of S. mansoni(Supplementary Fig. S1C, D). This modification was enough to seg-regate almost all groups in PCA analysis, except control male andfemale groups (Fig. 1). Most compounds were probably derivedfrom the host. providing very close similarities between bothsexes, but with different locations. PZQ, however, had differentialreach and action on each sex (Pica-Mattoccia and Cioli, 2004),which could explain the clear segregation of male worms treatedwith PZQ (MPZQ) and female worms treated with PZQ (FPZQ) inPCA.

The potential biomarkers obtained from PCA of MALDI-MSI datawere confirmed using high resolution ESI-MS analysis (Table 1).Among the identified chemical markers, phospholipids (PLs) repre-sent the major class, as also related by Young and Podesta (1982).In general, lipids play important roles in the lives of schistosomes.Apart from constituting biological membranes, they alsoparticipate in host recognition (Fusco et al., 1988), immuneresponse modulation and evasion (Abath and Werkhauser, 1996;McLaren, 1984), communication (Fried and Haseeb, 1991) anddevelopment (Hockley and McLaren, 1973; Shaw et al., 1977;Smith and Brooks, 1969).

Among PL classes, phosphatidylcholines (PCs), phospho-ethanolamines (PEs) and phosphatidic acids (PAs) were found incontrol groups. Interestingly, the same chemical markers pre-sented distinct spatial locations in MCT and FCT groups. For exam-ple, (PA(12:0/18:2)+H)+ (m/z 617) and (PA(13:0/17:1)+H)+ (m/z619) were located in the oral sucker in males, and in the reproduc-tive system and gut of female worms. However, the role of PA in S.mansoni is not yet known. The same occurs with (PC(12:0/20:5)+H)+ (m/z 724) and (PC(14:0/18:4)+H)+ (m/z 726), that werepresent in the oral sucker and gut of the MCT group, and in thetegument of the FCT group. Although the functional roles of thesemolecules in S. mansoni remain unknown, some studies alreadydemonstrated that PCs are the most abundant PL class present inS. mansoni (Brouwers et al., 1998; Young and Podesta, 1982).Furthermore, according to Brouwers et al. (1998), PCs presentedin bulk in schistosome tegument, corroborating our result obtained

ted with praziquantel. Pos, posterior portion; Ant, anterior portion; R, reproductive

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from the FCT group. Moreover, (PE(12:0/15:1)+H)+ (m/z 620) waslocated in the reproductive system and gut of females, and in theoral sucker and gut of males. Diacylglycerol (DG) (DG(17:2/18:3/0:0)+H)+ (m/z 601) was present in gut and tegument, respectively,of female and male bodies of the control group. This compoundclass was previously found as a biomarker in the tegument of adultBH strain schistosomes (Ferreira et al., 2014b). Furthermore, a pre-vious study suggested that DGs are cleaved from phosphoinositol(PI) to be used in the biosynthesis of acetylcholinesterase (AChE)in S. mansoni. AChE is an ectoenzyme that is released on the surfaceof the schistosome when the worm frees itself from the wall ororgan to which it is anchored in the host (Espinoza et al., 1991).

When female worms were treated with PZQ, there was a nota-ble change in its chemical markers: phosphoglycerol (PG(12:0/15:1)+H)+ (m/z 651), phosphatidylserine (PS(14:0/12:0)+H)+ (m/z652) and PE coupled with ceramide (PE-Cer(14:1/18:0)+H)+ (m/z649). All of those were located in different regions of the wormbody. For example, PG was located in the tegument, while PSwas more concentrated in the oral and ventral suckers and repro-ductive system. On the other hand, PE-Cer was concentrated in thegut and reproductive system. The latter is included in the sphingo-lipid category and may be produced in microsomes of rat liver andplasma membrane, and internalised by schistosomes, as they donot have a de novo synthesis pathway (Malgat et al., 1986).However, other studies demonstrated that this sphingolipid canbe synthesized by S. mansoni females via sphingomyelinase activ-ity, and plays a secondary messenger role (Redman et al., 1997;Testi, 1996; Wiest et al., 1992). This role has been established inseveral cell types (Hannun, 1994; Kolesnick and Golde, 1994;Obeid et al., 1993; Testi, 1996), being implicated in cell deathand differentiation. Sometimes, this secondary messenger role isactivated in response to external stimuli such as TNF-a, IFN-cand vitamin D3 (Andrieu et al., 1994; Belka et al., 1995; Hannun,1994; Kolesnick and Golde, 1994; Raines et al., 1993). TNF-a, inturn, is believed to play a preventive role in cases of superinfec-tions (Amiri et al., 1992; Garside et al., 1996; Hagan et al., 1993).PZQ also stimulates TNF-a expression in animals (Pinlaor et al.,2009). Thus, we suggest that PZQ may activate sphingolipid pro-duction in female adult worms and, more specifically, PE-Cer canalso be internalised from the host production; more experimentsare, however, required to test this hypothesis; likewise to under-stand this chemical marker location and the importance of thispathway in female worms.

Similar to females, male worms presented a different lipid pro-file with PZQ treatment. Three PI derivatives were found: (PI(12:0/22:4)+Na)+ (m/z 853), (PI(13:0/20:4)+K)+ (m/z 855) and (PI(12:0/20:3)+Na)+ (m/z 827). All of those were placed in the same spatiallocation in the body of the worm: oral sucker, gut and reproductivesystem. It is also important to note that all chemical markers wereidentified as sodium (Na+) or potassium (K+) adducts. This informa-tion could be supported by Pax et al. (1978), demonstrating thatPZQ stimulates influx of Na+ in S. mansoni male adult worms. Onthe other hand, some reports related that PZQ could induce modi-fication in membrane PLs, causing fluidity which may producealterations in permeability to ions or result in indirect effects onmembrane receptors and channels (Harder et al., 1988; Limaet al., 1994). Moreover, Cunha and Noël (1997) described thatPZQ had no effect on schistosome (Na+ K+)-ATPase. However, allof the previous tests were performed only in the tegument of theworm (Cunha and Noël, 1997). In contrast, a recent study using agene expression assay of male S. japonicum demonstrated thatadult worms exposed to PZQ up-regulated the (Na+ K+)-transport-ing ATPase gene (You et al., 2013), which may indicate that similarinteractions occur for S. mansoni when exploring PZQ actions notonly restricted to the tegument. Since chemical markers of MPZQwere located in internal structures of anatomical male worms,

and all PIs from different species were related with Na+ and K+,we could suggest that the tegument is not the only target involvedin worm death caused by PZQ.

In summary, our results suggest the activation of different path-ways for male and female worms in response to PZQ, since chemi-cal markers were distinct according to sex after drug treatment. Itcould be related to the resistance level of each schistosome sex, aswas also demonstrated by Pica-Mattoccia and Cioli (2004).Moreover, knowing the PZQ action sites in each sex may guidethe development of new drugs for specific targets. Specificity formale or female worm elimination by new drugs could cease repro-duction, prevent the liberation of new eggs in the faeces and elimi-nate the remaining worms of the other sex, since one could notsurvive without the other. In female worms, PZQ could inducedeath cell by sphingomyelinase activity. Furthermore, our resultsindicated that the activity of (Na+ K+)-ATPase in male worms couldpossibly be compromised when adult worms are exposed to PZQ,making this enzyme an important target in new drug developmentfor schistosomiasis treatment.

Acknowledgements

This work was financially supported by Coordination for theImprovement of Higher Level – or Education – Personnel(CAPES), São Paulo Research Foundation (FAPESP), Brazil processnumber 11/50400-0 and 14/00302-0 and National Science andTechnology Institutes (INCT), Brazil.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ijpara.2014.12.008.

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