International Journal for Parasitology xxx (2015) xxx–xxx
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Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni 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 b
Innovare Biomarkers Laboratory, Medicine and Experimental Surgery Nucleus, University of Campinas, Campinas, São Paulo, Brazil 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 2014 Received in revised form 21 October 2014 Accepted 19 December 2014 Available online xxxx Keywords: Schistosoma mansoni Mass spectrometry imaging Praziquantel Biomarkers Lipids
a b s t r a c t Finding specific molecular targets and the mechanism of action of praziquantel in the treatment of schistosomiasis remains a challenging task. Our efforts were focused on obtaining further information on worm composition before and after exposure to praziquantel in the treatment of schistosomiasis to elucidate the potential sites of action of this drug. Evidence indicates that the lipid bilayer is changed by treatment with praziquantel. Following this rationale, we employed a mass spectrometry imagingbased approach that helped to characterise lipids in specific locations, which are directly involved in the biochemical pathways of the BH strain of Schistosoma mansoni, as well as differentiating the molecular response that each worm sex presents in vivo. Our findings demonstrated significant differences between the chemical markers found in adult worms before and after praziquantel exposure, especially in phospholipids, which were predominantly identified as chemical markers in all samples. Results also indicate that distinct molecular pathways in both male and female worms could be differentially affected by praziquantel treatment. These data shine new light on the mechanism of action of praziquantel, taking a further step towards its full understanding. Ó 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Praziquantel (PZQ) is the most commonly prescribed drug for all forms of schistosomiasis, since it is equally effective against Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, Schistosoma intercalatum and Schistosoma haematobium (Fenwick et al., 2003; Webbe and James, 1977). However, it does not show full efficacy against schistosomula and the juveniles stages of S. mansoni, resulting in lower cure rates in areas with high endemicity (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; LoVerde et al., 2004); some physiological and morphological aspects have been understood for a relatively long time, such as rapid Ca2+ ion uptake (Pax et al., 1978) and vacuolation and blebbing near and on the surface of worms (Becker et al., 1980). In male worms, in ⇑ Corresponding authors at: Rua Cinco de Junho, 350 – Barão Geraldo, 13083-877 Campinas, SP, Brazil. Tel.: +55 19 3521 9138. E-mail addresses: [email protected] (S.M. Allegretti), [email protected] (R.R. Catharino). 1 These authors contributed equally to this work.
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 as well as in phospholipid (PL) composition, producing alterations in its permeability to ions or resulting in indirect effects on membrane receptors and channels (Harder et al., 1988; Lima et al., 1994). However, the full mechanism still remains unknown; for example, the pathway of Ca2+ homeostasis disruption by PZQ in adult schistosomes (Day et al., 1992; Redman et al., 1996) and the mechanism of PZQ binding to its molecular targets (Tallima and El Ridi, 2007; Troiani et al., 2007) are yet to be determined. To better understand some of these mechanisms, modern analytical approaches, e.g. chromatographic techniques combined with MS, have been employed for chemical characterisation of adult schistosomes and PZQ metabolites in the host (Meier and Blaschke, 2000, 2001; van Balkom et al., 2005). More recently, MALDI-MS has been applied as the main analytical tool (Frank et al., 2012). Approaches using MS Imaging (MALDI-MSI) (Cornett et al., 2007) were developed to identify the spatial distribution of compounds in any physical sample such as tissue sections (Solon, 2007), single cell (Ferreira et al., 2014a), drug tablets (Rodrigues et al., 2014) and cosmetic products (de Oliveira et al., 2013).
http://dx.doi.org/10.1016/j.ijpara.2014.12.008 0020-7519/Ó 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
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Moreover, MALDI-MSI was applied, for the first known time, in S. mansoni adult worms to demonstrate different chemical markers according to schistosome sex and strain (Ferreira et al., 2014b). In that study we localised each identified compound in the body of the worm. Since schistosomes show stage- and sex-dependent differences in susceptibility to PZQ (Pica-Mattoccia and Cioli, 2004), information on possible targets and pathways of the drug could be clarified by chemical marker localisation. Based on this, the present work has employed the metabolomic platform to characterise both sexes of S. mansoni adult worms (BH strain) treated with PZQ. This report identifies the spatial distribution of chemical markers using MALDI-MSI technology, aiding the search for the understanding of possible targets and pathways of 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 cercariae of the BH strain (from Belo Horizonte, MG, Brazil). At this stage, the utilised procedure was caudal immersion for 2 h, with light exposure and controlled temperature at 28 °C (Olivier and Stirewalt, 1952). After 45 days p.i., animals were divided into two groups (n = 5/group): (i) treated with PZQ (Merck, Darmstadt, Germany) and (ii) negative control. The first group received a single oral dose of 40 mg/kg of PZQ. The control group received 1% PBS solution. The oral dose of 40 mg/kg of PZQ used in the present study is considered curative in humans, as adopted by the schistosomiasis treatment and control programs in Brazil with a cure rate of 80–90%. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. The protocol was approved by the International 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 dislocation. Schistosoma mansoni adult worms (n = 2/mouse) were recovered by perfusion of the hepatic portal system and mesenteric veins (Pellegrino et al., 1962). All worms were recovered alive
and washed carefully in a 0.9% saline solution at a temperature between 28 and 30 °C. Samples were sequentially transferred to an Eppendorf tube containing 1 mL of MilliQ H2O at the same temperature. 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 airbrush, spraying a-cyano-4-hydroxycinnamic acid (Sigma–Aldrich, Pennsylvania, USA) (10 mg/mL in 1:1 Acetonitrile/Methanol solution). Images and MS were acquired in a MALDI-LTQ-XL instrument equipped with an imaging feature (Thermo Scientific, California, USA). The instrument uses a nitrogen laser as the ionisation source and a quadrupole-ion-trap analysing system. All data were acquired in the positive ion mode. For image acquisition, a 50 lm raster width was selected, with three shots taken per spectrum. Fragmentation data (MS/MS) were acquired by setting the collision-induced normalised energy to 40. Helium was used as the collision gas. Each ion was fragmented in triplicate. All imaging data were then processed using ImageQuest software v.1.0.1 (Thermo Scientific). Both spectral and imaging data were normalised 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 in the Principal Component Analysis (PCA), which was performed using Unscrambler v.9.7 (CAMO Software, Trondheim, Norway). After discrimination by PCA, potential biomarkers were selected and, to identify the analytes, MS/MS reactions were performed to generate their fragmentation pattern. In addition to the spectrum, MALDI-MSI generated a chemical image allowing us to observe the spatial distribution of the analyte precursors, which were previously characterised as lipids. For lipid identification, MS/MS patterns and error values were considered, with the assistance of online databases such as Lipid MAPS (University of California, San Diego, CA, USA – www.lipidmaps.org) and METLIN (Scripps Center for Metabolomics, La Jolla, CA, USA), in order to guide the choice for potential lipid markers. Their structures were later proposed using Mass Frontier software v.6.0 (Thermo Scientific) (Urayama et al., 2010).
Fig. 1. Principal Component Analysis of all compounds of Schistosoma mansoni adult worms. Ion biomarkers for each group were separated by Principal Component Analysis (n = 5/group). The explained variances (X-expl) are shown below the figure. N, male negative control; d, female negative control; , male treated with praziquantel; j, female treated with praziquantel.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
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2.5. High resolution electrospray ionisation-MS (ESI-MS) analysis
of them presented clear differences in their spectra when compared with each other (Supplementary Fig. S1).
To confirm the chemical markers identities, male and female worms, treated or not with PZQ, were submitted to Bligh–Dyer extraction (Bligh and Dyer, 1959). Lipid extracts were resuspended in 50 lL of MilliQ H2O and 10 lL was diluted in 990 lL of methanol and 0.1% formic acid. Data acquisition was performed in an LTQ-XL Orbitrap Discovery instrument (30,000 FWHM, Thermo Scientific, Bremen, Germany) in the positive ion mode and at the m/z range of 600–2000 for complex lipid identification. Structural propositions were performed using high resolution as the main parameter. Mass accuracy was calculated and expressed in terms of parts per million (ppm) chemical shifts, according to Machado et al. (2013). 3. Results 3.1. Metabolic fingerprints of adult worms Male and female adult worms, treated or not with PZQ, were subjected to MALDI-MSI analysis, as described in Section 2.1. All
3.2. Statistical analysis and biomarker identification Preliminary analyses on MALDI-MSI were performed at the m/z range of 50 and 2000 in both positive and negative ion modes (data not shown). Accurate results were bound to the m/z range of 600– 2000 in the positive ion mode, since they exhibited clear discrimination in PCA. Statistical analyses were performed by the comparison between male and female worms, treated or not with PZQ. As shown in Fig. 1, worm control groups (MCT, male control and FCT, female control) were clearly separated from worms submitted to PZQ treatment, with an accuracy of 99%. The identified ions compose the final model of optimised PCA. Table 1 presents chemical markers identified in each adult worm, as well as the precursor ion fragmentation and the mass errors for each signal observed in high resolution Fourier transform mass spectrometry (HR-FTMS), measured in ppm (with all results presenting a deviation of less than 2 ppm).
Table 1 Lipid chemical markers identified via MS imaging (MALDI-MSI) of Schistosoma mansoni adult worms (positive ion mode). The assigned IDs are for general structures and can be any of the positional isomers. Identification is based on MS/MS data, exact mass of each compound and Lipid Maps and METLIN databases. Adult worms
Parental ion (m/z)
Product ions (m/z)
Molecule
LM ID
Theoretical mass
Experimental mass
FCT/MCT
601 617 619 620 724 726 651 652 649 853 855 827
557, 558, 448, 475, 680, 682, 584, 463, 460, 809, 666, 768,
(DG(17:2/18:3/0:0)+H)+ (PA(12:0/18:2)+H)+ (PA(13:0/17:1)+H)+ (PE(12:0/15:1)+H)+ (PC(12:0/20:5)+H)+ (PC(14:0/18:4)+H)+ (PG(12:0/15:1)+H)+ (PS(14:0/12:0)+H)+ (PE-Cer(14:1/18:0)+H)+ (PI(12:0/22:4)+Na)+ (PI(13:0/20:4)+K)+ (PI(12:0/20:3)+Na)+
LMGL02010051 LMGP10010051 LMGP10010072 LMGP02010361 LMGP01011333 LMGP01010499 LMGP04010048 LMGP03010931 LMSP03020059 LMGP06010035 LMGP06010054 LMGP06010029
601,4826 617,4177 619,4333 620,4286 724,4912 726,5068 651,4232 652,4184 649,4915 853,4837 855,442 827,4681
601,4818 617,417 619,433 620,4291 724,4899 726,506 651,424 652,4175 649,4905 853,4833 855,4431 827,4688
FPZQ
MPZQ
583 457, 430, 502, 606, 581, 462, 585, 605, 708, 811, 682,
413 560 576 579, 706 710 480 608 582 664 684 783
Error (ppm) 1,330046788 1,133754345 0,484313646 0,805894506 1,79436272 1,101159686 1,228080302 1,379482859 1,539666031 0,468667416 1,285884958 0,845954061
MID 4357 81,217 81,238 76,596 75,613 59,327 78,873 78,595 103,098 80,057 80,076 80,051
LM ID, Lipid MAPS ID (online database); MID, METLIN ID (online database); ppm, parts per million; FCT, female worm control; MCT, male control worm; FPZQ, female worm treated with praziquantel (PZQ); MPZQ, male worm treated with PZQ; DG, diacylglycerol; PA, phosphatidic acid; PE, phosphoethanolamine; PC, phosphatidylcholine; PG, phosphoglycerol; PS, phosphatidylserine; PI, phosphoinositol; Cer, ceramide.
Fig. 2. Schistosoma mansoni male control adult worm images generated by MS precursors. Pos, posterior portion; Ant, anterior portion; T, tegument; OS, oral sucker; G, gut.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
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3.3. Spatial distribution of biomarkers in adult worms
4. Discussion
Individual worms were submitted to MS/MS analysis. In Figs. 2–5, images clearly revealed differential spatial distribution of chemical markers, where the darker the scale, the higher the concentration in the site. It was possible to distinguish some characteristic anatomical structures of the worms, e.g. suckers, gut, reproductive system and tegument. The increased intensity in grey-scale indicates higher concentrations of these biomarkers in each location.
In the present report, we applied the same tools to identify metabolic alterations caused by PZQ treatment in the S. mansoni BH strain (in vivo). The results demonstrate the specificity of the method for schistosomes, identifying and differentiating each sex (Supplementary Fig. S1A, B). Recently, a study performed in our laboratory demonstrated the spatial distribution of lipids in adult worms of two Brazilian strains (BH and SE) of S. mansoni
Fig. 3. Schistosoma mansoni female control adult worm images generated by MS precursors. Pos, posterior portion; Ant, anterior portion; R, reproductive system; G, gut; T, tegument.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
M.S. Ferreira et al. / International Journal for Parasitology xxx (2015) xxx–xxx
Fig. 4. MS precursor-generated images of a Schistosoma mansoni male adult worm treated with praziquantel. Pos, posterior portion; Ant, anterior portion; R, Reproductive system; OS, Oral sucker; G, Gut.
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(in vitro) (Ferreira et al., 2014b) using MALDI-MSI and the lipidomics approach (Ferreira et al., 2014b). However, as expected due to biochemical interactions with the host, the in vivo lipid profile differed from the in vitro profile, as it has been described that schistosomes use compounds from the host in lipid synthesis (Furlong, 1991). Furthermore, in addition to tegumental damage, PZQ significantly changed the metabolic profile of both sexes of S. mansoni (Supplementary Fig. S1C, D). This modification was enough to segregate almost all groups in PCA analysis, except control male and female groups (Fig. 1). Most compounds were probably derived from the host. providing very close similarities between both sexes, but with different locations. PZQ, however, had differential reach and action on each sex (Pica-Mattoccia and Cioli, 2004), which could explain the clear segregation of male worms treated with PZQ (MPZQ) and female worms treated with PZQ (FPZQ) in PCA. The potential biomarkers obtained from PCA of MALDI-MSI data were confirmed using high resolution ESI-MS analysis (Table 1). Among the identified chemical markers, phospholipids (PLs) represent 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 also participate in host recognition (Fusco et al., 1988), immune response modulation and evasion (Abath and Werkhauser, 1996; McLaren, 1984), communication (Fried and Haseeb, 1991) and development (Hockley and McLaren, 1973; Shaw et al., 1977; Smith and Brooks, 1969). Among PL classes, phosphatidylcholines (PCs), phosphoethanolamines (PEs) and phosphatidic acids (PAs) were found in control groups. Interestingly, the same chemical markers presented distinct spatial locations in MCT and FCT groups. For example, (PA(12:0/18:2)+H)+ (m/z 617) and (PA(13:0/17:1)+H)+ (m/z 619) were located in the oral sucker in males, and in the reproductive 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 were present in the oral sucker and gut of the MCT group, and in the tegument of the FCT group. Although the functional roles of these molecules in S. mansoni remain unknown, some studies already demonstrated that PCs are the most abundant PL class present in S. mansoni (Brouwers et al., 1998; Young and Podesta, 1982). Furthermore, according to Brouwers et al. (1998), PCs presented in bulk in schistosome tegument, corroborating our result obtained
Fig. 5. MS precursor-generated images of a Schistosoma mansoni female adult worm treated with praziquantel. Pos, posterior portion; Ant, anterior portion; R, reproductive system; T, tegument; G, gut; OS, oral sucker; VS, ventral sucker.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
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from the FCT group. Moreover, (PE(12:0/15:1)+H)+ (m/z 620) was located in the reproductive system and gut of females, and in the oral 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 compound class was previously found as a biomarker in the tegument of adult BH strain schistosomes (Ferreira et al., 2014b). Furthermore, a previous 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 surface of the schistosome when the worm frees itself from the wall or organ to which it is anchored in the host (Espinoza et al., 1991). When female worms were treated with PZQ, there was a notable change in its chemical markers: phosphoglycerol (PG(12:0/ 15:1)+H)+ (m/z 651), phosphatidylserine (PS(14:0/12:0)+H)+ (m/z 652) and PE coupled with ceramide (PE-Cer(14:1/18:0)+H)+ (m/z 649). All of those were located in different regions of the worm body. For example, PG was located in the tegument, while PS was more concentrated in the oral and ventral suckers and reproductive system. On the other hand, PE-Cer was concentrated in the gut and reproductive system. The latter is included in the sphingolipid category and may be produced in microsomes of rat liver and plasma membrane, and internalised by schistosomes, as they do not have a de novo synthesis pathway (Malgat et al., 1986). However, other studies demonstrated that this sphingolipid can be synthesized by S. mansoni females via sphingomyelinase activity, and plays a secondary messenger role (Redman et al., 1997; Testi, 1996; Wiest et al., 1992). This role has been established in several cell types (Hannun, 1994; Kolesnick and Golde, 1994; Obeid et al., 1993; Testi, 1996), being implicated in cell death and differentiation. Sometimes, this secondary messenger role is activated in response to external stimuli such as TNF-a, IFN-c and vitamin D3 (Andrieu et al., 1994; Belka et al., 1995; Hannun, 1994; Kolesnick and Golde, 1994; Raines et al., 1993). TNF-a, in turn, is believed to play a preventive role in cases of superinfections (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 production in female adult worms and, more specifically, PE-Cer can also be internalised from the host production; more experiments are, however, required to test this hypothesis; likewise to understand this chemical marker location and the importance of this pathway in female worms. Similar to females, male worms presented a different lipid profile 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 spatial location in the body of the worm: oral sucker, gut and reproductive system. It is also important to note that all chemical markers were identified as sodium (Na+) or potassium (K+) adducts. This information could be supported by Pax et al. (1978), demonstrating that PZQ stimulates influx of Na+ in S. mansoni male adult worms. On the other hand, some reports related that PZQ could induce modification in membrane PLs, causing fluidity which may produce alterations in permeability to ions or result in indirect effects on membrane receptors and channels (Harder et al., 1988; Lima et al., 1994). Moreover, Cunha and Noël (1997) described that PZQ had no effect on schistosome (Na+ K+)-ATPase. However, all of the previous tests were performed only in the tegument of the worm (Cunha and Noël, 1997). In contrast, a recent study using a gene expression assay of male S. japonicum demonstrated that adult worms exposed to PZQ up-regulated the (Na+ K+)-transporting ATPase gene (You et al., 2013), which may indicate that similar interactions occur for S. mansoni when exploring PZQ actions not only restricted to the tegument. Since chemical markers of MPZQ were 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 involved in worm death caused by PZQ. In summary, our results suggest the activation of different pathways for male and female worms in response to PZQ, since chemical markers were distinct according to sex after drug treatment. It could be related to the resistance level of each schistosome sex, as was also demonstrated by Pica-Mattoccia and Cioli (2004). Moreover, knowing the PZQ action sites in each sex may guide the development of new drugs for specific targets. Specificity for male or female worm elimination by new drugs could cease reproduction, prevent the liberation of new eggs in the faeces and eliminate the remaining worms of the other sex, since one could not survive without the other. In female worms, PZQ could induce death cell by sphingomyelinase activity. Furthermore, our results indicated that the activity of (Na+ K+)-ATPase in male worms could possibly be compromised when adult worms are exposed to PZQ, making this enzyme an important target in new drug development for schistosomiasis treatment. Acknowledgements This work was financially supported by Coordination for the Improvement of Higher Level – or Education – Personnel (CAPES), São Paulo Research Foundation (FAPESP), Brazil process number 11/50400-0 and 14/00302-0 and National Science and Technology Institutes (INCT), Brazil. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijpara.2014.12. 008. References Abath, F.G., Werkhauser, R.C., 1996. The tegument of Schistosoma mansoni: functional and immunological features. Parasite Immunol. 18, 15–20. Amiri, P., Locksley, R.M., Parslow, T.G., Sadickt, M., Rector, E., Ritter, D., McKerrow, J.H., 1992. Tumour necrosis factor a restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice. Nature, 604–607. Andrews, P., 1985. Praziquantel: mechanisms of anti-schistosomal activity. Pharmacol. Ther. 29, 129–156. Andrieu, N., Salvayre, R., Levade, T., 1994. Evidence against involvement of the acid lysosomal sphingomyelinase in the tumor-necrosis-factor-and interleukin-1induced sphingomyelin cycle and cell proliferation in human fibroblasts. Biochem. J. 303, 341–345. Becker, B., Mehlhorn, H., Andrews, P., Thomas, H., Eckert, J., 1980. Light and electron microscopic studies on the effect of praziquantel on Schistosoma mansoni, Dicrocoelium dendriticum, and Fasciola hepatica (Trematoda) in vitro. Z. Parasitenkd. 63, 113–128. Belka, C., Wiegmann, K., Adam, D., Holland, R., Neuloh, M., Herrmann, F., Krönke, M., Brach, M., 1995. Tumor necrosis factor (TNF)-alpha activates c-raf-1 kinase via the p55 TNF receptor engaging neutral sphingomyelinase. EMBO J. 14, 1156. Bligh, E., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Phys. 37, 911–917. Brouwers, J.F., Van Hellemond, J.J., van Golde, L.M., Tielens, A.G., 1998. Ether lipids and their possible physiological function in adult Schistosoma mansoni. Mol. Biochem. Parasitol. 96, 49–58. Cornett, D.S., Reyzer, M.L., Chaurand, P., Caprioli, R.M., 2007. MALDI imaging mass spectrometry: molecular snapshots of biochemical systems. Nat. Methods 4, 828–833. Cunha, V., Noël, F., 1997. Praziquantel has no direct effect on (Na+/K+)-ATPases and (Ca2+/Mg2+) ATPases of Schistosoma mansoni. Life Sci. 60, PL289–PL294. Day, T., Bennett, J., Pax, R., 1992. Praziquantel: the enigmatic antiparasitic. Parasitol. Today 8, 342–344. de Oliveira, D.N., de Bona Sartor, S., Ferreira, M.S., Catharino, R.R., 2013. Cosmetic analysis using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Materials 6, 1000–1010. Doenhoff, M.J., Cioli, D., Utzinger, J., 2008. Praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis. Curr. Opin. Infect. Dis. 21, 659–667. Espinoza, B., Silman, I., Arnon, R., Tarrab-Hazdai, R., 1991. Phosphatidylinositolspecific phospholipase C induces biosynthesis of acetylcholinesterase via diacylglycerol in Schistosoma mansoni. Eur. J. Biochem. 195, 863–870.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
M.S. Ferreira et al. / International Journal for Parasitology xxx (2015) xxx–xxx Fenwick, A., Savioli, L., Engels, D., Robert Bergquist, N., Todd, M.H., 2003. Drugs for the control of parasitic diseases: current status and development in schistosomiasis. Trends Parasitol. 19, 509–515. Ferreira, M.S., de Oliveira, D.N., Gonçalves, R.F., Catharino, R.R., 2014a. Lipid characterization of embryo zones by silica plate laser desorption ionization mass spectrometry imaging (SP-LDI-MSI). Anal. Chim. Acta 807, 96–102. Ferreira, M.S., Oliveira, D.N., Oliveira, R.N., Allegretti, S.M., Vercesi, A.E., Catharino, R.R., 2014b. Mass spectrometry imaging: a new vision in differentiating Schistosoma mansoni strains. J. Mass Spectrom. 49, 86–92. Frank, S., van Die, I., Geyer, R., 2012. Structural characterization of Schistosoma mansoni adult worm glycosphingolipids reveals pronounced differences with those of cercariae. Glycobiology 22, 676–695. Fried, B., Haseeb, M.A., 1991. Role of lipids in Schistosoma mansoni. Parasitol. Today 7, 204, author reply 204. Furlong, S., 1991. Unique roles for lipids in Schistosoma mansoni. Parasitol. Today 7, 59–62. Fusco, A., Salafsky, B., Ellenberger, B., Li, L.-H., 1988. Schistosoma mansoni: correlations between mouse strain, skin eicosanoid production, and cercarial skin penetration. J. Parasitol. 73, 253–261. Garside, P., Sands, W.A., Kusel, J.R., Hagan, P., 1996. Is the induction of apoptosis the mechanism of the protective effects of TNF-a in helminth infection? Parasite Immunol. 18, 111–113. Gönnert, R., Andrews, P., 1977. Praziquantel, a new broad-spectrum antischistosomal agent. Z. Parasitenkd. 52, 129–150. Hagan, P., Garside, P., Kusel, J., 1993. Is tumour necrosis factor alpha the molecular basis of concomitant immunity in schistosomiasis? Parasite Immunol. 15, 553– 557. Hannun, Y.A., 1994. The sphingomyelin cycle and the second messenger function of ceramide. J. Biol. Chem. 269, 3125–3128. Harder, A., Goossens, J., Andrews, P., 1988. Influence of praziquantel and Ca2+ on the bilayer-isotropic-hexagonal transition of model membranes. Mol. Biochem. Parasitol. 29, 55–59. Hockley, D.J., McLaren, D.J., 1973. Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. Int. J. Parasitol. 3, 13–20. Hu, W., Brindley, P.J., McManus, D.P., Feng, Z., Han, Z.-G., 2004. Schistosome transcriptomes: new insights into the parasite and schistosomiasis. Trends Mol. Med. 10, 217–225. Kolesnick, R., Golde, D.W., 1994. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 77, 325–328. Lima, S., Vieira, L., Harder, A., Kusel, J., 1994. Effects of culture and praziquantel on membrane fluidity parameters of adult Schistosoma mansoni. Parasitology 109, 57. LoVerde, P.T., Hirai, H., Merrick, J.M., Lee, N.H., El-Sayed, N., 2004. Schistosoma mansoni genome project: an update. Parasitol. Int. 53, 183–192. Machado, D., Shishido, S.M., Queiroz, K.C.S., Oliveira, D.N., Faria, A.L.C., Catharino, R.R., Spek, C.A., Ferreira, C.V., 2013. Irradiated riboflavin diminishes the aggressiveness of melanoma in vitro and in vivo. PLoS One 8, e54269. Malgat, M., Maurice, A., Baraud, J., 1986. Sphingomyelin and ceramidephosphoethanolamine synthesis by microsomes and plasma membranes from rat liver and brain. J. Lipid Res. 27, 251–260. McLaren, D.J., 1984. Disguise as an evasive stratagem of parasitic organisms. Parasitology 88, 597–611. Meier, H., Blaschke, G., 2000. Capillary electrophoresis–mass spectrometry, liquid chromatography–mass spectrometry and nanoelectrospray-mass spectrometry of praziquantel metabolites. J. Chrom. B Biomed. Sci. Appl. 748, 221–231. Meier, H., Blaschke, G., 2001. Investigation of praziquantel metabolism in isolated rat hepatocytes. J. Pharm. Biomed. Anal. 26, 409–415. Obeid, L.M., Linardic, C.M., Karolak, L.A., Hannun, Y.A., 1993. Programmed cell death induced by ceramide. Science 259, 1769–1771. Olivier, L., Stirewalt, M., 1952. An efficient method for exposure of mice to cercariae of Schistosoma mansoni. J. Parasitol. 38, 19–23.
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Pax, R., Bennett, J., Fetterer, R., 1978. A benzodiazepine derivative and praziquantel: effects on musculature of Schistosoma mansoni and Schistosoma japonicum. Naunyn Schmiedebergs Arch Pharmacol. 304, 309–315. Pellegrino, J., Oliveira, C.A., Faria, J., Cunha, A.S., 1962. New approach to the screening of drugs in experimental schistosomiasis mansoni in mice. Am. J. Trop. Med. Hyg. 11, 201. Pica-Mattoccia, L., Cioli, D., 2004. Sex-and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment. Int. J. Parasitol. 34, 527– 533. Pinlaor, S., Prakobwong, S., Boonmars, T., Wongkham, C., Pinlaor, P., Hiraku, Y., 2009. Effect of praziquantel treatment on the expression of matrix metalloproteinases in relation to tissue resorption during fibrosis in hamsters with acute and chronic Opisthorchis viverrini infection. Acta Trop. 111, 181–191. Raines, M., Kolesnick, R., Golde, D., 1993. Sphingomyelinase and ceramide activate mitogen-activated protein kinase in myeloid HL-60 cells. J. Biol. Chem. 268, 14572–14575. Redman, C., Robertson, A., Fallon, P., Modha, J., Kusel, J., Doenhoff, M., Martin, R., 1996. Praziquantel: an urgent and exciting challenge. Parasitol. Today 12, 14– 20. Redman, C.A., Kennington, S., Spathopoulou, T., Kusel, J.R., 1997. Interconversion of sphingomyelin and ceramide in adult Schistosoma mansoni. Mol. Biochem. Parasitol. 90, 145–153. Rodrigues, L.R., Oliveira, D.N.D., Ferreira, M.S., Catharino, R.R., 2014. In situ assessment of atorvastatin impurity using MALDI mass spectrometry imaging (MALDI-MSI). Anal. Chim. Acta 818, 32–38. Sabah, A., Fletcher, C., Webbe, G., Doenhoff, M., 1986. Schistosoma mansoni: chemotherapy of infections of different ages. Exp. Parasitol. 61, 294–303. Shaw, J., Marshall, I., Erasmus, D., 1977. Schistosoma mansoni. In vitro stimulation of vitelline cell development by extracts of male worms. Exp. Parasitol. 42, 14– 20. Smith, T.M., Brooks, T.J., 1969. Lipid fractions in adult Schistosoma mansoni. Parasitology 59, 293–298. Solon, E.G., 2007. Autoradiography: high-resolution molecular imaging in pharmaceutical discovery and development. Expert Opin. Drug Discov. 2, 503–514. Tallima, H., El Ridi, R., 2007. Praziquantel binds Schistosoma mansoni adult worm actin. Int. J. Antimicrob. Agents 29, 570–575. Testi, R., 1996. Sphingomyelin breakdown and cell fate. Trends Biochem. Sci. 21, 468–471. Troiani, A.R., Pica-Mattoccia, L., Valle, C., Cioli, D., Mignogna, G., Ronketti, F., Todd, M., 2007. Is actin the praziquantel receptor? Int. J. Antimicrob. Agents 30, 280– 281. Urayama, S., Zou, W., Brooks, K., Tolstikov, V., 2010. Comprehensive mass spectrometry based metabolic profiling of blood plasma reveals potent discriminatory classifiers of pancreatic cancer. Rapid Commun. Mass Spectrom. 24, 613–620. van Balkom, B.W., van Gestel, R.A., Brouwers, J.F., Krijgsveld, J., Tielens, A.G., Heck, A.J., van Hellemond, J.J., 2005. Mass spectrometric analysis of the Schistosoma mansoni tegumental sub-proteome. J. Prot. Res. 4, 958–966. Webbe, G., James, C., 1977. A comparison of the susceptibility to praziquantel of Schistosoma haematobium, S. japonicum, S. mansoni, S. intercalatum and S. mattheei in hamsters. Z. Parasitenkd. 52, 169–177. Wiest, P.M., Burnham, D.C., Olds, G.R., Bowen, W.D., 1992. Developmental expression of protein kinase C activity in Schistosoma mansoni. Am. J. Trop. Med. Hyg. 46, 358–365. You, H., McManus, D.P., Hu, W., Smout, M.J., Brindley, P.J., Gobert, G.N., 2013. Transcriptional responses of in vivo praziquantel exposure in schistosomes identifies a functional role for calcium signalling pathway member CamKII. PLoS Pathol. 9, e1003254. Young, B.W., Podesta, R.B., 1982. Major phospholipids and phosphatidylcholine synthesis in adult Schistosoma mansoni. Mol. Biochem. Parasitol. 5, 165–172.
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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni 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 b
Innovare Biomarkers Laboratory, Medicine and Experimental Surgery Nucleus, University of Campinas, Campinas, São Paulo, Brazil 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 2014 Received in revised form 21 October 2014 Accepted 19 December 2014 Available online xxxx Keywords: Schistosoma mansoni Mass spectrometry imaging Praziquantel Biomarkers Lipids
a b s t r a c t Finding specific molecular targets and the mechanism of action of praziquantel in the treatment of schistosomiasis remains a challenging task. Our efforts were focused on obtaining further information on worm composition before and after exposure to praziquantel in the treatment of schistosomiasis to elucidate the potential sites of action of this drug. Evidence indicates that the lipid bilayer is changed by treatment with praziquantel. Following this rationale, we employed a mass spectrometry imagingbased approach that helped to characterise lipids in specific locations, which are directly involved in the biochemical pathways of the BH strain of Schistosoma mansoni, as well as differentiating the molecular response that each worm sex presents in vivo. Our findings demonstrated significant differences between the chemical markers found in adult worms before and after praziquantel exposure, especially in phospholipids, which were predominantly identified as chemical markers in all samples. Results also indicate that distinct molecular pathways in both male and female worms could be differentially affected by praziquantel treatment. These data shine new light on the mechanism of action of praziquantel, taking a further step towards its full understanding. Ó 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Praziquantel (PZQ) is the most commonly prescribed drug for all forms of schistosomiasis, since it is equally effective against Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, Schistosoma intercalatum and Schistosoma haematobium (Fenwick et al., 2003; Webbe and James, 1977). However, it does not show full efficacy against schistosomula and the juveniles stages of S. mansoni, resulting in lower cure rates in areas with high endemicity (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; LoVerde et al., 2004); some physiological and morphological aspects have been understood for a relatively long time, such as rapid Ca2+ ion uptake (Pax et al., 1978) and vacuolation and blebbing near and on the surface of worms (Becker et al., 1980). In male worms, in ⇑ Corresponding authors at: Rua Cinco de Junho, 350 – Barão Geraldo, 13083-877 Campinas, SP, Brazil. Tel.: +55 19 3521 9138. E-mail addresses: [email protected] (S.M. Allegretti), [email protected] (R.R. Catharino). 1 These authors contributed equally to this work.
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 as well as in phospholipid (PL) composition, producing alterations in its permeability to ions or resulting in indirect effects on membrane receptors and channels (Harder et al., 1988; Lima et al., 1994). However, the full mechanism still remains unknown; for example, the pathway of Ca2+ homeostasis disruption by PZQ in adult schistosomes (Day et al., 1992; Redman et al., 1996) and the mechanism of PZQ binding to its molecular targets (Tallima and El Ridi, 2007; Troiani et al., 2007) are yet to be determined. To better understand some of these mechanisms, modern analytical approaches, e.g. chromatographic techniques combined with MS, have been employed for chemical characterisation of adult schistosomes and PZQ metabolites in the host (Meier and Blaschke, 2000, 2001; van Balkom et al., 2005). More recently, MALDI-MS has been applied as the main analytical tool (Frank et al., 2012). Approaches using MS Imaging (MALDI-MSI) (Cornett et al., 2007) were developed to identify the spatial distribution of compounds in any physical sample such as tissue sections (Solon, 2007), single cell (Ferreira et al., 2014a), drug tablets (Rodrigues et al., 2014) and cosmetic products (de Oliveira et al., 2013).
http://dx.doi.org/10.1016/j.ijpara.2014.12.008 0020-7519/Ó 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
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Moreover, MALDI-MSI was applied, for the first known time, in S. mansoni adult worms to demonstrate different chemical markers according to schistosome sex and strain (Ferreira et al., 2014b). In that study we localised each identified compound in the body of the worm. Since schistosomes show stage- and sex-dependent differences in susceptibility to PZQ (Pica-Mattoccia and Cioli, 2004), information on possible targets and pathways of the drug could be clarified by chemical marker localisation. Based on this, the present work has employed the metabolomic platform to characterise both sexes of S. mansoni adult worms (BH strain) treated with PZQ. This report identifies the spatial distribution of chemical markers using MALDI-MSI technology, aiding the search for the understanding of possible targets and pathways of 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 cercariae of the BH strain (from Belo Horizonte, MG, Brazil). At this stage, the utilised procedure was caudal immersion for 2 h, with light exposure and controlled temperature at 28 °C (Olivier and Stirewalt, 1952). After 45 days p.i., animals were divided into two groups (n = 5/group): (i) treated with PZQ (Merck, Darmstadt, Germany) and (ii) negative control. The first group received a single oral dose of 40 mg/kg of PZQ. The control group received 1% PBS solution. The oral dose of 40 mg/kg of PZQ used in the present study is considered curative in humans, as adopted by the schistosomiasis treatment and control programs in Brazil with a cure rate of 80–90%. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. The protocol was approved by the International 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 dislocation. Schistosoma mansoni adult worms (n = 2/mouse) were recovered by perfusion of the hepatic portal system and mesenteric veins (Pellegrino et al., 1962). All worms were recovered alive
and washed carefully in a 0.9% saline solution at a temperature between 28 and 30 °C. Samples were sequentially transferred to an Eppendorf tube containing 1 mL of MilliQ H2O at the same temperature. 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 airbrush, spraying a-cyano-4-hydroxycinnamic acid (Sigma–Aldrich, Pennsylvania, USA) (10 mg/mL in 1:1 Acetonitrile/Methanol solution). Images and MS were acquired in a MALDI-LTQ-XL instrument equipped with an imaging feature (Thermo Scientific, California, USA). The instrument uses a nitrogen laser as the ionisation source and a quadrupole-ion-trap analysing system. All data were acquired in the positive ion mode. For image acquisition, a 50 lm raster width was selected, with three shots taken per spectrum. Fragmentation data (MS/MS) were acquired by setting the collision-induced normalised energy to 40. Helium was used as the collision gas. Each ion was fragmented in triplicate. All imaging data were then processed using ImageQuest software v.1.0.1 (Thermo Scientific). Both spectral and imaging data were normalised 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 in the Principal Component Analysis (PCA), which was performed using Unscrambler v.9.7 (CAMO Software, Trondheim, Norway). After discrimination by PCA, potential biomarkers were selected and, to identify the analytes, MS/MS reactions were performed to generate their fragmentation pattern. In addition to the spectrum, MALDI-MSI generated a chemical image allowing us to observe the spatial distribution of the analyte precursors, which were previously characterised as lipids. For lipid identification, MS/MS patterns and error values were considered, with the assistance of online databases such as Lipid MAPS (University of California, San Diego, CA, USA – www.lipidmaps.org) and METLIN (Scripps Center for Metabolomics, La Jolla, CA, USA), in order to guide the choice for potential lipid markers. Their structures were later proposed using Mass Frontier software v.6.0 (Thermo Scientific) (Urayama et al., 2010).
Fig. 1. Principal Component Analysis of all compounds of Schistosoma mansoni adult worms. Ion biomarkers for each group were separated by Principal Component Analysis (n = 5/group). The explained variances (X-expl) are shown below the figure. N, male negative control; d, female negative control; , male treated with praziquantel; j, female treated with praziquantel.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
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2.5. High resolution electrospray ionisation-MS (ESI-MS) analysis
of them presented clear differences in their spectra when compared with each other (Supplementary Fig. S1).
To confirm the chemical markers identities, male and female worms, treated or not with PZQ, were submitted to Bligh–Dyer extraction (Bligh and Dyer, 1959). Lipid extracts were resuspended in 50 lL of MilliQ H2O and 10 lL was diluted in 990 lL of methanol and 0.1% formic acid. Data acquisition was performed in an LTQ-XL Orbitrap Discovery instrument (30,000 FWHM, Thermo Scientific, Bremen, Germany) in the positive ion mode and at the m/z range of 600–2000 for complex lipid identification. Structural propositions were performed using high resolution as the main parameter. Mass accuracy was calculated and expressed in terms of parts per million (ppm) chemical shifts, according to Machado et al. (2013). 3. Results 3.1. Metabolic fingerprints of adult worms Male and female adult worms, treated or not with PZQ, were subjected to MALDI-MSI analysis, as described in Section 2.1. All
3.2. Statistical analysis and biomarker identification Preliminary analyses on MALDI-MSI were performed at the m/z range of 50 and 2000 in both positive and negative ion modes (data not shown). Accurate results were bound to the m/z range of 600– 2000 in the positive ion mode, since they exhibited clear discrimination in PCA. Statistical analyses were performed by the comparison between male and female worms, treated or not with PZQ. As shown in Fig. 1, worm control groups (MCT, male control and FCT, female control) were clearly separated from worms submitted to PZQ treatment, with an accuracy of 99%. The identified ions compose the final model of optimised PCA. Table 1 presents chemical markers identified in each adult worm, as well as the precursor ion fragmentation and the mass errors for each signal observed in high resolution Fourier transform mass spectrometry (HR-FTMS), measured in ppm (with all results presenting a deviation of less than 2 ppm).
Table 1 Lipid chemical markers identified via MS imaging (MALDI-MSI) of Schistosoma mansoni adult worms (positive ion mode). The assigned IDs are for general structures and can be any of the positional isomers. Identification is based on MS/MS data, exact mass of each compound and Lipid Maps and METLIN databases. Adult worms
Parental ion (m/z)
Product ions (m/z)
Molecule
LM ID
Theoretical mass
Experimental mass
FCT/MCT
601 617 619 620 724 726 651 652 649 853 855 827
557, 558, 448, 475, 680, 682, 584, 463, 460, 809, 666, 768,
(DG(17:2/18:3/0:0)+H)+ (PA(12:0/18:2)+H)+ (PA(13:0/17:1)+H)+ (PE(12:0/15:1)+H)+ (PC(12:0/20:5)+H)+ (PC(14:0/18:4)+H)+ (PG(12:0/15:1)+H)+ (PS(14:0/12:0)+H)+ (PE-Cer(14:1/18:0)+H)+ (PI(12:0/22:4)+Na)+ (PI(13:0/20:4)+K)+ (PI(12:0/20:3)+Na)+
LMGL02010051 LMGP10010051 LMGP10010072 LMGP02010361 LMGP01011333 LMGP01010499 LMGP04010048 LMGP03010931 LMSP03020059 LMGP06010035 LMGP06010054 LMGP06010029
601,4826 617,4177 619,4333 620,4286 724,4912 726,5068 651,4232 652,4184 649,4915 853,4837 855,442 827,4681
601,4818 617,417 619,433 620,4291 724,4899 726,506 651,424 652,4175 649,4905 853,4833 855,4431 827,4688
FPZQ
MPZQ
583 457, 430, 502, 606, 581, 462, 585, 605, 708, 811, 682,
413 560 576 579, 706 710 480 608 582 664 684 783
Error (ppm) 1,330046788 1,133754345 0,484313646 0,805894506 1,79436272 1,101159686 1,228080302 1,379482859 1,539666031 0,468667416 1,285884958 0,845954061
MID 4357 81,217 81,238 76,596 75,613 59,327 78,873 78,595 103,098 80,057 80,076 80,051
LM ID, Lipid MAPS ID (online database); MID, METLIN ID (online database); ppm, parts per million; FCT, female worm control; MCT, male control worm; FPZQ, female worm treated with praziquantel (PZQ); MPZQ, male worm treated with PZQ; DG, diacylglycerol; PA, phosphatidic acid; PE, phosphoethanolamine; PC, phosphatidylcholine; PG, phosphoglycerol; PS, phosphatidylserine; PI, phosphoinositol; Cer, ceramide.
Fig. 2. Schistosoma mansoni male control adult worm images generated by MS precursors. 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
4. Discussion
Individual worms were submitted to MS/MS analysis. In Figs. 2–5, images clearly revealed differential spatial distribution of chemical markers, where the darker the scale, the higher the concentration in the site. It was possible to distinguish some characteristic anatomical structures of the worms, e.g. suckers, gut, reproductive system and tegument. The increased intensity in grey-scale indicates higher concentrations of these biomarkers in each location.
In the present report, we applied the same tools to identify metabolic alterations caused by PZQ treatment in the S. mansoni BH strain (in vivo). The results demonstrate the specificity of the method for schistosomes, identifying and differentiating each sex (Supplementary Fig. S1A, B). Recently, a study performed in our laboratory demonstrated the spatial distribution of lipids in adult worms of two Brazilian strains (BH and SE) of S. mansoni
Fig. 3. Schistosoma mansoni female control adult worm images generated by MS precursors. Pos, posterior portion; Ant, anterior portion; R, reproductive system; G, gut; T, tegument.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
M.S. Ferreira et al. / International Journal for Parasitology xxx (2015) xxx–xxx
Fig. 4. MS precursor-generated images of a Schistosoma mansoni male adult worm treated with praziquantel. Pos, posterior portion; Ant, anterior portion; R, Reproductive system; OS, Oral sucker; G, Gut.
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(in vitro) (Ferreira et al., 2014b) using MALDI-MSI and the lipidomics approach (Ferreira et al., 2014b). However, as expected due to biochemical interactions with the host, the in vivo lipid profile differed from the in vitro profile, as it has been described that schistosomes use compounds from the host in lipid synthesis (Furlong, 1991). Furthermore, in addition to tegumental damage, PZQ significantly changed the metabolic profile of both sexes of S. mansoni (Supplementary Fig. S1C, D). This modification was enough to segregate almost all groups in PCA analysis, except control male and female groups (Fig. 1). Most compounds were probably derived from the host. providing very close similarities between both sexes, but with different locations. PZQ, however, had differential reach and action on each sex (Pica-Mattoccia and Cioli, 2004), which could explain the clear segregation of male worms treated with PZQ (MPZQ) and female worms treated with PZQ (FPZQ) in PCA. The potential biomarkers obtained from PCA of MALDI-MSI data were confirmed using high resolution ESI-MS analysis (Table 1). Among the identified chemical markers, phospholipids (PLs) represent 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 also participate in host recognition (Fusco et al., 1988), immune response modulation and evasion (Abath and Werkhauser, 1996; McLaren, 1984), communication (Fried and Haseeb, 1991) and development (Hockley and McLaren, 1973; Shaw et al., 1977; Smith and Brooks, 1969). Among PL classes, phosphatidylcholines (PCs), phosphoethanolamines (PEs) and phosphatidic acids (PAs) were found in control groups. Interestingly, the same chemical markers presented distinct spatial locations in MCT and FCT groups. For example, (PA(12:0/18:2)+H)+ (m/z 617) and (PA(13:0/17:1)+H)+ (m/z 619) were located in the oral sucker in males, and in the reproductive 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 were present in the oral sucker and gut of the MCT group, and in the tegument of the FCT group. Although the functional roles of these molecules in S. mansoni remain unknown, some studies already demonstrated that PCs are the most abundant PL class present in S. mansoni (Brouwers et al., 1998; Young and Podesta, 1982). Furthermore, according to Brouwers et al. (1998), PCs presented in bulk in schistosome tegument, corroborating our result obtained
Fig. 5. MS precursor-generated images of a Schistosoma mansoni female adult worm treated with praziquantel. Pos, posterior portion; Ant, anterior portion; R, reproductive system; T, tegument; G, gut; OS, oral sucker; VS, ventral sucker.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
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from the FCT group. Moreover, (PE(12:0/15:1)+H)+ (m/z 620) was located in the reproductive system and gut of females, and in the oral 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 compound class was previously found as a biomarker in the tegument of adult BH strain schistosomes (Ferreira et al., 2014b). Furthermore, a previous 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 surface of the schistosome when the worm frees itself from the wall or organ to which it is anchored in the host (Espinoza et al., 1991). When female worms were treated with PZQ, there was a notable change in its chemical markers: phosphoglycerol (PG(12:0/ 15:1)+H)+ (m/z 651), phosphatidylserine (PS(14:0/12:0)+H)+ (m/z 652) and PE coupled with ceramide (PE-Cer(14:1/18:0)+H)+ (m/z 649). All of those were located in different regions of the worm body. For example, PG was located in the tegument, while PS was more concentrated in the oral and ventral suckers and reproductive system. On the other hand, PE-Cer was concentrated in the gut and reproductive system. The latter is included in the sphingolipid category and may be produced in microsomes of rat liver and plasma membrane, and internalised by schistosomes, as they do not have a de novo synthesis pathway (Malgat et al., 1986). However, other studies demonstrated that this sphingolipid can be synthesized by S. mansoni females via sphingomyelinase activity, and plays a secondary messenger role (Redman et al., 1997; Testi, 1996; Wiest et al., 1992). This role has been established in several cell types (Hannun, 1994; Kolesnick and Golde, 1994; Obeid et al., 1993; Testi, 1996), being implicated in cell death and differentiation. Sometimes, this secondary messenger role is activated in response to external stimuli such as TNF-a, IFN-c and vitamin D3 (Andrieu et al., 1994; Belka et al., 1995; Hannun, 1994; Kolesnick and Golde, 1994; Raines et al., 1993). TNF-a, in turn, is believed to play a preventive role in cases of superinfections (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 production in female adult worms and, more specifically, PE-Cer can also be internalised from the host production; more experiments are, however, required to test this hypothesis; likewise to understand this chemical marker location and the importance of this pathway in female worms. Similar to females, male worms presented a different lipid profile 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 spatial location in the body of the worm: oral sucker, gut and reproductive system. It is also important to note that all chemical markers were identified as sodium (Na+) or potassium (K+) adducts. This information could be supported by Pax et al. (1978), demonstrating that PZQ stimulates influx of Na+ in S. mansoni male adult worms. On the other hand, some reports related that PZQ could induce modification in membrane PLs, causing fluidity which may produce alterations in permeability to ions or result in indirect effects on membrane receptors and channels (Harder et al., 1988; Lima et al., 1994). Moreover, Cunha and Noël (1997) described that PZQ had no effect on schistosome (Na+ K+)-ATPase. However, all of the previous tests were performed only in the tegument of the worm (Cunha and Noël, 1997). In contrast, a recent study using a gene expression assay of male S. japonicum demonstrated that adult worms exposed to PZQ up-regulated the (Na+ K+)-transporting ATPase gene (You et al., 2013), which may indicate that similar interactions occur for S. mansoni when exploring PZQ actions not only restricted to the tegument. Since chemical markers of MPZQ were 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 involved in worm death caused by PZQ. In summary, our results suggest the activation of different pathways for male and female worms in response to PZQ, since chemical markers were distinct according to sex after drug treatment. It could be related to the resistance level of each schistosome sex, as was also demonstrated by Pica-Mattoccia and Cioli (2004). Moreover, knowing the PZQ action sites in each sex may guide the development of new drugs for specific targets. Specificity for male or female worm elimination by new drugs could cease reproduction, prevent the liberation of new eggs in the faeces and eliminate the remaining worms of the other sex, since one could not survive without the other. In female worms, PZQ could induce death cell by sphingomyelinase activity. Furthermore, our results indicated that the activity of (Na+ K+)-ATPase in male worms could possibly be compromised when adult worms are exposed to PZQ, making this enzyme an important target in new drug development for schistosomiasis treatment. Acknowledgements This work was financially supported by Coordination for the Improvement of Higher Level – or Education – Personnel (CAPES), São Paulo Research Foundation (FAPESP), Brazil process number 11/50400-0 and 14/00302-0 and National Science and Technology Institutes (INCT), Brazil. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijpara.2014.12. 008. References Abath, F.G., Werkhauser, R.C., 1996. The tegument of Schistosoma mansoni: functional and immunological features. Parasite Immunol. 18, 15–20. Amiri, P., Locksley, R.M., Parslow, T.G., Sadickt, M., Rector, E., Ritter, D., McKerrow, J.H., 1992. Tumour necrosis factor a restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice. Nature, 604–607. Andrews, P., 1985. Praziquantel: mechanisms of anti-schistosomal activity. Pharmacol. Ther. 29, 129–156. Andrieu, N., Salvayre, R., Levade, T., 1994. Evidence against involvement of the acid lysosomal sphingomyelinase in the tumor-necrosis-factor-and interleukin-1induced sphingomyelin cycle and cell proliferation in human fibroblasts. Biochem. J. 303, 341–345. Becker, B., Mehlhorn, H., Andrews, P., Thomas, H., Eckert, J., 1980. Light and electron microscopic studies on the effect of praziquantel on Schistosoma mansoni, Dicrocoelium dendriticum, and Fasciola hepatica (Trematoda) in vitro. Z. Parasitenkd. 63, 113–128. Belka, C., Wiegmann, K., Adam, D., Holland, R., Neuloh, M., Herrmann, F., Krönke, M., Brach, M., 1995. Tumor necrosis factor (TNF)-alpha activates c-raf-1 kinase via the p55 TNF receptor engaging neutral sphingomyelinase. EMBO J. 14, 1156. Bligh, E., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Phys. 37, 911–917. Brouwers, J.F., Van Hellemond, J.J., van Golde, L.M., Tielens, A.G., 1998. Ether lipids and their possible physiological function in adult Schistosoma mansoni. Mol. Biochem. Parasitol. 96, 49–58. Cornett, D.S., Reyzer, M.L., Chaurand, P., Caprioli, R.M., 2007. MALDI imaging mass spectrometry: molecular snapshots of biochemical systems. Nat. Methods 4, 828–833. Cunha, V., Noël, F., 1997. Praziquantel has no direct effect on (Na+/K+)-ATPases and (Ca2+/Mg2+) ATPases of Schistosoma mansoni. Life Sci. 60, PL289–PL294. Day, T., Bennett, J., Pax, R., 1992. Praziquantel: the enigmatic antiparasitic. Parasitol. Today 8, 342–344. de Oliveira, D.N., de Bona Sartor, S., Ferreira, M.S., Catharino, R.R., 2013. Cosmetic analysis using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Materials 6, 1000–1010. Doenhoff, M.J., Cioli, D., Utzinger, J., 2008. Praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis. Curr. Opin. Infect. Dis. 21, 659–667. Espinoza, B., Silman, I., Arnon, R., Tarrab-Hazdai, R., 1991. Phosphatidylinositolspecific phospholipase C induces biosynthesis of acetylcholinesterase via diacylglycerol in Schistosoma mansoni. Eur. J. Biochem. 195, 863–870.
Please cite this article in press as: Ferreira, M.S., et al. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to Schistosoma mansoni. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.12.008
M.S. Ferreira et al. / International Journal for Parasitology xxx (2015) xxx–xxx Fenwick, A., Savioli, L., Engels, D., Robert Bergquist, N., Todd, M.H., 2003. Drugs for the control of parasitic diseases: current status and development in schistosomiasis. Trends Parasitol. 19, 509–515. Ferreira, M.S., de Oliveira, D.N., Gonçalves, R.F., Catharino, R.R., 2014a. Lipid characterization of embryo zones by silica plate laser desorption ionization mass spectrometry imaging (SP-LDI-MSI). Anal. Chim. Acta 807, 96–102. Ferreira, M.S., Oliveira, D.N., Oliveira, R.N., Allegretti, S.M., Vercesi, A.E., Catharino, R.R., 2014b. Mass spectrometry imaging: a new vision in differentiating Schistosoma mansoni strains. J. Mass Spectrom. 49, 86–92. Frank, S., van Die, I., Geyer, R., 2012. Structural characterization of Schistosoma mansoni adult worm glycosphingolipids reveals pronounced differences with those of cercariae. Glycobiology 22, 676–695. Fried, B., Haseeb, M.A., 1991. Role of lipids in Schistosoma mansoni. Parasitol. Today 7, 204, author reply 204. Furlong, S., 1991. Unique roles for lipids in Schistosoma mansoni. Parasitol. Today 7, 59–62. Fusco, A., Salafsky, B., Ellenberger, B., Li, L.-H., 1988. Schistosoma mansoni: correlations between mouse strain, skin eicosanoid production, and cercarial skin penetration. J. Parasitol. 73, 253–261. Garside, P., Sands, W.A., Kusel, J.R., Hagan, P., 1996. Is the induction of apoptosis the mechanism of the protective effects of TNF-a in helminth infection? Parasite Immunol. 18, 111–113. Gönnert, R., Andrews, P., 1977. Praziquantel, a new broad-spectrum antischistosomal agent. Z. Parasitenkd. 52, 129–150. Hagan, P., Garside, P., Kusel, J., 1993. Is tumour necrosis factor alpha the molecular basis of concomitant immunity in schistosomiasis? Parasite Immunol. 15, 553– 557. Hannun, Y.A., 1994. The sphingomyelin cycle and the second messenger function of ceramide. J. Biol. Chem. 269, 3125–3128. Harder, A., Goossens, J., Andrews, P., 1988. Influence of praziquantel and Ca2+ on the bilayer-isotropic-hexagonal transition of model membranes. Mol. Biochem. Parasitol. 29, 55–59. Hockley, D.J., McLaren, D.J., 1973. Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. Int. J. Parasitol. 3, 13–20. Hu, W., Brindley, P.J., McManus, D.P., Feng, Z., Han, Z.-G., 2004. Schistosome transcriptomes: new insights into the parasite and schistosomiasis. Trends Mol. Med. 10, 217–225. Kolesnick, R., Golde, D.W., 1994. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 77, 325–328. Lima, S., Vieira, L., Harder, A., Kusel, J., 1994. Effects of culture and praziquantel on membrane fluidity parameters of adult Schistosoma mansoni. Parasitology 109, 57. LoVerde, P.T., Hirai, H., Merrick, J.M., Lee, N.H., El-Sayed, N., 2004. Schistosoma mansoni genome project: an update. Parasitol. Int. 53, 183–192. Machado, D., Shishido, S.M., Queiroz, K.C.S., Oliveira, D.N., Faria, A.L.C., Catharino, R.R., Spek, C.A., Ferreira, C.V., 2013. Irradiated riboflavin diminishes the aggressiveness of melanoma in vitro and in vivo. PLoS One 8, e54269. Malgat, M., Maurice, A., Baraud, J., 1986. Sphingomyelin and ceramidephosphoethanolamine synthesis by microsomes and plasma membranes from rat liver and brain. J. Lipid Res. 27, 251–260. McLaren, D.J., 1984. Disguise as an evasive stratagem of parasitic organisms. Parasitology 88, 597–611. Meier, H., Blaschke, G., 2000. Capillary electrophoresis–mass spectrometry, liquid chromatography–mass spectrometry and nanoelectrospray-mass spectrometry of praziquantel metabolites. J. Chrom. B Biomed. Sci. Appl. 748, 221–231. Meier, H., Blaschke, G., 2001. Investigation of praziquantel metabolism in isolated rat hepatocytes. J. Pharm. Biomed. Anal. 26, 409–415. Obeid, L.M., Linardic, C.M., Karolak, L.A., Hannun, Y.A., 1993. Programmed cell death induced by ceramide. Science 259, 1769–1771. Olivier, L., Stirewalt, M., 1952. An efficient method for exposure of mice to cercariae of Schistosoma mansoni. J. Parasitol. 38, 19–23.
7
Pax, R., Bennett, J., Fetterer, R., 1978. A benzodiazepine derivative and praziquantel: effects on musculature of Schistosoma mansoni and Schistosoma japonicum. Naunyn Schmiedebergs Arch Pharmacol. 304, 309–315. Pellegrino, J., Oliveira, C.A., Faria, J., Cunha, A.S., 1962. New approach to the screening of drugs in experimental schistosomiasis mansoni in mice. Am. J. Trop. Med. Hyg. 11, 201. Pica-Mattoccia, L., Cioli, D., 2004. Sex-and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment. Int. J. Parasitol. 34, 527– 533. Pinlaor, S., Prakobwong, S., Boonmars, T., Wongkham, C., Pinlaor, P., Hiraku, Y., 2009. Effect of praziquantel treatment on the expression of matrix metalloproteinases in relation to tissue resorption during fibrosis in hamsters with acute and chronic Opisthorchis viverrini infection. Acta Trop. 111, 181–191. Raines, M., Kolesnick, R., Golde, D., 1993. Sphingomyelinase and ceramide activate mitogen-activated protein kinase in myeloid HL-60 cells. J. Biol. Chem. 268, 14572–14575. Redman, C., Robertson, A., Fallon, P., Modha, J., Kusel, J., Doenhoff, M., Martin, R., 1996. Praziquantel: an urgent and exciting challenge. Parasitol. Today 12, 14– 20. Redman, C.A., Kennington, S., Spathopoulou, T., Kusel, J.R., 1997. Interconversion of sphingomyelin and ceramide in adult Schistosoma mansoni. Mol. Biochem. Parasitol. 90, 145–153. Rodrigues, L.R., Oliveira, D.N.D., Ferreira, M.S., Catharino, R.R., 2014. In situ assessment of atorvastatin impurity using MALDI mass spectrometry imaging (MALDI-MSI). Anal. Chim. Acta 818, 32–38. Sabah, A., Fletcher, C., Webbe, G., Doenhoff, M., 1986. Schistosoma mansoni: chemotherapy of infections of different ages. Exp. Parasitol. 61, 294–303. Shaw, J., Marshall, I., Erasmus, D., 1977. Schistosoma mansoni. In vitro stimulation of vitelline cell development by extracts of male worms. Exp. Parasitol. 42, 14– 20. Smith, T.M., Brooks, T.J., 1969. Lipid fractions in adult Schistosoma mansoni. Parasitology 59, 293–298. Solon, E.G., 2007. Autoradiography: high-resolution molecular imaging in pharmaceutical discovery and development. Expert Opin. Drug Discov. 2, 503–514. Tallima, H., El Ridi, R., 2007. Praziquantel binds Schistosoma mansoni adult worm actin. Int. J. Antimicrob. Agents 29, 570–575. Testi, R., 1996. Sphingomyelin breakdown and cell fate. Trends Biochem. Sci. 21, 468–471. Troiani, A.R., Pica-Mattoccia, L., Valle, C., Cioli, D., Mignogna, G., Ronketti, F., Todd, M., 2007. Is actin the praziquantel receptor? Int. J. Antimicrob. Agents 30, 280– 281. Urayama, S., Zou, W., Brooks, K., Tolstikov, V., 2010. Comprehensive mass spectrometry based metabolic profiling of blood plasma reveals potent discriminatory classifiers of pancreatic cancer. Rapid Commun. Mass Spectrom. 24, 613–620. van Balkom, B.W., van Gestel, R.A., Brouwers, J.F., Krijgsveld, J., Tielens, A.G., Heck, A.J., van Hellemond, J.J., 2005. Mass spectrometric analysis of the Schistosoma mansoni tegumental sub-proteome. J. Prot. Res. 4, 958–966. Webbe, G., James, C., 1977. A comparison of the susceptibility to praziquantel of Schistosoma haematobium, S. japonicum, S. mansoni, S. intercalatum and S. mattheei in hamsters. Z. Parasitenkd. 52, 169–177. Wiest, P.M., Burnham, D.C., Olds, G.R., Bowen, W.D., 1992. Developmental expression of protein kinase C activity in Schistosoma mansoni. Am. J. Trop. Med. Hyg. 46, 358–365. You, H., McManus, D.P., Hu, W., Smout, M.J., Brindley, P.J., Gobert, G.N., 2013. Transcriptional responses of in vivo praziquantel exposure in schistosomes identifies a functional role for calcium signalling pathway member CamKII. PLoS Pathol. 9, e1003254. Young, B.W., Podesta, R.B., 1982. Major phospholipids and phosphatidylcholine synthesis in adult Schistosoma mansoni. Mol. Biochem. Parasitol. 5, 165–172.
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