Volume11,Number1(ene-jun2017)

ÓrganooficialdelaAsociaciónPeruanadeHelmintologíaeInvertebradosAfines(APHIA)

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Neotropical Helminthology, 2017, 11(1), jan-jun: 115-128.

ORIGINAL ARTICLE / ARTÍCULO ORIGINAL

DEVELOPMENT, DIFFERENTIAL PROTEIN EXPRESSIONS AND GENE NETWORK

ON SCHISTOSOMA MANSONI SUBMITTED TO EUPHORBIA MILII LATEX ON

CERCARIAL STAGE

DESARROLLO, EXPRESIÓN DIFERENCIAL DE PROTEÍNAS Y REDES GÉNICAS EN

SCHISTOSOMA MANSONI EXPUESTOS A EUPHORBIA MILII LATEX EN ESTADO DE

CERCARIA

1Laboratório de Avaliação e Promoção da Saúde Ambiental, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz,

Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ, 21040-360, Brasil.

²Université de Perpignan Via Domitia, IHPE UMR 5244, CNRS, Perpignan, France.

3Laboratório de Parasitologia e Epidemiologia Molecular, Universidade Federal de Viçosa, Minas Gerais, MG, Brasil.

4Núcleo de Análises de Biomoléculas, Universidade Federal de Viçosa, Minas Gerais, MG, Brasil.

* Corresponding author: Tel.: +55 21 25621526

E-mail: ronaldodecarvalhoaugusto@gmail.com

Neotropical Helminthology

115

ABSTRACT

We tested a new approach to describe how a sublethal dose of the Euphorbia milii Des Moul. latex can

affect cercariae, inducing changes in the adult Schistosoma mansoni Sambon 1907. We analyzed the

effects on biology and used proteomic tools to identify the expression of different proteins. The treatment

reduced the development of adults (males, 74.1%; females, 76.3%) and decreased the number of eggs

released in 89.9%. The protein profile between adult males of S. mansoni from the control and exposed

groups detected 1,020 spots, 26 were differently expressed and 10 were identified and validated. The

proteins downregulated after exposition included actin, 14-3-3 epsilon and aldehyde dehydrogenase. The

hsp70 was the only protein that was upregulated. The changes in the proteomic profile of adult males of S.

mansoni may be related to a muscular fragility of the body. This work provides new insight for

schistosomiasis control using a natural, water-soluble product at sublethal concentrations for mollusks.

This approach is inexpensive, ecological and efficient as an indirect therapy for schistosomiasis.

Keywords: Schistosomiasis – molluscicide – Euphorbia milii – parasitic disease control – proteomic – gene network

1,2* 3,4 4

Ronaldo de Carvalho Augusto ; Edvaldo Barros ; Pedro Marcus Pereira Vidigal ; Cynthia Mantovani³;

4 1 3 1

Humberto Josué de Oliveira Ramos ; Clélia Christina Mello-Silva ; Cláudio Mafra & Claudia Portes Santos

116

Neotropical Helminthology, 2017, 11(1), jan-jun

RESUMEN

Palabras-clave: Esquistosomiasis – molusquicida – Euphorbia milii – control de enfermedades parasitarias – proteoma – redes génicas

Hemos probado un nuevo enfoque para describir cómo una dosis subletal del látex de Euphorbia milii Des

Moul. puede afectar a las cercarias, induciendo cambios en el Schistosoma mansoni Sambon 1907 adulto.

Se analizaron los efectos sobre la biología y se utilizaron herramientas proteómicas para identificar la

expresión de diferentes proteínas. El tratamiento redujo el desarrollo de adultos (machos 74,1%, hembras

76,3%) y disminuyó el número de huevos liberados en 89,9%. El perfil proteico entre machos adultos de S.

mansoni de los grupos control y expuestos detectó 1.020 spots, 26 se expresaron de forma diferente y 10

fueron identificados y validados. Las proteínas menos abundante después de la exposición incluyeron

actina, 14-3-3 epsilon y aldehído deshidrogenasa. La hsp70 fue la única proteína que se más abundante.

Los cambios en el perfil proteómico de los machos adultos de S. mansoni pueden estar relacionados con

una fragilidad muscular del cuerpo. Este trabajo proporciona una nueva visión para el control de la

esquistosomiasis utilizando un producto natural, soluble en agua a concentraciones subletales para

moluscos. Este enfoque es barato, ecológico y eficiente como terapia indirecta para la esquistosomiasis.

INTRODUCTION Upregulated genes identified in the cercariae

transcriptome are necessary to sustain its

swimming behavior and infection of the definitive

host (Jolly et al., 2007). These genes encode

proteins related to the energy metabolism, such as

NADH dehydrogenase, subunits of cytochrome C

and its homologues, ATP/ADP carriers and

motility genes such as actin and fibrillin. In adult

parasites, the transcriptomes show the intense

expression of genes linked to the escape of the host

immune system and motility, to acquire and

metabolize a variety of nutrients, including nutrient

transporters, such as the glucose transporter (Jolly

et al., 2007). Roquis et al. (2015) showed the

influence of environmental factors on changes in

gene expression on schistosomula and its effects on

adult worms. The effect of these factors can be

mitotically heritable and have consequences in the

ecology of adult parasites, making them a

therapeutic target.

Among the research topics recommended by the

WHO (2002) is the development of effective and

practical measures for micro-focal transmission,

including the use of molluscicides. The latex of

Euphorbia milii var. hislopii Des Moul., tested

under laboratory and field conditions, meets these

recommendations due to its potential as a natural

molluscicide (Lima et al., 2012; Augusto et al.,

2015). De-Carvalho et al. (1988) tested high

-1

concentrations of E. milii latex (10-100 mg·L for 2

Digenetic trematodes have complex life cycles

characterized by a series of striking morphological

and biochemical transitions between the aquatic

environment and molluscan and mammalian hosts

(Evans & miller, 1987; Jolly et al., 2007). In the

aquatic environment, Schistosoma mansoni

Sambon, 1907 takes the form of two free-

swimming larvae – miracidia and cercariae – in a

relatively short-lived 'transitional' stage into

freshwater. They must swim to find their hosts

before energy sources are exhausted. However,

many soluble compounds and different forms of

pollution can disrupt their interaction with the next

host. The acquisition of soluble macromolecules

through the tegument or by ingestion has been

reported to induce changes in the genome

expression of cercariae of S. mansoni, which affect

its growth and development in its definitive host

(Lenzi et al., 2008; Thornhill et al., 2009).

Therefore, the use of the latex of Euphorbia milii

(N.E.Br.) (Ursch & Leandri, 1955) in the control of

schistosomiasis could also elicit changes in the

gene expression of S. mansoni.

The success of the host-parasite relationship

depends, among other factors, on the expression,

interaction and modulation of proteins for the co-

existence of both organisms (Johnston et al., 2009).

Augusto et al.

Neotropical Helminthology, 2017, 11(1), jan-jun

were assessed per group, which was repeated once

a week for three weeks.

Preparation of a total extract of S. mansoni and

two-dimensional electrophoresis - The protein

extracts were obtained by the sonication of

approximately 15 adult male and 20 adult female

parasites per group in extraction solution (7 M

urea, 2 M thiourea, 4% CHAPS). Three replicates

for each sex and each group were performed.

The total protein concentration was determined

using the Bradford method (Bradford, 1976), and

the samples were stored at -80 °C until use. The

protein concentration from female adult worms

was not sufficient to allow for two-dimensional

electrophoresis analysis. From male adult worms,

the protein concentration allowed separate aliquots

containing 150 µg per sample that were diluted to a

final volume of 125 µL in Destreak solution (GE

Healthcare, Sweden) and 2% IPG buffer (pH 3–10)

(GE Healthcare). Seven-centimeter strips

(Immobiline, GE Healthcare, Sweden) with an

immobilized pH gradient in the range of 3–10 were

rehydrated with the protein extract for 20 h using

IPGBox (GE Healthcare, Sweden).

Isoelectric focusing was initiated immediately

after rehydration. Isoelectric focusing was

performed with an automated system (Ettan

IPGphor III, GE Healthcare, Sweden) at 20 °C with

a constant current of 50 µA/strip and a total of 11.7

kVh following a four-step program: 300 V for 15

hours; linear gradient to 1000 V for 300 vh; linear

gradient to 5000 V for 4000 vh and 5000 V for 2000

vh. After isoelectric focusing, the strips were

reduced in equilibration buffer (6 M urea, 0.075 M

Tris HCl (pH 8.8), 29.3% glycerol, 2% SDS, and

0.002% bromophenol blue) containing 2%

dithiothreitol (DTT) for 60 min and then alkylated

for 60 min in equilibration buffer containing 5%

iodoacetamide.

For the second dimension, the strips were placed on

a 12.5% polyacrylamide gel in a Mini Protean Cell

system (Bio-Rad, USA). Electrophoresis was

performed at a constant 80 V for 2:40 h. The gels

were stained with Coomassie Blue G-250 solution

for 72 hours under stirring and scanned by

ImageScanner III (GE Healthcare, Sweden) using

Labscan software (GE Healthcare, Sweden). The

spots were quantitatively analyzed using the

h) against S. mansoni cercariae, and no changes

were observed in cercarial survival or the

percentage of penetration into the skin of mice.

Therefore, the aim of the current study was to

describe how a sublethal dose of the E. milii latex

can affect cercariae, inducing changes in the adult

S. mansoni, using proteomic tools to identify the

different proteins expressed. Additionally, the

development of worms was evaluated using the

parasite rate and the number of eggs released.

Latex - The E. milii var. hislopii latex was collected

a t I l h a d o G o v e r n a d o r d i s t r i c t

(22°48´09´´S/43°12´35´´W), Rio de Janeiro,

Brazil. The collected latex sample was pre-frozen

in dry ice and absolute ethanol and subsequently

-1

lyophilized at -52 °C on 8 x10 mBar for three 12-h

cycles in a Modulyo 4K Freeze Dryer with an

acrylic chamber (Edwards High Vacuum Int., UK).

The lyophilized pellet obtained with this process

was diluted in distilled water and homogenized by

sonication for 20 min. The sublethal doses of the

powdered lyophilized latex of E. milii used to

exposed cercariae were equivalent to the mean,

-1

LC =1.4 mg·L , described by Schall et al. (1992)

for Biomphalaria glabrata (Say, 18118).

Animals - The cercariae of S. mansoni (LE strain)

were collected and separated into two groups. The

first group was exposed to a solution of E. milii

-1

lyophilized latex in distilled water (1.4 mg·L ) for

one hour. The second (control) cercarial group was

kept in distilled water for the same time period.

Then, 30 female Swiss-Webster mice were infected

with 150 exposed cercariae per mouse, and another

30 mice were infected with 150 normal cercariae

per mouse, all using percutaneous inoculation.

Water and food were given ad libitum. At 65 days

post-infection, the adult parasites were collected

for proteomics analysis.

Parasitological analysis - Three analyses of the

number of eggs per gram of stool (EPG) by Kato-

Katz were performed in each experimental group.

Feces were collected in a one-hour period between

10:00 and 12:00 a.m. For egg counts, three slides

Schistosoma submitted to Euphorbia milii latex

MATERIALS AND METHODS

117

118

The spectra were analyzed using FlexAnalysis 3.3

software (Bruker Daltonics, Germany) for the

determination of peaks.

The list of peptide and fragment mass values

generated by the mass spectrometer for each spot

were submitted to a MS/ MS ion search using

Mascot Daemon version 2.4.0 software (Matrix

Science, London, UK) to search in the

Schistosomatidae Protein DataBase from the

UniProt DataBase (download in 03/27/2015 with

40,657 proteins). The search parameters were set as

follows: up to one missed cleavage site; 0.15 Da of

error for the identification of peptides;

carbamidomethylation of cysteine as a fixed

modification and the oxidation of methionine as a

variable modification.

Scaffold software 4.4.1.1 (Proteome Software,

USA) was used to visualize and statistically

validate the MS/MS results based in the peptides

and proteins identified by Mascot Daemon. The

peptide identifications were accepted if they

exhibited probability values greater than 90% by

the Peptide Prophet algorithm (Keller et al., 2002),

and the protein identifications were accepted if

they exhibited probability values greater than 90%

by the Protein Prophet algorithm (Nesvizhskii et

al., 2003). Peaks software 7.0 (Bioinformatics

Solutions Inc., Canada) was used for the de novo

sequencing of the peptides (Ma et al., 2003), and

the identified proteins were statistically and

manually validated. To determine the biological

processes in which the identified molecules were

associated, the Gene Ontology (GO) databases

through the UniProt, the KEGG Pathway and

Schistodb were used.

Gene network analysis - The STRING 10.0

database (Franceschini ., 2013) was also used et al

to analyze the protein-protein interaction in each

group, as it is one of the largest databases of known

and predicted protein-protein interactions

according to Emily (2009). The interactions et al.

included direct (physical) and indirect (functional)

associations derived from four sources: genomic

context, high throughput, co-expression and

previous knowledge. The inference on STRING is

predicted by the confidence score, and we focused

only on highest-confidence interactions (i.e.,

interactions with a score larger than 0.9) in our

results. The identified proteins were grouped into

ImageMaster 2D Platinum 7.5 software (GE

Healthcare, Sweden) and the abundance of each

protein spot was calculated by the percentage

volume (vol%) with the following parameters:

smoothness greater than 2, saliency greater than

50, and area greater than 50. Only those spots that

were significantly different at a significance level

of p< 0.05 based on one-way ANOVA analysis

(assuming equal variance) and a ratio above 1.5

were selected for the next step.

In-gel tryptic digestion and mass spectrometry -

The significantly different spots were manually

excised, treated with washing solution (50%

acetonitrile and 0.0025 M ammonium

bicarbonate), and dehydrated in 100% acetonitrile

in a vacuum centrifuge at room temperature. The

proteins were subsequently subjected to reduction

(65 mM DTT) and alkylation (100 mM

iodoacetamide).

The samples were digested at 37 °C overnight with

proteomic-grade trypsin from the porcine pancreas

(SIGMA, USA) in 40 mM ammonium bicarbonate

and 10% acetonitrile (final concentration: 25

ng/µL). Tryptic peptides were extracted from the

gel solution with 50% acetonitrile in 5% formic

acid. The extracted peptides were transferred to a

sterile tube, dried in a vacuum centrifuge, and

resuspended in a solution of 50% acetonitrile and

0.1% trifluoroacetic acid.

One-microliter aliquots of each sample were

applied to a steel plate at a 1:1 ratio with a α-cyano-

4-hydroxycinnamic acid matrix (Bruker Daltonics,

Germany). After crystallization, the plate was

inserted into the mass spectrometer for analysis.

The first search for proteins was performed by

PMF (peptide mass fingerprint) homology in

comparison with the NCBI [National Center for

Biotechnology Information (NCBInr)] public

database with the aid of the online version of

Mascot (Matrix Science, London, UK). The

taxonomic parameter for the search was restricted

to Schistosomatidae considering up to two lost

cleavage sites, an error of 0.1 Da in peptide

identification, cysteine carbamidomethylation as a

fixed modification and methionine oxidation as a

variable modification. A second search for proteins

was performed with all MS spectra obtained in the

m/z range of 800–4000 kDa using a MALDI-TOF-

TOF spectrometer (Bruker Daltonics, Germany).

Neotropical Helminthology, 2017, 11(1), jan-jun Augusto et al.

Neotropical Helminthology, 2017, 11(1), jan-jun

reduction of: (i) the parasite rate (number of adult

parasites) and (ii) the number of eggs released in

the feces. In the exposed group, the number of adult

parasites recovered decreased by 74.1% for males

and 76.3% for females and the quantity of eggs

released was reduced in 89.9% (Table 1).

The quantity of protein extracted from females

-1

(0.75 µg·µl per replicate) was not sufficient for

-1

proteomic analysis. In the males (4.7 µg·µl per

replicate) with two-dimensional electrophoresis

we detected a total of 1,020 spots with 26

differentially expressed between the control and

exposed groups. Of these, only 10 spots were

detected by the MALDI TOF-TOF spectrometer

and subsequently identified by PMF and/or

MASCOT to be statistically validated by the

Scaffold software and manually validated by Peaks

(Table 2).

The most significant cellular components of the 10

valid peptide sequences in the control and exposed

groups were: myofibril (p-value ≥0.00018),

contractile fiber (p-value ≥0.00018), actin

biological processes and molecular functions

according to their ontology. The peptide sequences

were submitted to the UniProt database.

Statistical analysis - The results were expressed as

the mean number of parasites per mouse and were

submitted to Student's t test (α = 5%) performed

using the R program (R Development Core Team,

2012). The graphics were constructed using

GraphPad Prism software (GraphPad V.4.00,

Prism, GraphPad, vol. 3.02, Prism Inc.).

Ethics - This research was approved by the Animal

Ethics Committee of the Oswaldo Cruz Foundation

(CEUA-FIOCRUZ LW-07/13) in accordance with

the guidelines of the Brazilian College for Animal

Experiments (COBEA).

The striking effects observed in the development of

S. mansoni due to the exposition of cercariae to a

low dose of the latex included a significant E. milii

Schistosoma submitted to Euphorbia milii latex

RESULTS

119

Figure 1. Gene ontology analysis using the String database. Analysis of the cellular components of ten valid peptide sequences

from control adult males of Schistosoma mansoni and adult males originating from cercariae exposed to Euphorbia millii latex.

120

Neotropical Helminthology, 2017, 11(1), jan-jun

Figure 2. Upregulation of biological processes in adult male Schistosoma mansoni from cercariae exposed to latex; Figure 3. Upregulation

molecular function in adult male Schistosoma mansoni from cercariae exposed to latex; Figure 4. Downregulation of biological processes in

adult male S. mansoni from cercariae exposed to latex; Figure 5. Downregulation of molecular functions in adult male S. mansoni from cercariae

exposed to latex.

Augusto et al.

Neotropical Helminthology, 2017, 11(1), jan-jun Schistosoma submitted to Euphorbia milii latex

(p-value ≥0.000017), response to heat (p-value

≥0.000017), response to a temperature stimulus (p-

value ≥0.000017), response to stress (p-value

≥0.000044), cellular metabolism (p-value

≥0.00085), organic substance metabolism (p-value

≥0.0021) and cellular process (p-value ≥0.0065)

(Fig. 3). However, on S. mansoni from cercariae

exposed to the latex, four molecular functions were

downregulated: oxidoreductase activity (p-value

≥0.00089), hydrolase activity (p-value ≥0.0025),

transferase activity (p-value ≥0.011) and catalytic

activity (p-value ≥0.021) (Fig. 4). After exposition,

significant biological processes included the

oxidation-reduction process (p-value ≥0.001),

cellular component organization or biogenesis (p-

value ≥0.02), single-organism organelle

organization (p-value ≥0.02), single-organism

processes (p-value ≥0.02) and single-organism

metabolism (p-value ≥0.01) (Fig. 5).

Analyzing the upregulated and downregulated

proteins reported in Table 2 all together by

STRING , Sm p - 1 98 2 9 0 (para m y o s i n ) ,

Smp_046590 (actin), Smp_179810 (troponin t),

Smp_044010.2_mRNA (tropomyosin-1) and the

multifunctional protein hsp70 appear all forming a

closed interactome with a clustering coefficient =

0.81 and PPI enrichment p-value of 0.000206. Two

proteins were unconnected: Smp_050390

(aldehyde dehydrogenase) and Smp_034840.2

(14-3-3 epsilon) (Fig. 6).

cytoskeleton (p-value ≥0.00017), cytoskeletal

portion (p-value ≥0.0028), cytoskeleton (p-value

≥0.0046), non-membrane-bounded organelle (p-

value ≥0.01), intracellular non-membrane-

bounded organelle (p-value ≥0.01), cytoplasmic

portion (p-value ≥0.02), intracellular portion (p-

value ≥0.02), organelle portion (p-value ≥0.02),

protein complex (p-value ≥0.03) and cytoplasm (p-

value ≥0.04) (Fig. 1). The differential analysis

showed that five spots were not expressed (not

seen, not altered in expression) in the exposed

group (actin-R5, troponin-R8, tropomyosin-R10,

paramyosin-R11 and paramyosin-R15). The four

proteins that appeared downregulated in the

exposed . control group were actin-R4 (-1.55) vs

and R7 (-4.04), 14-3-3 epsilon-R23 (-1.73) and

aldehyde dehydrogenase-R25 (-1.86). The spot

protein upregulated in the exposed control vs.

group was the heat shock protein hsp70 (R16 +

1.71) (Table 2).

The input of the latex on cercariae changed the

distribution of protein abundance in adult male S.

mansoni samples. The phytochemical compounds

of the latex were responsible for the upregulation of

the peptide sequence involved in three molecular

functions: Heat shock protein binding (p-value

≥0.000017), chaperone binding (p-value

≥0.00025), and calcium ion binding (p-value

≥0.0089) (Figure 2). These sequences are involved

in many biological processes in the adult parasites,

including the cellular protein metabolism process

-1

Table 1. Effect of exposure to 1.4 mg·L of Euphorbia milii latex on the survival rate of denitive host, male and

female worms recovered and total Schistosoma mansoni recovered in Swiss Webster infected in the laboratory.

-1

Group 1 = cercariae kept in distilled water for a period of one hour and Group 2 = cercariae exposed to 1.4 mg·L of

E. milli latex during the same period. Different letters indicate signicant differences between the means (α = 5%).

Survival rate

of denitive

host

OPG

Male

Parasites

Female

Parasites

Total

Parasites

Group 1 62% 708±28.8a76.6±5.1a89.8±6.1a166.6±11.3a

Group 2 85% 71.4±42.2 b19.8±7.6b21.2±2.1b41.1±4b

121

122

Neotropical Helminthology, 2017, 11(1), jan-jun

K.LTLEIKDLQSEIESLSLENSELIR

R.RVDELTIEVNTLTSQNSQLESEN

LR.L

Spot

number

Protein

Fold

Changes

Anova

Schistosoma

mansoni

Gene

names

PMF Peptide sequence

Mascot Peptide sequence

Mascot

Score

Protein

Sequence

Coverage

(Da)

R04 Actin -1.55

0.0001

Smp_046590

-.MCVPK.H

-.MCVPK.H + Oxidation (M)

K.HTHWKTER.R

R.ISEYVSK.C

R.ISEYVSKCLR.L

R.LKGTTALNSAISR.H

K.SYELPDGQVITIGNER.F

67% 41992 5.30

33%

R05 Actin * * Smp_046590 -.QGVMVGMGQK.D + 2

Oxidation

(M)

K.DSYVGDEAQSK.R

R.VAPEEHPVLLTEAPLNPK.A

R.TTGIVLDSGDGVTHTVPIYEGY

ALPHAILR.L

R.LDLAGR.D

K.SYELPDGQVITIGNER.F

R.AVFPSIVGRPR.H

K.SYELPDGQVITIGNER.F

45865 5.90

R07

Actin

-4.04

0.0044

Smp_046590

Non-identied

R.VAPEEHPVLLTEAPLNPK.A

K.SYELPDGQVITIGNER.F

203

9% 41992 5.03

R08

Troponin t,

invertebrate

Smp_179810

Non-identied

R.TVFTGANYAEEYQR.I

4% 37472 5.89

R10

Tropomyosin

Smp_044010.2

Non-identied

K.ALEISEQESAQREESYEETIR.

7% 97815 4.5

R11

Paramyosin

Smp_021920.1

M.NHDTESHVK.I

R.HAADLGFQVDALSERLDEAGG

STTQTQELLK.R

K.SKFER.E

K.TEEFEEMKR.K

R.ITELEDTAERER.L

R.VKDLETFLDEER.R

R.VSELTIQVNTLTNDKR.R

R.EITVKLEEAEAFATR.E

5% 113247 5.4

Table 2. Differentially expressed proteins of adult males of Schistosoma mansoni and parasites originated from cercariae exposed to the latex of Euphorbia

milii var. hilopii. Proteins were separated by two-dimensional electrophoresis and identied by Maldi TOF-TOF.

Augusto et al.

Neotropical Helminthology, 2017, 11(1), jan-jun Schistosoma submitted to Euphorbia milii latex

R.ENRQMNDQVK.E + Oxidation

(M)

R.KSTTR.T

K.RYESNIADLEIQLDTANK.A

R.VKDLETFLDEER.R

R.LQLANEIEEIRSTLENLER.L

R.VSELTIQVNTLTNDKR.R

R.EITVKLEEAEAFATR.E

R.KFER.Q

K.MKAYK.R + Oxidation (M)

Spot

number

Protein

Fold

Changes

Anova

Schistosoma

mansoni

Gene

names

PMF Peptide sequence

Mascot Peptide sequence

Mascot

Score

Protein

Sequence

Coverage

(Da)

R15

Paramyosin

Smp_021920.1 Non-identied

K.DLQSEIESLSLENSELIR.R

2% 111600 5.5

R16

Heat shock

protein 70

1.71

0.0236

Smp_106930

Non-identied

R.ARFEELNADLFR.G

R.FEELNADLFR.G

1% 80012 5.9

R23

14-3-3 epsilon

-1.73

0.0304

Smp_034840.2 Non-identied

R.VFSAVEQTEGNR.G

K.FREVLESELDR.V

K.DILELIDKYLIK.S

R.YMAEFSVDPQR.K +

Oxidation (M)

K.AYQEASEIAATQLFPTHPIR.L

198

26% 32042 5.0

R25

Aldehyde

dehydrogenase

-1.86

0.0506 Smp_050390

R.IRQLEDELESTETR.L

R.QLEDELESTETR.L

R.TFADEER.I

R.TFADEERINQLEEQLK.E

R.INQLEEQLK.E

R.INQLEEQLKESTFMAEDADR.K

Oxidation (M)

K.ESTFMAEDADR.K + Oxidation

(M)

K.YDEAARK.L

K.LAITEVELER.A

K.ITELEEELR.I

K.SLEISEQEAAQR.E

K.SLEISEQEAAQREEAYEENIR.D

R.EEAYEENIR.D

R.EEAYEENIRDLTER.L

R.LVNTLQADADRLEDELVTEK.E

K.FREVLESELDR.V

K.TVESALGDVFFAAQTTR.Y

30 3% 54412 5.76

Note: The symbol “*” indicates expression only in control group. MW = molecular weight IP = isoelectric point.

123

124

Neotropical Helminthology, 2017, 11(1), jan-jun

Figure 6. Protein Network interaction for up and downregulated proteins of Schistosoma mansoni using the STRING database.

DISCUSSION of Bakry & Mohamed (2011), who used E. milii

latex at a concentration 73.6% higher than the

concentration used in the present study. In our

study, the exposition of cercariae to a low

-1

concentration (1.4 mg·L for 1 h) produced

striking effects in the adult worms, with a reduction

of the parasite rate, number of eggs released and

changes in the male tegument.

The exposition of cercariae to the latex changed the

biology of the parasites showing their effectiveness

as a control strategy influencing the transmission

(reduction of adult worms and the number of eggs

released). The proteomic analysis of adult males of

S. mansoni from exposed cercariae showed protein

targets that produced remarkable changes,

including the upregulation of three molecular

functions (heat shock protein binding, chaperone

binding and calcium ion binding) and the

downregulation of another four molecular

functions (oxidoreductase activity, hydrolase

activity, transferase activity and catalytic activity).

These data confirm the influence of latex on the

development and survival of the parasites.

It is important to draw attention to the influence of

water-soluble compounds in the development of a

parasite's life cycle. The input of soluble

macromolecules or environmental factors

(chemical pollutants, dietary components,

temperature changes and other external stresses) in

the free larval stages of the parasite could be

responsible for changes in genome expression

(long-lasting effects on gene expression and

chromatin), which modify the phenotype of the

worm and thus affect the parasite's biology in ways

that could be heritable over the parasite's life span

(Thornhil ., 2009; Feil & Fraga, 2012; Roquis et al

et al., 2015).

De-Carvalho et al. (1988) tested high

concentrations of E. milii latex against S. mansoni

-1

cercariae (10-100 mg·L for 2 h), and no changes

were observed in cercarial survival or viability.

However, our results indicated a reduction of

parasite development of 74.5% in males and 76.5%

in females. These results were similar to the results

Augusto et al.

Neotropical Helminthology, 2017, 11(1), jan-jun

Troponin T, an invertebrate protein, and

paramyosin were exclusive to the control group.

The genes Smp_179810 (troponin T) and

Smp_0198290 (paramyosin) are involved in the

actin cytoskeleton and muscle system and are

important targets for the mobility of S. mansoni.

High levels of expression of paramyosin are also

observed in adult male Schistosoma japonicum

(Gobert et al., 2009). The functions of paramyosin

in schistosomes are well documented as a

structural component of smooth muscle fibers and

an immunomodulator of the host immune response

through its binding to the immunoglobulin Fc

region and its inhibition of complement activation

(Loukas et al., 2001). Previous studies on the

multiple functions of paramyosin showed that it

may be a potential vaccine candidate, as it has been

suggested to play an important role in immune

stimulation, while schistosome paramyosin is

postulated to play a role in immune evasion (Jones

et al., 2004).

Some proteins were downregulated after

exposition, such as actin, 14-3-3 epsilon and

aldehyde dehydrogenase. The decrease of the 14-3-

3 epsilon protein can reveal a decrease in the cell's

life cycle on exposed parasites because its proteins

are involved in the cell cycle and the PI3K-Akt

signaling pathway (Protasio et al., 2012). The

PI3K-Akt signaling pathway mediates survival

signals in a wide range of neuronal cell types and

can be involved with the metabolic pathways that

regulate cell survival (Brunet et al., 2001). The

downregulation of aldehyde dehydrogenase

reinforces the hypothesis that the parasite likely

produces a molecular compensatory mechanism

against the stress caused by the latex, as this protein

is important in the signaling of stress in glycolysis,

pyruvate and arginine metabolism and fatty acid

degradation. The changes in the proteomic profile

of adult S. mansoni from cercariae exposed to the

latex (exposed group) were significant, and an

increase of heat shock 70 kDa protein (hsp70), a

major immunogen in S. mansoni infections, was

detected. Many phases of Schistosoma's life cycle

are specific to increased levels of hsp70 mRNA,

and it is more commonly expressed in cercaria-

schistosomula transformation and heat-shocked

adult parasites (Neumann et al., 1993). These data

reflect a compensatory mechanism against the

host-parasite relationship caused by latex.

To support this evidence, 26 differentially

expressed proteins in both experimental groups

were d e t e c t e d with two-dimensional

electrophoresis. It is interesting that five spots were

not detected in the exposed group (actin-R5,

troponin-R8, tropomyosin-R10, paramyosin-R11,

and paramyosin-R15). The actin gene has a strong

association with the surface membrane and is very

important for the dynamic activity of parasites and

other organisms (Braschi et al., 2006). This protein

is the most abundant protein found in eukaryotic

cells and is involved in many biological processes,

such as ATP and nucleotide binding, the regulation

of the actin cytoskeleton, tissue remodeling and

apoptosis (Gourlay & Ayscough, 2005; Watanabe

et al., 2015). The proteome of S. mansoni's surface

presents molecular motor activity, and the layer

immediately beneath the plasma membrane is

largely composed of actin homologues in a

macromolecular complex involved in the shuttling

of vesicles to the surface plasma membrane

(Braschi et al., 2006).

There are several actin isoforms expressed in

schistosomes involved in various types of parasite

motility (Abbas & Cain, 1987; Abbas et al., 1997).

In the present study, actin spots R4 and R7 were

downregulated, mostly R7 (-4.04) that was the

more downregulated spot seen in this work. Actin

spot R5 was not seen in E. milii-exposed vs.

unexposed cercariae (Table 2). In the future, it

would be interesting to identify the molecular type

of the expression-altered actins. These results

clearly indicate that actin is an important target for

the E. milli phytochemical activity, remembering

that S. mansoni surface spines are “crystals” of

actin (Cohen et al., 1982). Furthermore, it has been

reported that actin is a target for Praziquantel

(Tallima & El Ridi, 2007), the actual drug of choice

for schistosomiasis treatment. Actin is also a

circulating parasite antigen (together with other

muscular proteins) in human S. mansoni infections,

and it is able to induce an immunoprotective

response in experimental animals (Sulbarán et al.,

2013; Ludolf et al., 2014). All this information

might provide an explanation for the reduction in

the number of adult male worms found in animals

infected with E. milii-exposed cercariae vs.

unexposed cercariae. Actin downregulation would

al s o a ffe c t i t s in t e r act i o n w i t h t h e

troponin/tropomyosin complex, troponin being an

actin-binding motor protein.

Schistosoma submitted to Euphorbia milii latex

125

126

This study was funded by Fundação de Amparo à

Pesquisa do Estado de Minas Gerais (Fapemig),

Financiadora de Estudos e Projetos (Finep),

Sistema Nacional de Laboratórios em

Nanotecnologias (SisNANO)/Ministério da

Ciência, Tecnologia e Informação (MCTI),

Coordenação de Aperfeiçoamento de Pessoal de

Nível Superior (CAPES Parasitologia

Básica/2011), Fundação Carlos Chagas Filho de

Amparo à Pesquisa no Rio de Janeiro

(FAPERJ–BIOTA 2012), Conselho Nacional de

Pesquisa (CNPq) – Edital Universal 2014 and

PAEF/Fiocruz and was sponsored by fellowships

from the CNPq and CAPES. In addition, we are

greatful to the Núcleo de Análise de Biomoléculas

of the Universidade Federal de Viçosa for

providing the facilities for the conduction of the

experiments. The funders did not participate in the

study design, data collection and analysis, decision

to publish, or preparation of the manuscript. This

study is part of the doctoral thesis of the first author,

under the Programa de Pós-Graduação em

Biologia Parasitária, Instituto Oswaldo Cruz,

Fiocruz.

One spot was identified for tropomyosin

(Smp_044010.2). The sequence was not detected

in adult male parasites after exposition, and the

absence and/or low expression of this protein (and

homologues) may be dangerous for the survival of

S. mansoni because it interferes with the constant

renewal of the tegument, immune modulation with

the host, osmoregulation, the absorption and

secretion of substances, and reproduction (Faghiri

et al., 2010).

The protein network significantly indicated that the

analyzed proteins have more interactions among

themselves than what would be expected for a

random set of proteins of similar size, drawn from

the genome at - value = 0.000206. Such an p

enrichment indicated that the proteins are at least

partially biologically connected, as a group,

according to STRING definition. The unconnected

proteins probably form interactomes with proteins

in different pathways, not established in the present

study. Smp_050390 (aldehyde dehydrogenase),

although multifunctional, is probably mainly

involved in oxido-reduction metabolic process in

S. mansoni.

The present work may also provide new insight on

targets for the development of new interventions

for future control of schistosomiasis. The changes

observed in the proteomic profile of adult S.

mansoni males occurred by the exposition of

cercariae to the var latex, which E. milii . hislopii

induced the significant muscular fragility of the

body. We could consider that it may prevent males

from maintaining females inside with consequent

reduction of the parasite load. The proteins that

were not expressed or that were downregulated

affected the muscle contraction function in

exposed parasites, probably interfering with its

intrahost migration.

In conclusion, this study suggests a route for the

control of schistosomiasis in endemic areas using

natural water-soluble products in sublethal

concentrations for mollusks, which reduces the

parasite rate in definitive hosts. This indirect

therapy is inexpensive, ecological and efficient.

The authors declare no conflicts of interest.

Neotropical Helminthology, 2017, 11(1), jan-jun

CONFLICT OF INTEREST

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