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Fasciola gigantica enolase is a major component of worm tegumental fraction protective against sheep fasciolosis
Accepted Manuscript Title: Fasciola gigantica enolase is a major component of worm tegumental fraction protective against sheep fasciolosis Author: N. Mahana H.A.-S. Abd-Allah M. Salah H. Tallima R.El Ridi PII: DOI: Reference:
S0001-706X(16)30099-7 http://dx.doi.org/doi:10.1016/j.actatropica.2016.03.009 ACTROP 3881
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
15-10-2015 6-3-2016 7-3-2016
Please cite this article as: Mahana, N., Abd-Allah, H.A.-S., Salah, M., Tallima, H., Ridi, R.El, Fasciola gigantica enolase is a major component of worm tegumental fraction protective against sheep fasciolosis.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.03.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fasciola gigantica enolase is a major component of worm tegumental fraction protective against sheep fasciolosis N. Mahana a, H.A.-S. Abd-Allah a, M. Salah b, H. Tallima c, R. El Ridi a* a
Zoology Department, Faculty of Science, Cairo University, Cairo 12613, Egypt
b
Schistosoma Biological Materials Supply Program, Theodore Bilharz Research
Institute, Giza 12411, Egypt c
Department of Chemistry, School of Science and Engineering, American University
in Cairo, New Cairo 11835, Cairo, Egypt *Corresponding author. Tel.: +202 3567 6708; fax: +202 3760 3735 E-mail address: [email protected] (R. El Ridi)
Graphical abstract
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Percentage
50 40 30 20 10 0
Enolase‐containing 60 kDa F. gigantica tegument band induces in sheep antibodies that bind to juvenile flukes and mediate their attrition by complement‐ and cell‐mediated cytotoxicity associated with anti‐fluke challenge infection protection.
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Highlights
The Fasciola gigantica Triton-soluble surface membrane and tegumental proteins of 60, 32 and 28 kDa, we have previously shown to be immune protective in mice, were used for vaccination of sheep against challenge F. gigantica.
The 60 kDa proteins induced antibodies able to bind to the surface membrane of newly excysted flukes and mediate their attrition.
The 60 kDa fraction elicited significant (P < 0.05) protection and was found to consist essentially of F. gigantica enolase.
F. gigantica enolase showed 95% identity with F. hepatica enolase at the amino acid level.
Abbreviations: FABP, fatty acid binding protein; GST, glutathione-S-transferase; TSMTP, Triton-soluble surface membrane and tegumental proteins; Mr, molecular mass; IF, indirect membrane immunofluorescence; PRX, 2-Cys peroxiredoxin.
ABSTRACT Infection of cattle and sheep with the parasite Fasciola gigantica is a cause of important economic losses throughout Asia and Africa. Many of the available anthelmintics have undesirable side effects, and the parasite may acquire drug resistance as a result of mass and repeated treatments of livestock. Accordingly, the need for developing a vaccine is evident. Triton-soluble surface membrane and tegumental proteins (TSMTP) of 60, 32, and 28 kDa previously shown to elicit protective immunity in mice against challenge F. gigantica infection were found to be strongly immunogenic in sheep eliciting vigorous specific antibody responses to a titer > 1:16,000 as assessed by enzyme-linked immunosorbent assay. Furthermore, 2
the 60 kDa fraction induced production of antibodies able to bind to the surface membrane of newly excysted juvenile flukes and mediate their attrition in antibodydependent complement- and cell-mediated cytotoxicity assays, and significant (P < 0.05) 40% protection of sheep against F. gigantica challenge infection. Amino acid micro sequencing of the 60 kDa-derived tryptic peptides revealed the fraction predominantly consists of F. gigantica enolase. The cDNA nucleotide and translated amino acid sequences of F. gigantica enolase showed homology of 92% and 95%, respectively to F. hepatica enolase, suggesting that a fasciolosis vaccine might be effective against both tropical and temperate liver flukes. Keywords: Fasciola gigantica; Fasciolosis; Vaccination; Enolase
1.
Introduction Fasciolosis is a neglected tropical disease despite it infects ruminant livestock in
developed countries such as Australia, Ireland, Spain, Portugal, and the United States of America. The disease is caused by hermaphroditic trematodes of the genus, Fasciola, of which F. hepatica (temperate liver fluke) and F. gigantica (tropical liver fluke) are responsible for infection of > 600 million animals and loss of > 3 billion USD annually, through decrease in the productivity of domestic ruminants (cattle, buffaloes, sheep and goats), mortality, ill-thrift, decrease in body weight, condemnation of livers at the abattoir, predisposition to other diseases and associated veterinary costs (Robinson and Dalton, 2009; Morales and Espino, 2012). Fasciolosis 3
has recently been shown to be an important public health problem with 2.4 million human cases reported from 70 countries in five continents, the level of endemicity ranging from hypo- to hyperendemic, and a further 180 million at risk of infection (Mas-Coma, 2005; Mas-Coma et al., 2014). Over-reliance of the effective and broad-spectrum flukicide, triclabendazole, led to the development of resistance to the drug in fluke populations worldwide (reviewed in Hanna et al., 2015). Accordingly, the emphasis in control schemes should be shifted to new drugs and preferably to vaccine development (McManus and Dalton, 2006; Molina-Hernández et al., 2015). Vaccine trials were conducted in cattle evaluating the efficacy of F. gigantica native glutathione-S-transferase (FgGST), cathepsin L, paramyosin, fatty acid binding protein (FABP), and a recombinant FABP expressed in E. coli (rFABP), combined with various adjuvants, including Montanide 80 and Freund's, against challenge F. gigantica infection. While all tested molecules induced detectable antibody responses, low but significant reductions in fluke burdens (31%, P < 0.05) were only observed in cattle vaccinated with the native FABP in Freund's adjuvant (Estuningsih et al., 1997). Fasciola gigantica rFABP emulsified in Freund's adjuvant used to vaccinate buffalo (Bubalus bubalis) calves evoked reduction of 36% in challenge fluke burden (Nambi et al., 2005). Additionally, a maximum protection level of 35% against F. gigantica was achieved in buffaloes immunized with a mixture of rFABP and GST combined with montanide 70 M-VGO as adjuvant (Kumar et al., 2012). Of note, sheep were immunized with native FgGST alone or in combination with either aluminum hydroxide or saponin and then challenged with 120 metacercariae of F. gigantica. The results indicated that anti-GST IgG was not elevated after challenge. The highest fluke burden reduction was observed in the group vaccinated with GST4
saponin (32%), but this reduction was not statistically significant in comparison with the control group (Paykari et al., 2002). Likewise, F. gigantica leucine aminopeptidase and peroxiredoxin (PRX) alone or in combination also failed to protect buffaloes against challenge infection with 400 metacercariae (Raina et al., 2011); yet, it showed when combined with Freund's adjuvant significant protective efficacy (approximately 40% reduction in challenge worm burden) in mice (Changklungmoa et al., 2013). Additionally, F. gigantica recombinant cathepsin L was effective in protecting mice against homologous challenge infection (Kueakhai et al., 2015; Meemon and Sobhon, 2015; Sansri et al., 2015), and recombinant cathepsin B and cathepsin L5 in combination elicited in rat protection of 83% against challenge F. hepatica infection (Jayaraj et al., 2009). There is consensus that antibody-dependent complement- and cell-mediated cytotoxicity directed to juvenile flukes are of preponderant importance to vaccinerelated protective immunity against fasciolosis, thus implying parasite surface membrane antigens are potential vaccine candidates (Toet et al., 2014). Accordingly, the analysis of F. hepatica tegument proteome was undertaken (Wilson et al., 2011), and identified enolase, aldolase, GST, and FABP as the most immunoreactive components (Morales and Espino, 2012). In parallel, we isolated and electroseparated the Triton-soluble surface membrane and tegumental proteins (TSMTP) of adult F. gigantica worms. Resolved bands of 110, 70, 60, 47, 45, 40, 32, 30, 28, and 20 kDa were electroeluted and the eluates used to immunize outbred mice. The data conclusively showed that the bands of approximately 60, 32, and 28 kDa were the only bands to contain proteins that elicited protection (100%, 50%, and 83%, respectively) in mice against oral challenge with 25 metacercariae (El Ridi et al., 2001). Since ample data in the literature demonstrated that anti-fasciolosis vaccine 5
efficacy of parasite molecules in rodents is often not reproduced in ruminants, we have herein undertaken to examine whether the TSMTP of 60, 32, and 28 kDa are also capable of protecting sheep against infection with F. gigantica and to identify the putative protective molecule(s).
2. Materials and methods 2.1.
Ethics statement
All animal experiments were performed following the recommendations of the current edition of the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, Washington D.C., USA, and were approved by the Institutional Animal Care and Use Committee of Theodore Bilharz Research Institute (TBRI), Giza, Egypt. 2.2. Animals and parasites Twenty male, healthy, parasite-free sheep (El Farafra breed) were maintained throughout experimentation at the TBRI. Inbred female BALB/c mice and metacercariae and adult worms of F. gigantica were obtained from the Schistosome Biological Supply Program, TBRI. 2.3. Fasciola gigantica antigen preparation Adult worms (12 week-old), recovered from sheep experimentally infected at TBRI with 400 F. gigantica metacercariae, were washed repeatedly (10x) in Hank's buffer to remove host serum and proteins and worm regurgitates, incubated for 20 min on ice in Hank's buffer supplemented with protease inhibitors (1 mM phenyl methyl sulfonyl fluoride and 2 g/ml leupeptin obtained from Sigma-Aldrich, St. Louis, MO) and 0.2% Triton X-100 (Bio-Rad Laboratories, Inc., Hercules, CA), vortexed for 1 min,
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and centrifuged at 400 x g. The supernatant containing Triton-soluble surface membrane and tegumental proteins (TSMTP) was stored at -76oC until use. 2.4. Antigen electro separation and elution Fasciola gigantica TSMTP were electro separated by preparative sodium dodecyl sulfate-polyacrylamide gel (10%) electrophoresis (SDS-PAGE) under non-reducing conditions, and the molecules in the electro separated bands eluted using Mini Whole Gel Eluter (Bio-Rad), following the manufacturer's instructions. The profile and molecular mass (Mr) of the eluates were assessed by SDS-PAGE followed by Coomassie blue staining. Eluates of approximately 60, 32, and 28 kDa were dialyzed against Dulbecco's phosphate-buffered saline, pH 7.1 (D-PBS), assessed for protein concentration using the Bradford's assay (Bio-Rad), and freed from SDS using SDSOut Precipitating Reagent of Pierce (Rockford, IL). 2.5. Sheep immunization and bleeding Sheep were divided into 4 groups of 4 each, and were injected intramuscularly three times with adjuvant-free D-PBS or 25 g/sheep of molecules of 60, 32, and 28 kDa, respectively. The immunogens were combined with complete and incomplete Freund's adjuvant for the first and second immunization, respectively. Blood was obtained from each sheep via the jugular vein, one week after the last immunization (adjuvant-free), and sera stored at -20oC until use. 2.6. Assessment of sheep humoral responses Sera of control and vaccinated sera were examined, on a sheep individual basis, for antibody reactivity to the parasite immunogens and metacercariae. 2.6.1. ELISA Sera serially diluted (1:500-1:16,000) were assessed in duplicates for antibody binding to F. gigantica TSMTP (500 ng/well) by enzyme-linked immunosorbent
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assay (ELISA) as described (Jezek et al., 2008; Raina et al., 2011) using horseradish peroxidase-labeled anti sheep IgG (H+L) conjugate (Pierce). Reactivity was estimated spectrophotometrically at 405 nm after adding 2, 2'-Azino-bis (3ethylbenzothiazzoline-sulfonic acid) (ABTS) substrate (Pierce). 2.6.2. Excystment of metacercariae Excystment of juvenile flukes is an active process, occurring in two stages, activation and emergence. Activation appears to be initiated by high concentrations of CO2, reducing conditions, and a temperature of about 390C. The larva emerges from a small hole in the ventral side of the cyst provided exposure to bile or taurocholic acid. Excystment of F. gigantica juvenile flukes was performed via adopting established procedures previously described (Dixon, 1966; Smith and Clegg, 1981; Tielens et al., 1981). 2.6.3. Indirect membrane immunofluorescence (IF) Juvenile F. gigantica flukes were washed after excystment and incubated overnight at 37oC with 100 l/well of control and test sera diluted 1:500 in RPMI medium/5% fetal calf serum (FCS), then thoroughly washed, incubated at room temperature for 1 h in the dark with 100 l/well with fluorescein isothiocyanate-labeled goat anti-sheep IgG (H+L) conjugate (Pierce) diluted 1:100, washed again, and then inspected by alternate and ultraviolet (UV) microscopy (Olympus, Tokyo, Japan). 2.6.4. Antibody-dependent complement-mediated cytotoxicity Excysted Juvenile F. gigantica (8 to 10 per well) were washed in sterile medium and incubated at 37oC/5% CO2 with 100 l/well heat-inactivated (30 min at 56oC), 1:500-diluted (final dilution 1:1000) and 0.45 m membrane- (Corning Costar, Cambridge, MA) sterilized control and immune sheep sera and 20 l/well (final
8
concentration 10%) fresh rabbit serum as a source of complement, and examined for viability and motility under inverted microscope (Olympus) after 0, 2, 24 and 48 h. 2.6.5. Antibody-dependent cell-mediated cytotoxicity Peritoneal cells were obtained from the peritoneal cavity of naïve inbred BALB/c mice under sterile conditions and cultured at a concentration of 105 cells/well of tissue culture flat-bottomed microplates (Costar) in 100 l/well Rosewell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 100 U/ml penicillinstreptomycin, 50 g/ml gentamycin, 1 mM sodium pyruvate, 2 mM L-glutamine, and 5% FCS (Invitrogen, Carlsbad, CA) (RPMI medium). Parallel wells were seeded with peripheral blood mononuclear cells (PBMC, 106 cells/well) isolated from venous blood of naïve, parasite-free sheep by Ficoll-Paque (Sigma-Aldrich) gradient centrifugation (400 x g for 20 min). All wells were added 2 h later with 100 l 1:100diluted (final dilution 1:200), membrane-sterilized control and test sera in duplicates and 8-10 freshly excysted, thoroughly washed juvenile flukes. After 24 and 28 h incubation at 37oC/5% CO2, the number and proportion of viable contractile larvae were recorded for each well following examination by inverted light microscopy. 2.7. Sheep challenge and evaluation of worm burden All control and test sheep were challenged orally, 5 weeks after the last immunization each with 150 viable metacercariae of F. gigantica in water. Twelve weeks after challenge, the sheep were slaughtered humanely, the liver removed, and flukes collected from liver slices were counted. Percentage protection in each of the test groups was calculated as C – T/C x 100 where C = mean worm count in control sheep and T = mean worm count in vaccinated sheep. 2.8. Amino acid microsequencing
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Eluted F. gigantica TSMTP of 60 kDa were resolved by SDS-PAGE (preparative 10% gel, reducing conditions). A single band of ca. 60 kDa was seen, excised from the gel and analyzed by tryptic peptides amino acid microsequencing, which was performed at the Harvard Microchemistry and Proteomics Analysis Facility (Cambridge, MA) by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry (µLC/MS/MS) on a Thermo LTQ-Orbitrap mass spectrometer. These instruments are capable of acquiring individual sequence (MS/MS) spectra on-line at high sensitivity (<<<1 femtomole) for multiple peptides in the chromatographic run. The MS/MS spectra were then correlated with known sequences using the algorithm Sequest developed at the University of Washington (Eng et al., 1994) and programs developed at Harvard laboratory (Chittum et al., 1998). MS/MS peptide sequences were then reviewed by a scientist at Harvard Laboratory for consensus with known proteins and the results manually confirmed for fidelity. Searches were performed on the National Center for Biotechnology Information (NCBI) non-redundant protein database (NCBInr) and NCBI database EST_Others. 2.9. Fasciola gigantica enolase gene cloning and sequencing The coding sequence for enolase was obtained from adult F. gigantica worm total RNA by reverse transcription-polymerase chain reaction (RT-PCR) amplification using synthetic oligonucleotides obtained from published sequences (Davis et al., 1994), GenAmp EZ rTth one-step RNA PCR Kit (Applied Biosystems, Foster City, CA), and GeneAmp PCR instrument System 9600 (Perkin-Elmer Corporation, Norwalk, CT). Amplicon purity and size were determined by UV visualization of ethidium bromide-stained 2% agarose gels (UV Transilluminator TI 3, Biometra, Germany), in parallel with 50-2000 bp Ladder (AmpliSize Molecular Ruler, Bio-
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Rad). The RT-PCR products were sequenced in both directions at Molecular Cloning Laboratories (MCLAB, South San Francisco, CA). 2.10. Statistical analyses Student's t- test was used to analyze the statistical significance of differences between mean values from experimental and control values.
3. Results 3.1. Profile of electro- separated and eluted antigens The SDS-PAGE profile of adult F. gigantica TSMTP revealed about 20 bands of 106 to 10 kDa; the bands of 60, 32, and 28 kDa were particularly evident (Fig. 1A). Following whole (mini) gel elution, equal protein amounts of each fraction were separated by SDS-PAGE. Figure 1B reveals the Mr and profile of the eluates, and indicate the fractions used for sheep immunization, namely of 60, 32, and 28 kDa, shown previously to generate protective immunity against F. gigantica challenge infection in outbred mice (El Ridi, 2001). 3.2. Serum antibody reactivity Sheep sera from adjuvant control and vaccinated sheep were assessed by ELISA for serum titer reactivity against TSMTP on a sheep individual basis. The molecules of 60, 32, and 28 kDa appeared all strongly immunogenic in sheep eliciting specific serum antibody titers of >16,000 (Fig. 2A). Antibody from control and 28 kDa proteins-vaccinated sheep failed to bind to the surface of excysted larvae in IF, while serum antibodies from 60 > 32 kDa proteins-immunized sheep consistently bound to the entire surface of the juvenile flukes (Fig. 2B). For 4 independent experiments, serum antibodies from 60 kDa proteins-vaccinated sheep induced significantly higher complement-dependent antibody-mediated cytotoxicity of freshly excysted flukes as compared to serum antibodies from adjuvant control (P < 0.0001) and 32 and 28 kDa 11
(P <0.05) fraction-immunized sheep (Fig. 2C). The percentage of juvenile flukes killed by antibody-dependent cell-mediated cytotoxicity was lower than for complement mediated cytotoxicity as it never exceeded 16% on using mouse peritoneal cells and 27% with sheep PBMC, provided test sera were from 60 kDa proteins-immunized sheep. Percentage killing dependent on sera from control and 28 kDa fraction-immunized sheep was equally low (Fig. 2D). 3.3. Protective potential The data demonstrated that the TSMTP of 32 and 28 kDa failed to evoke protection against challenge infection. Conversely, the 60 kDa fraction appeared to include potential vaccine candidate(s) as it induced significant (P = 0.020) reduction of 41.3% in challenge fluke burden (Table 1). 3.4. Amino acid micro sequencing Amino acid micro sequencing revealed that a substantial percentage of the 60 kDa fraction-derived tryptic peptides showed 96%-100% homology with the predicted amino acid sequence of F. hepatica enolase (Accession: AAA57450) (Fig. 3), while the rest preponderantly showed homology to actin and tubulin (Table 2). Since actin is a highly conserved molecule, and Fasciola alpha and beta tubulin show up to 99% homology with the sheep counterparts, it was assumed that the molecule responsible for immunological responses and protection is enolase. 3.5. cDNA sequencing Alignment of our sequence was done using the National Center of Biotechnology information (NCBI) nucleotide blast versus Fasciola hepatica mRNA for enolase (eno gene), Length=1296 bp (Accession: emb|AM279156.1|; GI: 190350154), and yielded 92% Identities (Fig. 4A). 3.6. cDNA translation
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Fasciola gigantica partial cDNA was translated using the Six-Frame program and yielded amino acid sequence, which showed 184/192 (95%) identities with those of F. hepatica (Accession: AAA57450.1) (Fig. 4B). The translated sequence of F. gigantica enolase partial cDNA was found to be related to Enolase-superfamily, characterized by the presence of an enolate anion intermediate, which is generated by abstraction of the alpha-proton of the carboxylate substrate by an active site residue and is stabilized by coordination to the essential Mg2+ ion (Fig. 4C).
4. Discussion The F. gigantica TSMTP of 28, 32, and 60 kDa appeared strongly immunogenic in sheep evoking serum antibody titers of >16,000 as assessed by ELISA. High serum antibody titers to the immunogens appeared to not necessarily correlate with protective immunity against fasciolosis as sheep immunized with TSMTP of 32 and 28 kDa did not develop resistance to the challenge infection. Likewise, vaccination of buffaloes with leucine aminopeptidase and PRX evoked vigorous antibody responses to the immunogens yet failed to elicit protection against F. gigantica challenge infection (Raina et al., 2011). Additionally, goats immunized with F. hepatica recombinant cathepsin L, PRX, and S. mansoni FABP (Sm14) alone or in combination together with Quil A adjuvant developed high levels of antibodies specific to the immunogens but failed to resist challenge infection with 200 metacercariae (Buffoni et al., 2012). Conversely, induction of antibodies able to access and interact with the surface membrane antigens of juvenile flukes could correlate with protective immunity. In this regard, TMSTP of 60 kDa evoked antibodies that strongly bound to the surface of larvae in IF, and mediated the highest level of in vitro antibody-dependent complement- and cell-mediated killing of newly excysted flukes as compared to the 32 and 28 kDa species, that was associated with 13
significant protection against challenge infection. Accordingly, it is recommended to examine whether the ability of vaccine-generated antibodies to recognize the surface membrane antigens of juvenile flukes is a prognostic marker of immune protection. The TSMTP of 32 and 28 kDa failed to protect sheep against oral infection with 150 metacercariae of F. gigantica, suggesting they might be enriched in those molecules, such as cathepsin B, cathepsin L, and PRX, which are able to protect rodents, but not ruminant, against fasciolosis (El Ridi et al., 2007; Jayaraj et al., 2009; Mendes et al., 2010; Raina et al., 2011; Changklungmoa et al., 2013; Zafra et al., 2013; Kueakhai et al., 2015; Meemon and Sobhon, 2015; Sansri et al., 2015). Significant (P < 0.05) protection of approximately 40% was only obtained with the 60 kDa fraction, which was shown be enriched with a protein showing 96-100% homology with F. hepatica enolase. Enolase of F. hepatica has an apparent Mr of 47 kDa, has been identified in the excretory-secretory products (Bernal et al., 2004; Morphew et al., 2007) and tegument (Morales and Espino, 2012) of adult worms, and shows about 70% amino acid sequence homology with sheep, cow, buffalo, and goat enzyme. No reports are available regarding F. gigantica enolase. Therefore, we proceeded to cloning and sequencing of F. gigantica enolase, which revealed 92% and 95% identity with F. hepatica enolase nucleotide and amino acid sequences, respectively (Davis et al., 1994). Homology between cDNA sequences of F. gigantica and F. hepatica cathepsin L fragments was found to reach 87% to 99% (Grams et al., 2001). The 70 kDa heat-shock proteins of F. hepatica and F. gigantica nucleotide and protein sequences were found to be 98% and 99% identical, respectively (Smith et al., 2008). Gene coding for leucine aminopeptidase in F. gigantica also showed a close homology (98.6%) with leucine aminopeptidase of F. hepatica (Raina et al., 2011).
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Enolase is localized in the surface membrane of numerous pathogens including Fasciola (Bernal et al., 2004; Morales and Espino, 2012), and may, thus, be targeted by major immune effector mechanisms such as antibody-dependent, complement- or cellmediated cytotoxicity. Additionally, enolase is a prominent moonlighting (multifunctional) protein that participates in numerous non-glycolytic functions (Jung et al. 2014; Paludo et al., 2015). Enolase role as plasminogen-binding protein has been documented (Bernal et al., 2004). Moonlighting proteins are promising anti-parasite candidate vaccines as they would induce interference with a plethora of pathogen physiological functions. In conclusion, enolase has been identified as a major component of worm tegumental fraction protective against sheep fasciolosis, whose potential use as therapeutic target should be investigated in the future. Acknowledgment. The study was supported, in part, by the Academy of Scientific Research and Technology, Science and Technology Centre, Cairo, Egypt; Grant No. 20 to R. El Ridi. The Funding Agency has no role in the design of the study; in collection, analysis and interpretation of the data; in writing the report; or in the decision to submit the article for publication.
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Kueakhai, P., Changklungmoa, N., Chaichanasak, P., Jaikua,W., Itagaki, T., Sobhon, P., 2015. Vaccine potential of recombinant pro- and mature cathepsinL1 against fasciolosis gigantica in mice. Acta Trop. 150, 71-78. Kumar, N., Anju, V., Gaurav, N., Chandra, D., Samanta, S., Gupta, S.C., Adeppa, J., Raina, O.K., 2012. Vaccination of buffaloes with Fasciola gigantica recombinant glutathione S-transferase and fatty acid binding protein. Parasitol. Res. 110, 419426. Mas-Coma, S., 2005. Epidemiology of fascioliasis in human endemic areas. J. Helminthol. 79:207-216. Review. Mas-Coma, S., Valero, M,A,, Bargues, M,D., 2014. Fascioliasis. Adv. Exp. Med. Biol. 766, 77-114. McManus, D.P., Dalton, J.P., 2006. Vaccines against the zoonotic trematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica. Parasitology. 133 Suppl, S43-61. Review. Meemon, K,, Sobhon, P., 2015. Juvenile-specific cathepsin proteases in Fasciola spp.: their characteristics and vaccine efficacies. Parasitol. Res. 114, 2807-2813. Mendes, R.E., Pérez-Ecija, R.A., Zafra, R., Buffoni, L., Martínez-Moreno, A., Dalton, J.P., Mulcahy, G., Pérez, J., 2010. Evaluation of hepatic changes and local and systemic immune responses in goats immunized with recombinant Peroxiredoxin (Prx) and challenged with Fasciola hepatica. Vaccine 28, 2832-2840. Molina-Hernández, V., Mulcahy, G., Pérez, J., Martínez-Moreno, Á., Donnelly, S., O'Neill, S.M., Dalton, J.P., Cwiklinski, K., 2015. Fasciola hepatica vaccine: we may not be there yet but we're on the right road. Vet. Parasitol. 208, 101-111. Morphew, R.M., Wright H.A., LaCourse E.J., Woods, D.J., Brophy, P.M., 2007. Comparative proteomics of excretory-secretory proteins released by the liver fluke
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Fasciola hepatica in sheep host bile and during in vitro culture ex host. Mol. Cell. Proteomics 6, 963-972. Morales, A., Espino, A.M., 2012. Evaluation and characterization of Fasciola hepatica tegument protein extract for serodiagnosis of human fascioliasis. Clin. Vaccine Immunol. 19, 1870-1878. Nambi, P.A., Yadav, S.C., Raina, O.K., Sriveny, D., Saini, M., 2005. Vaccination of buffaloes with Fasciola gigantica recombinant fatty acid binding protein. Parasitol. Res. 97, 129-135. Paludo, G.P., Lorenzatto, K.R., Bonatto, D., Ferreira, H.B., 2015. Systems biology approach reveals possible evolutionarily conserved moonlighting functions for enolase. Comput. Biol. Chem. 58, 1-8. Paykari, H,, Dalimi, A,, Madani, R., 2002. Immunization of sheep against Fasciola gigantica with glutathione S-transferase. Vet. Parasitol. 105, 153-159. Raina, O.K., Naga,r G., Varghese, A., Prajitha, G., Alex, A., Maharana, B.R., Joshi, P., 2011. Lack of protective efficacy in buffaloes vaccinated with Fasciola gigantica leucine aminopeptidase and peroxiredoxin recombinant proteins. Acta Trop. 118, 217-222. Robinson, M.W., Dalton, J.P., 2009. Zoonotic helminth infections with particular emphasis on fasciolosis and other trematodiases. Philos. Trans. R .Soc. Lond. B Biol. Sci. 364, 2763-2776. Sansri, V., Meemon, K., Changklungmoa, N., Kueakhai, P., Chantree, P., Chaichanasak, P., Lorsuwannarat, N., Itagaki, T., Sobhon, P., 2015. Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L. Vaccine 33, 1596-1601.
19
Smith, M.A., Clegg, J.A., 1981. Improved culture of Fasciola hepatica in vitro. Z. Parasitenkd. 66, 9-15. Smith, R.E., Spithill, T.W., Pike, R.N., Meeusen, E.N., Piedrafita, D., 2008. Fasciola hepatica and Fasciola gigantica: cloning and characterisation of 70 kDa heatshock proteins reveals variation in HSP70 gene expression between parasite species recovered from sheep. Exp. Parasitol. 118, 536-542. Tielens, A.G., Van der Meer, P., Van den Bergh, S.G., 1981. Fasciola hepatica: simple, large-scale, in vitro excystment of metacercariae and subsequent isolation of juvenile liver flukes. Exp. Parasitol. 51, 8-12. Toet, H., Piedrafita, D.M., Spithill, T.W., 2014. Liver fluke vaccines in ruminants: strategies, progress and future opportunities. Int. J. Parasitol. 44, 915-927. Wilson, R.A., Wright, J.M., de Castro-Borges, W., Parker-Manuel, S.J., Dowle, A.A., Ashton, P.D., Young, N.D., Gasser, R.B., Spithill, T.W., 2011. Exploring the Fasciola hepatica tegument proteome. Int. J. Parasitol. 41, 1347-1359. Zafra, R, Pérez-Écija, RA, Buffoni, L, Moreno, P, Bautista, MJ, Martínez-Moreno, A, Mulcahy, G, Dalton, JP, Pérez, J., 2013. Early and late peritoneal and hepatic changes in goats immunized with recombinant cathepsin L1 and infected with Fasciola hepatica. J. Comp. Pathol. 148, 373-384.
20
Explanation of figures Fig. 1. Gel profile of adult F. gigantica TSMTP examined after SDS-PAGE (10% gel, non-reducing conditions) followed by Coomassie blue staining. On the left, migration position and Mr of SDS-PAGE protein standards (Bio-Rad). (A) Electroseparation of protein species of 2 preparations. The arrows show the position and approximate Mr of the bands selected because of protective potential against F. gigantica challenge infection in outbred mice. (B) Profile of eluted proteins. Arrows point to the fractions used for sheep immunization. Fig. 2. Sheep humoral responses. (A) Reactivity of sera from adjuvant control sheep ( and sheep immunized with 60 (
), 32 (
) and 28 (
),
) kDa proteins were tested against
TSMTP by ELISA. Each point represents mean absorbance of 4 sera individually tested and vertical bars denote the SD about the mean. (B) Sera from adjuvant control sheep, and sheep immunized with 60, 32 and 28 kDa species were diluted 1:500 and tested for reactivity against newly excysted juvenile flukes in IF. (C) Sera diluted 1:1000 were tested, on an individual sheep basis, for antibody-dependent complement-mediated cytotoxicity against F. gigantica excysted juveniles over 4 independent experiments. Each column represents mean percent killed larvae and the vertical bars denote the SD about the mean. (D) Sera diluted 1:200 were tested, on an individual sheep basis, for antibody21
dependent cell-mediated cytotoxicity against F. gigantica excysted juveniles using peritoneal cells from naïve BALB/c mice (white columns) or peripheral blood mononuclear cells from naïve, healthy, parasite-free sheep (greyish columns), over 2 independent experiments. Each column represents mean percent killed larvae and the vertical bars denote the SD about the mean. Star and asterisks indicate P values of respectively <0.0001 and <0.05, respectively as assessed by the Student's t- test. Fig. 3. Homology of F. gigantica 60 kDa-derived tryptic peptides with predicted amino acid sequence of F. hepatica (Fh) enolase (ACCESSION: AAA57450.1; Davis et al., 1994).
Fig. 4. (A) Homology between F. gigantica (Fg) and F. hepatica (Fh) enolase cDNA nucleotide sequence. (B) Homology between predicted amino acid sequences of F. gigantica (Fg) and F. hepatica (Fh) enolase. (C) Conserved domains in F. gigantica enolase (partial length).
22
A
B
Fig 1
Mean absorbance (405 nm)
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
A
B
500 1000 2000 4000 8000 16000 Reciprocal serum dilution
Control 60 32 28 Sera pooled from control or 60, 32, or 28 kDa protein‐immunized sheep Mean percent antibody‐ dependent cell‐mediated cytotoxicity
Mean percent antibody‐ dependent complement‐mediated cytotoxicity
D C 80 60 40 20 0
Sera from control or 60, 32, or 28 kDa protein‐immunized sheep
Fig. 2.
60 kDa SGETEDNFIADLVVGLR Fh enolase 379 SGETEDNFIADLVVGLR 389 60 kDa IQIVGDDLTVTNPLR Fh enolase 313 IQIVGDDLTVTNPLR 327 60 kDa AVANVNSQIAPNLIK Fh enolase 64 AVANVNSQIAPNLIK 78 60 kDa
AAVPSGAETGVHEALELR
35 30 25 20 15 10 5 0
Sera from control or 60, 32, or 28 kDa protein‐immunized sheep
Fh enolase 33 AAVPSGAETGVHEALELR 50 60 kDa YGLDACNVGDEGGFAPSIQDNLHGLELLR Fh enolase 199 YGLDACNVGDEGGFAPSIQDNLEGLELLR 227 60 kDa IGSEVYHNLR Fh enolase 183 IGSEVYHNLR 192 60 kDa EVIM*PVPSFNVINGGSHAGNK Fh enolase 141 EVIM PVPSFNVINGGSHAGNK 161 60 kDa SGINVTDQAAVDKFM*LDLDGTPNK Fh enolase 79 SGINVTDQAAVDKFM LDLDGTPNK 102 60 kDa IEEDLGGAAK Fh enolase 413 IEEDLGGAAK 422 60 kDa VNQIGSVSESIK Fh enolase 344 VNQIGSVSESIK 355
Fig. 3
Fig. 4A.
Fg
1
Fh
55
Fg
58
Fh
115
Fg
118
Fh
175
Fg 178
ACCGATGAAGATGATGTCACGACTGCGAA-GG-TTATTCCGTGCGG-NGTTCCAAGCGGT |||| ||||| |||||||||||| ||||| || ||||||||||||| |||||||||||| ACCGTTGAAGTTGATGTCACGACCGCGAAAGGTTTATTCCGTGCGGCAGTTCCAAGCGGT
57
GCTTCTACTGGTGTTCNNNAANCTNTGGAATTGCGNGATGGCCCTCCCGGCTATANGGGA ||||| |||||||||| || || |||||||||| ||||||||||||||||||| ||| GCTTCCACTGGTGTTCATGAAGCTCTGGAATTGCGTGATGGCCCTCCCGGCTATATGGGC
117
AAAGGTGTTCTGNAAGCGGNTGCAAACGTGANCAGCCAAATTGCTCCCNNCCTCATCAAA ||||||||||| |||||| ||||||||||| ||||||||| |||||| |||||||||| AAAGGTGTTCTAAAAGCGGTTGCAAACGTGAACAGCCAAATCGCTCCCAACCTCATCAAA
177
NGCGNAATAAATGTTACTGATCAAGCTGCGGTTGACAAATTCATGCTGGNCCTGGNCGGA ||| |||||||||||||||||||||||||||||||||||||||||| | ||||| |||| AGCGGAATAAATGTTACTGATCAAGCTGCGGTTGACAAATTCATGCTTGACCTGGACGGA
237
297
357
Fh
235
Fg
238
Fh
295
ACTCCCAACAAAGAAAAAACTCGGTGCGAAATGCAATCCTTNGGTGTGTCCCTCGCTGTC |||||||||||||||||| ||||||||| |||||||||||| |||||||| ||||||||| ACTCCCAACAAAGAAAAA-CTCGGTGCG-AATGCAATCCTT-GGTGTGTCTCTCGCTGTC
Fg
298
TGTAAAGCGGGTTGGCCGCTGAAGAAGGGTCTNCCCTTTGTACAAATACATCGCCACTCT
114
174
234
294
351
Fh
352
Fg
358
Fh
408
Fg
418
Fh
465
Fg
478
Fh
522
Fg
538
Fh
580
Fg
598
Fh
638
Fg
658
Fh
696
Fg
718
Fh
755
Fg
778
Fh
815
Fg
838
Fh
875
Fg
898
Fh
934
Fg
958
Fh
991
Fg
1018
Fh
1051
Fg
1078
Fh
1111
Fg
1138
Fh
1171
Fg
1198
Fh
1229
|||||||||||| ||||||||| |||||||| |||||||||||| |||||||||||||| TGTAAAGCGGGT--GCCGCTGAA-AAGGGTCT-CCCTTTGTACAAGTACATCGCCACTCT
407
TGCGGGAAATAAAGAAGGTTATAATGCCCGGGTGCCGTCTTTCAACGTCATCAATGGCGG ||||||||||||||| |||||||||| || |||||||||||||||||||||||||||||| TGCGGGAAATAAAGA-GGTTATAATG-CC-GGTGCCGTCTTTCAACGTCATCAATGGCGG
417
TAGTCACGCTTGGGAAACAAACTAGCGATGCAGGAGTTTTATGATAATGCCAACGGGTGC |||||||||| |||||||||||||||||||||||| ||||||||||||||||||||||| TAGTCACGCT--GGAAACAAACTAGCGATGCAGGAG-TTTATGATAATGCCAACGGGTGC
477
TAGTTCTNTTCACTGAAGCTATGGAAGATCGGAAGTGAGGTTTACCATAACCTCAGAGCT ||||||| |||||||||||||| ||||||||||||||||||||||||||||||||||||| TAGTTCT-TTCACTGAAGCTAT-GAAGATCGGAAGTGAGGTTTACCATAACCTCAGAGCT
537
GTTATCAAAAGCAAGTACGGTTTGGACGCCTGCAATGTGGGTGACGAGGGTGGTTTCNCC |||||||||||||||||||||||||||||||||||||||||||||||||||||||| | GTTATCAAAAGCAAGTACGGTTTGGACGCCTGCAATGTGGGTGACGAGGGTGGTTT--CG
597
CTCCAAGTATTCAAGATAATTTGGAAAAGGACTTGAGCTGCTTCGTACCGCAATAGACAA |||||||||||||||||||||||| |||||||||||||||||||||||||||||||||| CTCCAAGTATTCAAGATAATTTGG--AAGGACTTGAGCTGCTTCGTACCGCAATAGACAA
657
AGCTGGATATACGGGAAAAGTCAAGATTGCCATGGATTGCGCTGCCTCGGGAATTTTACA ||||||||||||||||||||||||||||||||||||||||||||| || ||||||||||| AGCTGGATATACGGGAAAAGTCAAGATTGCCATGGATTGCGCTGCTTC-GGAATTTTACA
717
AGGAAGGGAAATACGATTTGGATTTCAAAAACCCCAAGTCTCAGGCCAGTTCTTGGATCA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AGGAAGGGAAATACGATTTGGATTTCAAAAACCCCAAGTCTCAGGCCAGTTCTTGGATCA
777
CTTCGGATGCCATGGCTGATGTCTACAAGAAAATGATGTCTACTTACCCGATCGTTAGCA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTTCGGATGCCATGGCTGATGTCTACAAGAAAATGATGTCTACTTACCCGATCGTTAGCA
837
TCGAGGACCCGTTTCGATCAAGACGATTGGCCGGCTTGGACTAAACTGACGGGCGAATGT ||||||||||| ||||| |||||||||||||| |||||||||||||||||||| |||||| TCGAGGACCCG-TTCGACCAAGACGATTGGCCAGCTTGGACTAAACTGACGGGTGAATGT
897
AAAATCCAGATCGTTGGTGATGACTTAANCTGTCACCAANCCCCTCTTCGTGTGCAAAAA |||||||||||||||||||||||||||| |||||||||| |||| ||||||||||||||| AAAATCCAGATCGTTGGTGATGACTTAA-CTGTCACCAA-CCCC-CTTCGTGTGCAAAAA
957
GCTATCGACCAAAAGGCATGCCATTGCCTGTTGCTAAAAGTCAACCAGATCGGTTCGGGG ||||||||||||||||||||| |||||||||||||||||||||||||||| ||||||| | GCTATCGACCAAAAGGCATGCAATTGCCTGTTGCTAAAAGTCAACCAGATTGGTTCGGTG
1017
TCANANTCCATTNAGGCTTGCAAGATGGCCCAGGAAGCCGGTTGGGGTGTGATGGTATCA ||| | |||||| ||||||||||||||||||| ||||| ||||||||||||||||||||| TCAGAGTCCATTAAGGCTTGCAAGATGGCCCAAGAAGCTGGTTGGGGTGTGATGGTATCA
1077
464
521
579
637
695
754
814
874
933
990
1050
1110
CATCGTNCAGGATANACCGAAGATAANTTCATCGCTGATTTNGTGGNNGGNCTNNGNANC |||||| ||||| | || |||||||| |||||||||||||| |||| || || | | CATCGTTCAGGAGAAACTGAAGATAACTTCATCGCTGATTTGGTGGTTGGACTGCGTACT
1137
GGCCNGATTAAAACAGGTNCNCCTTGTCGTTCAGANCNTCTTNCGAAATACGAACCGAGT |||| ||||||||||||| | |||||||||||||| | |||| |||||||| |||| ||| GGCCAGATTAAAACAGGTGCACCTTGTCGTTCAGAACGTCTTGCGAAATAC-AACC-AGT
1197
TGCNGCGTNTTNAAG ||| |||| || ||| TGCTGCGTATTGAAG
1170
1228
1212 1243
Fig. 4A.
Fg
2
EDDVTTAX-VIPCGVPSGASTGVHEALELRDGPPGYMGKGVLKAVANVNSQIAPNLIKSG
60
Fh
21
Fg
61
Fh
81
Fg
121
Fh
141
Fg
181
Fh
201
E DVTTA + VPSGASTGVHEALELRDGPPGYMGKGVLKAVANVNSQIAPNLIKSG EVDVTTAKGLFRAAVPSGASTGVHEALELRDGPPGYMGKGVLKAVANVNSQIAPNLIKSG
80
INVTDQAAVDKFMLDLDGTPNKEKLGANAILGVSLAVCKAGAAEKGLPLYKYIATLAGNK INVTDQAAVDKFMLDLDGTPNKEKLGANAILGVSLA CKAGAAEKGLPLYKYIATLAGNK INVTDQAAVDKFMLDLDGTPNKEKLGANAILGVSLAXCKAGAAEKGLPLYKYIATLAGNK
120
EVIMPVPSFNVINGGSHAGNKLAMQEFMIMPTGASSFTEAMKIGSEVYHNLRAVIKSKYG EVIMPVPSFNVINGGSHAGNKLAMQEFMIMPTGASSFTEAMKIGSEVYHNLRAVIKSKYG EVIMPVPSFNVINGGSHAGNKLAMQEFMIMPTGASSFTEAMKIGSEVYHNLRAVIKSKYG
180
LDACNVGDEGGF LDACNVGDEGGF LDACNVGDEGGF
140
200
192 212
Fig. 4B.
Fig. 4C.
Table 1 Effect of sheep vaccination with TSMTP species of 60, 32, and 28 kDa on protection against challenge Fasciola gigantica. _____________________________________________________________________ Fluke burden P value a
Mean + SD
Percent reduction b
Sheep group _____________________________________________________________________ Adjuvant controls
74.5 + 16.6
60 kDa
43.7 + 10.6
0.020
32 kDa
64.0 + 7.8
NS
28 kDa
60.2 + 12.8
NS
41.3
_____________________________________________________________________ Sheep (4 per group) administered with immunogen-free adjuvant (adjuvant controls) or adjuvanted TSMTP species of 60, 32, or 28 kDa were orally challenged 5 weeks after the last, adjuvant-free injection with 150 viable F. gigantica metacercariae, and assessed for fluke burden 12 weeks later. a
Two-tailed Student's t test.
23
b
Percentage protection was calculated as C – T/C x 100 where C = mean worm count
in adjuvant control sheep and T = mean worm count in vaccinated sheep. Table 2 Fractionation of TSMTP of 60 kDa by amino acid microsequencing. _____________________________________________________________________ Peptides identity
Number
percentage
_____________________________________________________________________ Enolase (gi|3023708)
10
32.2
Actin (gi|113230)
8
25.8
Actin (gi|3328)
3
9.6
Tubulin beta chain (gi|109431)
6
19.3
Alpha tubulin (gi|1223784)
2
6.4
Thiol-specific anti-oxidant protein (gi|3549894) 2
6.4
_____________________________________________________________________
24
S0001-706X(16)30099-7 http://dx.doi.org/doi:10.1016/j.actatropica.2016.03.009 ACTROP 3881
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
15-10-2015 6-3-2016 7-3-2016
Please cite this article as: Mahana, N., Abd-Allah, H.A.-S., Salah, M., Tallima, H., Ridi, R.El, Fasciola gigantica enolase is a major component of worm tegumental fraction protective against sheep fasciolosis.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.03.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fasciola gigantica enolase is a major component of worm tegumental fraction protective against sheep fasciolosis N. Mahana a, H.A.-S. Abd-Allah a, M. Salah b, H. Tallima c, R. El Ridi a* a
Zoology Department, Faculty of Science, Cairo University, Cairo 12613, Egypt
b
Schistosoma Biological Materials Supply Program, Theodore Bilharz Research
Institute, Giza 12411, Egypt c
Department of Chemistry, School of Science and Engineering, American University
in Cairo, New Cairo 11835, Cairo, Egypt *Corresponding author. Tel.: +202 3567 6708; fax: +202 3760 3735 E-mail address: [email protected] (R. El Ridi)
Graphical abstract
60
Percentage
50 40 30 20 10 0
Enolase‐containing 60 kDa F. gigantica tegument band induces in sheep antibodies that bind to juvenile flukes and mediate their attrition by complement‐ and cell‐mediated cytotoxicity associated with anti‐fluke challenge infection protection.
1
Highlights
The Fasciola gigantica Triton-soluble surface membrane and tegumental proteins of 60, 32 and 28 kDa, we have previously shown to be immune protective in mice, were used for vaccination of sheep against challenge F. gigantica.
The 60 kDa proteins induced antibodies able to bind to the surface membrane of newly excysted flukes and mediate their attrition.
The 60 kDa fraction elicited significant (P < 0.05) protection and was found to consist essentially of F. gigantica enolase.
F. gigantica enolase showed 95% identity with F. hepatica enolase at the amino acid level.
Abbreviations: FABP, fatty acid binding protein; GST, glutathione-S-transferase; TSMTP, Triton-soluble surface membrane and tegumental proteins; Mr, molecular mass; IF, indirect membrane immunofluorescence; PRX, 2-Cys peroxiredoxin.
ABSTRACT Infection of cattle and sheep with the parasite Fasciola gigantica is a cause of important economic losses throughout Asia and Africa. Many of the available anthelmintics have undesirable side effects, and the parasite may acquire drug resistance as a result of mass and repeated treatments of livestock. Accordingly, the need for developing a vaccine is evident. Triton-soluble surface membrane and tegumental proteins (TSMTP) of 60, 32, and 28 kDa previously shown to elicit protective immunity in mice against challenge F. gigantica infection were found to be strongly immunogenic in sheep eliciting vigorous specific antibody responses to a titer > 1:16,000 as assessed by enzyme-linked immunosorbent assay. Furthermore, 2
the 60 kDa fraction induced production of antibodies able to bind to the surface membrane of newly excysted juvenile flukes and mediate their attrition in antibodydependent complement- and cell-mediated cytotoxicity assays, and significant (P < 0.05) 40% protection of sheep against F. gigantica challenge infection. Amino acid micro sequencing of the 60 kDa-derived tryptic peptides revealed the fraction predominantly consists of F. gigantica enolase. The cDNA nucleotide and translated amino acid sequences of F. gigantica enolase showed homology of 92% and 95%, respectively to F. hepatica enolase, suggesting that a fasciolosis vaccine might be effective against both tropical and temperate liver flukes. Keywords: Fasciola gigantica; Fasciolosis; Vaccination; Enolase
1.
Introduction Fasciolosis is a neglected tropical disease despite it infects ruminant livestock in
developed countries such as Australia, Ireland, Spain, Portugal, and the United States of America. The disease is caused by hermaphroditic trematodes of the genus, Fasciola, of which F. hepatica (temperate liver fluke) and F. gigantica (tropical liver fluke) are responsible for infection of > 600 million animals and loss of > 3 billion USD annually, through decrease in the productivity of domestic ruminants (cattle, buffaloes, sheep and goats), mortality, ill-thrift, decrease in body weight, condemnation of livers at the abattoir, predisposition to other diseases and associated veterinary costs (Robinson and Dalton, 2009; Morales and Espino, 2012). Fasciolosis 3
has recently been shown to be an important public health problem with 2.4 million human cases reported from 70 countries in five continents, the level of endemicity ranging from hypo- to hyperendemic, and a further 180 million at risk of infection (Mas-Coma, 2005; Mas-Coma et al., 2014). Over-reliance of the effective and broad-spectrum flukicide, triclabendazole, led to the development of resistance to the drug in fluke populations worldwide (reviewed in Hanna et al., 2015). Accordingly, the emphasis in control schemes should be shifted to new drugs and preferably to vaccine development (McManus and Dalton, 2006; Molina-Hernández et al., 2015). Vaccine trials were conducted in cattle evaluating the efficacy of F. gigantica native glutathione-S-transferase (FgGST), cathepsin L, paramyosin, fatty acid binding protein (FABP), and a recombinant FABP expressed in E. coli (rFABP), combined with various adjuvants, including Montanide 80 and Freund's, against challenge F. gigantica infection. While all tested molecules induced detectable antibody responses, low but significant reductions in fluke burdens (31%, P < 0.05) were only observed in cattle vaccinated with the native FABP in Freund's adjuvant (Estuningsih et al., 1997). Fasciola gigantica rFABP emulsified in Freund's adjuvant used to vaccinate buffalo (Bubalus bubalis) calves evoked reduction of 36% in challenge fluke burden (Nambi et al., 2005). Additionally, a maximum protection level of 35% against F. gigantica was achieved in buffaloes immunized with a mixture of rFABP and GST combined with montanide 70 M-VGO as adjuvant (Kumar et al., 2012). Of note, sheep were immunized with native FgGST alone or in combination with either aluminum hydroxide or saponin and then challenged with 120 metacercariae of F. gigantica. The results indicated that anti-GST IgG was not elevated after challenge. The highest fluke burden reduction was observed in the group vaccinated with GST4
saponin (32%), but this reduction was not statistically significant in comparison with the control group (Paykari et al., 2002). Likewise, F. gigantica leucine aminopeptidase and peroxiredoxin (PRX) alone or in combination also failed to protect buffaloes against challenge infection with 400 metacercariae (Raina et al., 2011); yet, it showed when combined with Freund's adjuvant significant protective efficacy (approximately 40% reduction in challenge worm burden) in mice (Changklungmoa et al., 2013). Additionally, F. gigantica recombinant cathepsin L was effective in protecting mice against homologous challenge infection (Kueakhai et al., 2015; Meemon and Sobhon, 2015; Sansri et al., 2015), and recombinant cathepsin B and cathepsin L5 in combination elicited in rat protection of 83% against challenge F. hepatica infection (Jayaraj et al., 2009). There is consensus that antibody-dependent complement- and cell-mediated cytotoxicity directed to juvenile flukes are of preponderant importance to vaccinerelated protective immunity against fasciolosis, thus implying parasite surface membrane antigens are potential vaccine candidates (Toet et al., 2014). Accordingly, the analysis of F. hepatica tegument proteome was undertaken (Wilson et al., 2011), and identified enolase, aldolase, GST, and FABP as the most immunoreactive components (Morales and Espino, 2012). In parallel, we isolated and electroseparated the Triton-soluble surface membrane and tegumental proteins (TSMTP) of adult F. gigantica worms. Resolved bands of 110, 70, 60, 47, 45, 40, 32, 30, 28, and 20 kDa were electroeluted and the eluates used to immunize outbred mice. The data conclusively showed that the bands of approximately 60, 32, and 28 kDa were the only bands to contain proteins that elicited protection (100%, 50%, and 83%, respectively) in mice against oral challenge with 25 metacercariae (El Ridi et al., 2001). Since ample data in the literature demonstrated that anti-fasciolosis vaccine 5
efficacy of parasite molecules in rodents is often not reproduced in ruminants, we have herein undertaken to examine whether the TSMTP of 60, 32, and 28 kDa are also capable of protecting sheep against infection with F. gigantica and to identify the putative protective molecule(s).
2. Materials and methods 2.1.
Ethics statement
All animal experiments were performed following the recommendations of the current edition of the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, Washington D.C., USA, and were approved by the Institutional Animal Care and Use Committee of Theodore Bilharz Research Institute (TBRI), Giza, Egypt. 2.2. Animals and parasites Twenty male, healthy, parasite-free sheep (El Farafra breed) were maintained throughout experimentation at the TBRI. Inbred female BALB/c mice and metacercariae and adult worms of F. gigantica were obtained from the Schistosome Biological Supply Program, TBRI. 2.3. Fasciola gigantica antigen preparation Adult worms (12 week-old), recovered from sheep experimentally infected at TBRI with 400 F. gigantica metacercariae, were washed repeatedly (10x) in Hank's buffer to remove host serum and proteins and worm regurgitates, incubated for 20 min on ice in Hank's buffer supplemented with protease inhibitors (1 mM phenyl methyl sulfonyl fluoride and 2 g/ml leupeptin obtained from Sigma-Aldrich, St. Louis, MO) and 0.2% Triton X-100 (Bio-Rad Laboratories, Inc., Hercules, CA), vortexed for 1 min,
6
and centrifuged at 400 x g. The supernatant containing Triton-soluble surface membrane and tegumental proteins (TSMTP) was stored at -76oC until use. 2.4. Antigen electro separation and elution Fasciola gigantica TSMTP were electro separated by preparative sodium dodecyl sulfate-polyacrylamide gel (10%) electrophoresis (SDS-PAGE) under non-reducing conditions, and the molecules in the electro separated bands eluted using Mini Whole Gel Eluter (Bio-Rad), following the manufacturer's instructions. The profile and molecular mass (Mr) of the eluates were assessed by SDS-PAGE followed by Coomassie blue staining. Eluates of approximately 60, 32, and 28 kDa were dialyzed against Dulbecco's phosphate-buffered saline, pH 7.1 (D-PBS), assessed for protein concentration using the Bradford's assay (Bio-Rad), and freed from SDS using SDSOut Precipitating Reagent of Pierce (Rockford, IL). 2.5. Sheep immunization and bleeding Sheep were divided into 4 groups of 4 each, and were injected intramuscularly three times with adjuvant-free D-PBS or 25 g/sheep of molecules of 60, 32, and 28 kDa, respectively. The immunogens were combined with complete and incomplete Freund's adjuvant for the first and second immunization, respectively. Blood was obtained from each sheep via the jugular vein, one week after the last immunization (adjuvant-free), and sera stored at -20oC until use. 2.6. Assessment of sheep humoral responses Sera of control and vaccinated sera were examined, on a sheep individual basis, for antibody reactivity to the parasite immunogens and metacercariae. 2.6.1. ELISA Sera serially diluted (1:500-1:16,000) were assessed in duplicates for antibody binding to F. gigantica TSMTP (500 ng/well) by enzyme-linked immunosorbent
7
assay (ELISA) as described (Jezek et al., 2008; Raina et al., 2011) using horseradish peroxidase-labeled anti sheep IgG (H+L) conjugate (Pierce). Reactivity was estimated spectrophotometrically at 405 nm after adding 2, 2'-Azino-bis (3ethylbenzothiazzoline-sulfonic acid) (ABTS) substrate (Pierce). 2.6.2. Excystment of metacercariae Excystment of juvenile flukes is an active process, occurring in two stages, activation and emergence. Activation appears to be initiated by high concentrations of CO2, reducing conditions, and a temperature of about 390C. The larva emerges from a small hole in the ventral side of the cyst provided exposure to bile or taurocholic acid. Excystment of F. gigantica juvenile flukes was performed via adopting established procedures previously described (Dixon, 1966; Smith and Clegg, 1981; Tielens et al., 1981). 2.6.3. Indirect membrane immunofluorescence (IF) Juvenile F. gigantica flukes were washed after excystment and incubated overnight at 37oC with 100 l/well of control and test sera diluted 1:500 in RPMI medium/5% fetal calf serum (FCS), then thoroughly washed, incubated at room temperature for 1 h in the dark with 100 l/well with fluorescein isothiocyanate-labeled goat anti-sheep IgG (H+L) conjugate (Pierce) diluted 1:100, washed again, and then inspected by alternate and ultraviolet (UV) microscopy (Olympus, Tokyo, Japan). 2.6.4. Antibody-dependent complement-mediated cytotoxicity Excysted Juvenile F. gigantica (8 to 10 per well) were washed in sterile medium and incubated at 37oC/5% CO2 with 100 l/well heat-inactivated (30 min at 56oC), 1:500-diluted (final dilution 1:1000) and 0.45 m membrane- (Corning Costar, Cambridge, MA) sterilized control and immune sheep sera and 20 l/well (final
8
concentration 10%) fresh rabbit serum as a source of complement, and examined for viability and motility under inverted microscope (Olympus) after 0, 2, 24 and 48 h. 2.6.5. Antibody-dependent cell-mediated cytotoxicity Peritoneal cells were obtained from the peritoneal cavity of naïve inbred BALB/c mice under sterile conditions and cultured at a concentration of 105 cells/well of tissue culture flat-bottomed microplates (Costar) in 100 l/well Rosewell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 100 U/ml penicillinstreptomycin, 50 g/ml gentamycin, 1 mM sodium pyruvate, 2 mM L-glutamine, and 5% FCS (Invitrogen, Carlsbad, CA) (RPMI medium). Parallel wells were seeded with peripheral blood mononuclear cells (PBMC, 106 cells/well) isolated from venous blood of naïve, parasite-free sheep by Ficoll-Paque (Sigma-Aldrich) gradient centrifugation (400 x g for 20 min). All wells were added 2 h later with 100 l 1:100diluted (final dilution 1:200), membrane-sterilized control and test sera in duplicates and 8-10 freshly excysted, thoroughly washed juvenile flukes. After 24 and 28 h incubation at 37oC/5% CO2, the number and proportion of viable contractile larvae were recorded for each well following examination by inverted light microscopy. 2.7. Sheep challenge and evaluation of worm burden All control and test sheep were challenged orally, 5 weeks after the last immunization each with 150 viable metacercariae of F. gigantica in water. Twelve weeks after challenge, the sheep were slaughtered humanely, the liver removed, and flukes collected from liver slices were counted. Percentage protection in each of the test groups was calculated as C – T/C x 100 where C = mean worm count in control sheep and T = mean worm count in vaccinated sheep. 2.8. Amino acid microsequencing
9
Eluted F. gigantica TSMTP of 60 kDa were resolved by SDS-PAGE (preparative 10% gel, reducing conditions). A single band of ca. 60 kDa was seen, excised from the gel and analyzed by tryptic peptides amino acid microsequencing, which was performed at the Harvard Microchemistry and Proteomics Analysis Facility (Cambridge, MA) by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry (µLC/MS/MS) on a Thermo LTQ-Orbitrap mass spectrometer. These instruments are capable of acquiring individual sequence (MS/MS) spectra on-line at high sensitivity (<<<1 femtomole) for multiple peptides in the chromatographic run. The MS/MS spectra were then correlated with known sequences using the algorithm Sequest developed at the University of Washington (Eng et al., 1994) and programs developed at Harvard laboratory (Chittum et al., 1998). MS/MS peptide sequences were then reviewed by a scientist at Harvard Laboratory for consensus with known proteins and the results manually confirmed for fidelity. Searches were performed on the National Center for Biotechnology Information (NCBI) non-redundant protein database (NCBInr) and NCBI database EST_Others. 2.9. Fasciola gigantica enolase gene cloning and sequencing The coding sequence for enolase was obtained from adult F. gigantica worm total RNA by reverse transcription-polymerase chain reaction (RT-PCR) amplification using synthetic oligonucleotides obtained from published sequences (Davis et al., 1994), GenAmp EZ rTth one-step RNA PCR Kit (Applied Biosystems, Foster City, CA), and GeneAmp PCR instrument System 9600 (Perkin-Elmer Corporation, Norwalk, CT). Amplicon purity and size were determined by UV visualization of ethidium bromide-stained 2% agarose gels (UV Transilluminator TI 3, Biometra, Germany), in parallel with 50-2000 bp Ladder (AmpliSize Molecular Ruler, Bio-
10
Rad). The RT-PCR products were sequenced in both directions at Molecular Cloning Laboratories (MCLAB, South San Francisco, CA). 2.10. Statistical analyses Student's t- test was used to analyze the statistical significance of differences between mean values from experimental and control values.
3. Results 3.1. Profile of electro- separated and eluted antigens The SDS-PAGE profile of adult F. gigantica TSMTP revealed about 20 bands of 106 to 10 kDa; the bands of 60, 32, and 28 kDa were particularly evident (Fig. 1A). Following whole (mini) gel elution, equal protein amounts of each fraction were separated by SDS-PAGE. Figure 1B reveals the Mr and profile of the eluates, and indicate the fractions used for sheep immunization, namely of 60, 32, and 28 kDa, shown previously to generate protective immunity against F. gigantica challenge infection in outbred mice (El Ridi, 2001). 3.2. Serum antibody reactivity Sheep sera from adjuvant control and vaccinated sheep were assessed by ELISA for serum titer reactivity against TSMTP on a sheep individual basis. The molecules of 60, 32, and 28 kDa appeared all strongly immunogenic in sheep eliciting specific serum antibody titers of >16,000 (Fig. 2A). Antibody from control and 28 kDa proteins-vaccinated sheep failed to bind to the surface of excysted larvae in IF, while serum antibodies from 60 > 32 kDa proteins-immunized sheep consistently bound to the entire surface of the juvenile flukes (Fig. 2B). For 4 independent experiments, serum antibodies from 60 kDa proteins-vaccinated sheep induced significantly higher complement-dependent antibody-mediated cytotoxicity of freshly excysted flukes as compared to serum antibodies from adjuvant control (P < 0.0001) and 32 and 28 kDa 11
(P <0.05) fraction-immunized sheep (Fig. 2C). The percentage of juvenile flukes killed by antibody-dependent cell-mediated cytotoxicity was lower than for complement mediated cytotoxicity as it never exceeded 16% on using mouse peritoneal cells and 27% with sheep PBMC, provided test sera were from 60 kDa proteins-immunized sheep. Percentage killing dependent on sera from control and 28 kDa fraction-immunized sheep was equally low (Fig. 2D). 3.3. Protective potential The data demonstrated that the TSMTP of 32 and 28 kDa failed to evoke protection against challenge infection. Conversely, the 60 kDa fraction appeared to include potential vaccine candidate(s) as it induced significant (P = 0.020) reduction of 41.3% in challenge fluke burden (Table 1). 3.4. Amino acid micro sequencing Amino acid micro sequencing revealed that a substantial percentage of the 60 kDa fraction-derived tryptic peptides showed 96%-100% homology with the predicted amino acid sequence of F. hepatica enolase (Accession: AAA57450) (Fig. 3), while the rest preponderantly showed homology to actin and tubulin (Table 2). Since actin is a highly conserved molecule, and Fasciola alpha and beta tubulin show up to 99% homology with the sheep counterparts, it was assumed that the molecule responsible for immunological responses and protection is enolase. 3.5. cDNA sequencing Alignment of our sequence was done using the National Center of Biotechnology information (NCBI) nucleotide blast versus Fasciola hepatica mRNA for enolase (eno gene), Length=1296 bp (Accession: emb|AM279156.1|; GI: 190350154), and yielded 92% Identities (Fig. 4A). 3.6. cDNA translation
12
Fasciola gigantica partial cDNA was translated using the Six-Frame program and yielded amino acid sequence, which showed 184/192 (95%) identities with those of F. hepatica (Accession: AAA57450.1) (Fig. 4B). The translated sequence of F. gigantica enolase partial cDNA was found to be related to Enolase-superfamily, characterized by the presence of an enolate anion intermediate, which is generated by abstraction of the alpha-proton of the carboxylate substrate by an active site residue and is stabilized by coordination to the essential Mg2+ ion (Fig. 4C).
4. Discussion The F. gigantica TSMTP of 28, 32, and 60 kDa appeared strongly immunogenic in sheep evoking serum antibody titers of >16,000 as assessed by ELISA. High serum antibody titers to the immunogens appeared to not necessarily correlate with protective immunity against fasciolosis as sheep immunized with TSMTP of 32 and 28 kDa did not develop resistance to the challenge infection. Likewise, vaccination of buffaloes with leucine aminopeptidase and PRX evoked vigorous antibody responses to the immunogens yet failed to elicit protection against F. gigantica challenge infection (Raina et al., 2011). Additionally, goats immunized with F. hepatica recombinant cathepsin L, PRX, and S. mansoni FABP (Sm14) alone or in combination together with Quil A adjuvant developed high levels of antibodies specific to the immunogens but failed to resist challenge infection with 200 metacercariae (Buffoni et al., 2012). Conversely, induction of antibodies able to access and interact with the surface membrane antigens of juvenile flukes could correlate with protective immunity. In this regard, TMSTP of 60 kDa evoked antibodies that strongly bound to the surface of larvae in IF, and mediated the highest level of in vitro antibody-dependent complement- and cell-mediated killing of newly excysted flukes as compared to the 32 and 28 kDa species, that was associated with 13
significant protection against challenge infection. Accordingly, it is recommended to examine whether the ability of vaccine-generated antibodies to recognize the surface membrane antigens of juvenile flukes is a prognostic marker of immune protection. The TSMTP of 32 and 28 kDa failed to protect sheep against oral infection with 150 metacercariae of F. gigantica, suggesting they might be enriched in those molecules, such as cathepsin B, cathepsin L, and PRX, which are able to protect rodents, but not ruminant, against fasciolosis (El Ridi et al., 2007; Jayaraj et al., 2009; Mendes et al., 2010; Raina et al., 2011; Changklungmoa et al., 2013; Zafra et al., 2013; Kueakhai et al., 2015; Meemon and Sobhon, 2015; Sansri et al., 2015). Significant (P < 0.05) protection of approximately 40% was only obtained with the 60 kDa fraction, which was shown be enriched with a protein showing 96-100% homology with F. hepatica enolase. Enolase of F. hepatica has an apparent Mr of 47 kDa, has been identified in the excretory-secretory products (Bernal et al., 2004; Morphew et al., 2007) and tegument (Morales and Espino, 2012) of adult worms, and shows about 70% amino acid sequence homology with sheep, cow, buffalo, and goat enzyme. No reports are available regarding F. gigantica enolase. Therefore, we proceeded to cloning and sequencing of F. gigantica enolase, which revealed 92% and 95% identity with F. hepatica enolase nucleotide and amino acid sequences, respectively (Davis et al., 1994). Homology between cDNA sequences of F. gigantica and F. hepatica cathepsin L fragments was found to reach 87% to 99% (Grams et al., 2001). The 70 kDa heat-shock proteins of F. hepatica and F. gigantica nucleotide and protein sequences were found to be 98% and 99% identical, respectively (Smith et al., 2008). Gene coding for leucine aminopeptidase in F. gigantica also showed a close homology (98.6%) with leucine aminopeptidase of F. hepatica (Raina et al., 2011).
14
Enolase is localized in the surface membrane of numerous pathogens including Fasciola (Bernal et al., 2004; Morales and Espino, 2012), and may, thus, be targeted by major immune effector mechanisms such as antibody-dependent, complement- or cellmediated cytotoxicity. Additionally, enolase is a prominent moonlighting (multifunctional) protein that participates in numerous non-glycolytic functions (Jung et al. 2014; Paludo et al., 2015). Enolase role as plasminogen-binding protein has been documented (Bernal et al., 2004). Moonlighting proteins are promising anti-parasite candidate vaccines as they would induce interference with a plethora of pathogen physiological functions. In conclusion, enolase has been identified as a major component of worm tegumental fraction protective against sheep fasciolosis, whose potential use as therapeutic target should be investigated in the future. Acknowledgment. The study was supported, in part, by the Academy of Scientific Research and Technology, Science and Technology Centre, Cairo, Egypt; Grant No. 20 to R. El Ridi. The Funding Agency has no role in the design of the study; in collection, analysis and interpretation of the data; in writing the report; or in the decision to submit the article for publication.
References Bernal, D., de la Rubia, J.E., Carrasco-Abad, A.M., Toledo, R., Mas-Coma, S., Marcilla, A., 2004. Identification of enolase as a plasminogen-binding protein in excretory-secretory products of Fasciola hepatica. FEBS Lett. 563, 203-206. Buffoni, L., Martínez-Moreno, F.J., Zafra, R., Mendes, R.E., Pérez-Écija, A., Sekiya, M., Mulcahy, G., Pérez, J., Martínez-Moreno, A., 2012. Humoral immune response in goats immunised with cathepsin L1, peroxiredoxin and Sm14 antigen
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and experimentally challenged with Fasciola hepatica. Vet. Parasitol. 185, 315321. Changklungmoa, N., Kueakhai, P., Riengrojpitak, S., Chaithirayanon, K., Chaichanasak, P., Preyavichyapugdee, N., Chantree, P., Sansri, V., Itagaki, T., Sobhon, P., 2013. Immunization with recombinant leucine aminopeptidase showed protection against Fasciola gigantica in mice. Parasitol. Res. 112, 3653-3659. Chittum, H.S., Lane, W.S., Carlson, B.A., Roller, P.P., Lung, F.T., Lee, B.I., Hatfield, D.L., 1998. Rabbit β-globin is extended beyond its UGA stop codon by multiple suppressions and translational reading gaps. Biochemistry 37, 10866-1087 Davis, R.E., Singh, H., Botka, C., Hardwick, C., Ashraf el Meanawy, M., Villanueva, J., 1994. RNA trans-splicing in Fasciola hepatica. Identification of a spliced leader (SL) RNA and SL sequences on mRNAs. J. Biol. Chem. 269, 2002620030. Dixon, K.E., 1966. The physiology of excystment of the metacercaria of Fasciola hepatica L. Parasitology 56, 431-456. El Ridi, R., 2001. Report to the Programs of the National Strategy for Biotechnology and Genetic Engineering, Ministry of Scientific Research, Academy of Scientific Research and Technology, Science and Technology Centre, Cairo, Egypt. El Ridi, R., Salah, M., Wagih, A., William, H., Tallima, H., El Shafie M.H., Abdel Khalek, T., El Amir, A., Abo Ammou, F.F., Motawi, H., 2007. Fasciola gigantica excretory-secretory products for immunodiagnosis and prevention of sheep fasciolosis. Vet. Parasitol. 149, 219-228. Eng, J.K., McCormack, A.L, Yates, J.R., 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass. Spectrom. 5, 976-989.
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Estuningsih, S.E., Smooker, P.M., Wiedosari, E., Widjajanti, S., Vaiano, S., Partoutomo, S., Spithill. T.W., 1997. Evaluation of antigens of Fasciola gigantica as vaccines against tropical fasciolosis in cattle. Int. J. Parasitol. 27, 1419-1428. Grams, R., Vichasri-Grams, S., Sobhon, P., Upatham, E.S., Viyanant, V., 2001. Molecular cloning and characterization of cathepsin L encoding genes from Fasciola gigantica. Parasitol. Int. 50, 105-114. Hanna, R.E., McMahon, C., Ellison, S., Edgar, H.W., Kajugu. P.E., Gordon. A., Irwin, D., Barley. J.P., Malone, F.E., Brennan, G.P., Fairweather. I., 2015. Fasciola hepatica: a comparative survey of adult fluke resistance to triclabendazole, nitroxynil and closantel on selected upland and lowland sheep farms in Northern Ireland using faecal egg counting, coproantigen ELISA testing and fluke histology. Vet. Parasitol. 207, 34-43. Jayaraj, R., Piedrafita, D., Dynon, K., Grams, R., Spithill, T.W., Smooker, P.M., 2009. Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens. Vet. Parasitol. 160, 230-236. Jezek, J., El Ridi, R., Salah, M., Wagih, A., Aziz, H.W., Tallima, H., El Shafie, M.H., Khalek, T.A., Ammou, F.F., Strongylis, C., Moussis, V., Tsikaris, V., 2008. Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis. J. Peptide Sci. 90, 349357. Jung, D.W., Kim, W.H., Williams, D.R., 2014. Chemical genetics and its application to moonlighting in glycolytic enzymes. Biochem. Soc. Trans. 42, 1756-1761.
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Kueakhai, P., Changklungmoa, N., Chaichanasak, P., Jaikua,W., Itagaki, T., Sobhon, P., 2015. Vaccine potential of recombinant pro- and mature cathepsinL1 against fasciolosis gigantica in mice. Acta Trop. 150, 71-78. Kumar, N., Anju, V., Gaurav, N., Chandra, D., Samanta, S., Gupta, S.C., Adeppa, J., Raina, O.K., 2012. Vaccination of buffaloes with Fasciola gigantica recombinant glutathione S-transferase and fatty acid binding protein. Parasitol. Res. 110, 419426. Mas-Coma, S., 2005. Epidemiology of fascioliasis in human endemic areas. J. Helminthol. 79:207-216. Review. Mas-Coma, S., Valero, M,A,, Bargues, M,D., 2014. Fascioliasis. Adv. Exp. Med. Biol. 766, 77-114. McManus, D.P., Dalton, J.P., 2006. Vaccines against the zoonotic trematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica. Parasitology. 133 Suppl, S43-61. Review. Meemon, K,, Sobhon, P., 2015. Juvenile-specific cathepsin proteases in Fasciola spp.: their characteristics and vaccine efficacies. Parasitol. Res. 114, 2807-2813. Mendes, R.E., Pérez-Ecija, R.A., Zafra, R., Buffoni, L., Martínez-Moreno, A., Dalton, J.P., Mulcahy, G., Pérez, J., 2010. Evaluation of hepatic changes and local and systemic immune responses in goats immunized with recombinant Peroxiredoxin (Prx) and challenged with Fasciola hepatica. Vaccine 28, 2832-2840. Molina-Hernández, V., Mulcahy, G., Pérez, J., Martínez-Moreno, Á., Donnelly, S., O'Neill, S.M., Dalton, J.P., Cwiklinski, K., 2015. Fasciola hepatica vaccine: we may not be there yet but we're on the right road. Vet. Parasitol. 208, 101-111. Morphew, R.M., Wright H.A., LaCourse E.J., Woods, D.J., Brophy, P.M., 2007. Comparative proteomics of excretory-secretory proteins released by the liver fluke
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Fasciola hepatica in sheep host bile and during in vitro culture ex host. Mol. Cell. Proteomics 6, 963-972. Morales, A., Espino, A.M., 2012. Evaluation and characterization of Fasciola hepatica tegument protein extract for serodiagnosis of human fascioliasis. Clin. Vaccine Immunol. 19, 1870-1878. Nambi, P.A., Yadav, S.C., Raina, O.K., Sriveny, D., Saini, M., 2005. Vaccination of buffaloes with Fasciola gigantica recombinant fatty acid binding protein. Parasitol. Res. 97, 129-135. Paludo, G.P., Lorenzatto, K.R., Bonatto, D., Ferreira, H.B., 2015. Systems biology approach reveals possible evolutionarily conserved moonlighting functions for enolase. Comput. Biol. Chem. 58, 1-8. Paykari, H,, Dalimi, A,, Madani, R., 2002. Immunization of sheep against Fasciola gigantica with glutathione S-transferase. Vet. Parasitol. 105, 153-159. Raina, O.K., Naga,r G., Varghese, A., Prajitha, G., Alex, A., Maharana, B.R., Joshi, P., 2011. Lack of protective efficacy in buffaloes vaccinated with Fasciola gigantica leucine aminopeptidase and peroxiredoxin recombinant proteins. Acta Trop. 118, 217-222. Robinson, M.W., Dalton, J.P., 2009. Zoonotic helminth infections with particular emphasis on fasciolosis and other trematodiases. Philos. Trans. R .Soc. Lond. B Biol. Sci. 364, 2763-2776. Sansri, V., Meemon, K., Changklungmoa, N., Kueakhai, P., Chantree, P., Chaichanasak, P., Lorsuwannarat, N., Itagaki, T., Sobhon, P., 2015. Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L. Vaccine 33, 1596-1601.
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Smith, M.A., Clegg, J.A., 1981. Improved culture of Fasciola hepatica in vitro. Z. Parasitenkd. 66, 9-15. Smith, R.E., Spithill, T.W., Pike, R.N., Meeusen, E.N., Piedrafita, D., 2008. Fasciola hepatica and Fasciola gigantica: cloning and characterisation of 70 kDa heatshock proteins reveals variation in HSP70 gene expression between parasite species recovered from sheep. Exp. Parasitol. 118, 536-542. Tielens, A.G., Van der Meer, P., Van den Bergh, S.G., 1981. Fasciola hepatica: simple, large-scale, in vitro excystment of metacercariae and subsequent isolation of juvenile liver flukes. Exp. Parasitol. 51, 8-12. Toet, H., Piedrafita, D.M., Spithill, T.W., 2014. Liver fluke vaccines in ruminants: strategies, progress and future opportunities. Int. J. Parasitol. 44, 915-927. Wilson, R.A., Wright, J.M., de Castro-Borges, W., Parker-Manuel, S.J., Dowle, A.A., Ashton, P.D., Young, N.D., Gasser, R.B., Spithill, T.W., 2011. Exploring the Fasciola hepatica tegument proteome. Int. J. Parasitol. 41, 1347-1359. Zafra, R, Pérez-Écija, RA, Buffoni, L, Moreno, P, Bautista, MJ, Martínez-Moreno, A, Mulcahy, G, Dalton, JP, Pérez, J., 2013. Early and late peritoneal and hepatic changes in goats immunized with recombinant cathepsin L1 and infected with Fasciola hepatica. J. Comp. Pathol. 148, 373-384.
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Explanation of figures Fig. 1. Gel profile of adult F. gigantica TSMTP examined after SDS-PAGE (10% gel, non-reducing conditions) followed by Coomassie blue staining. On the left, migration position and Mr of SDS-PAGE protein standards (Bio-Rad). (A) Electroseparation of protein species of 2 preparations. The arrows show the position and approximate Mr of the bands selected because of protective potential against F. gigantica challenge infection in outbred mice. (B) Profile of eluted proteins. Arrows point to the fractions used for sheep immunization. Fig. 2. Sheep humoral responses. (A) Reactivity of sera from adjuvant control sheep ( and sheep immunized with 60 (
), 32 (
) and 28 (
),
) kDa proteins were tested against
TSMTP by ELISA. Each point represents mean absorbance of 4 sera individually tested and vertical bars denote the SD about the mean. (B) Sera from adjuvant control sheep, and sheep immunized with 60, 32 and 28 kDa species were diluted 1:500 and tested for reactivity against newly excysted juvenile flukes in IF. (C) Sera diluted 1:1000 were tested, on an individual sheep basis, for antibody-dependent complement-mediated cytotoxicity against F. gigantica excysted juveniles over 4 independent experiments. Each column represents mean percent killed larvae and the vertical bars denote the SD about the mean. (D) Sera diluted 1:200 were tested, on an individual sheep basis, for antibody21
dependent cell-mediated cytotoxicity against F. gigantica excysted juveniles using peritoneal cells from naïve BALB/c mice (white columns) or peripheral blood mononuclear cells from naïve, healthy, parasite-free sheep (greyish columns), over 2 independent experiments. Each column represents mean percent killed larvae and the vertical bars denote the SD about the mean. Star and asterisks indicate P values of respectively <0.0001 and <0.05, respectively as assessed by the Student's t- test. Fig. 3. Homology of F. gigantica 60 kDa-derived tryptic peptides with predicted amino acid sequence of F. hepatica (Fh) enolase (ACCESSION: AAA57450.1; Davis et al., 1994).
Fig. 4. (A) Homology between F. gigantica (Fg) and F. hepatica (Fh) enolase cDNA nucleotide sequence. (B) Homology between predicted amino acid sequences of F. gigantica (Fg) and F. hepatica (Fh) enolase. (C) Conserved domains in F. gigantica enolase (partial length).
22
A
B
Fig 1
Mean absorbance (405 nm)
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
A
B
500 1000 2000 4000 8000 16000 Reciprocal serum dilution
Control 60 32 28 Sera pooled from control or 60, 32, or 28 kDa protein‐immunized sheep Mean percent antibody‐ dependent cell‐mediated cytotoxicity
Mean percent antibody‐ dependent complement‐mediated cytotoxicity
D C 80 60 40 20 0
Sera from control or 60, 32, or 28 kDa protein‐immunized sheep
Fig. 2.
60 kDa SGETEDNFIADLVVGLR Fh enolase 379 SGETEDNFIADLVVGLR 389 60 kDa IQIVGDDLTVTNPLR Fh enolase 313 IQIVGDDLTVTNPLR 327 60 kDa AVANVNSQIAPNLIK Fh enolase 64 AVANVNSQIAPNLIK 78 60 kDa
AAVPSGAETGVHEALELR
35 30 25 20 15 10 5 0
Sera from control or 60, 32, or 28 kDa protein‐immunized sheep
Fh enolase 33 AAVPSGAETGVHEALELR 50 60 kDa YGLDACNVGDEGGFAPSIQDNLHGLELLR Fh enolase 199 YGLDACNVGDEGGFAPSIQDNLEGLELLR 227 60 kDa IGSEVYHNLR Fh enolase 183 IGSEVYHNLR 192 60 kDa EVIM*PVPSFNVINGGSHAGNK Fh enolase 141 EVIM PVPSFNVINGGSHAGNK 161 60 kDa SGINVTDQAAVDKFM*LDLDGTPNK Fh enolase 79 SGINVTDQAAVDKFM LDLDGTPNK 102 60 kDa IEEDLGGAAK Fh enolase 413 IEEDLGGAAK 422 60 kDa VNQIGSVSESIK Fh enolase 344 VNQIGSVSESIK 355
Fig. 3
Fig. 4A.
Fg
1
Fh
55
Fg
58
Fh
115
Fg
118
Fh
175
Fg 178
ACCGATGAAGATGATGTCACGACTGCGAA-GG-TTATTCCGTGCGG-NGTTCCAAGCGGT |||| ||||| |||||||||||| ||||| || ||||||||||||| |||||||||||| ACCGTTGAAGTTGATGTCACGACCGCGAAAGGTTTATTCCGTGCGGCAGTTCCAAGCGGT
57
GCTTCTACTGGTGTTCNNNAANCTNTGGAATTGCGNGATGGCCCTCCCGGCTATANGGGA ||||| |||||||||| || || |||||||||| ||||||||||||||||||| ||| GCTTCCACTGGTGTTCATGAAGCTCTGGAATTGCGTGATGGCCCTCCCGGCTATATGGGC
117
AAAGGTGTTCTGNAAGCGGNTGCAAACGTGANCAGCCAAATTGCTCCCNNCCTCATCAAA ||||||||||| |||||| ||||||||||| ||||||||| |||||| |||||||||| AAAGGTGTTCTAAAAGCGGTTGCAAACGTGAACAGCCAAATCGCTCCCAACCTCATCAAA
177
NGCGNAATAAATGTTACTGATCAAGCTGCGGTTGACAAATTCATGCTGGNCCTGGNCGGA ||| |||||||||||||||||||||||||||||||||||||||||| | ||||| |||| AGCGGAATAAATGTTACTGATCAAGCTGCGGTTGACAAATTCATGCTTGACCTGGACGGA
237
297
357
Fh
235
Fg
238
Fh
295
ACTCCCAACAAAGAAAAAACTCGGTGCGAAATGCAATCCTTNGGTGTGTCCCTCGCTGTC |||||||||||||||||| ||||||||| |||||||||||| |||||||| ||||||||| ACTCCCAACAAAGAAAAA-CTCGGTGCG-AATGCAATCCTT-GGTGTGTCTCTCGCTGTC
Fg
298
TGTAAAGCGGGTTGGCCGCTGAAGAAGGGTCTNCCCTTTGTACAAATACATCGCCACTCT
114
174
234
294
351
Fh
352
Fg
358
Fh
408
Fg
418
Fh
465
Fg
478
Fh
522
Fg
538
Fh
580
Fg
598
Fh
638
Fg
658
Fh
696
Fg
718
Fh
755
Fg
778
Fh
815
Fg
838
Fh
875
Fg
898
Fh
934
Fg
958
Fh
991
Fg
1018
Fh
1051
Fg
1078
Fh
1111
Fg
1138
Fh
1171
Fg
1198
Fh
1229
|||||||||||| ||||||||| |||||||| |||||||||||| |||||||||||||| TGTAAAGCGGGT--GCCGCTGAA-AAGGGTCT-CCCTTTGTACAAGTACATCGCCACTCT
407
TGCGGGAAATAAAGAAGGTTATAATGCCCGGGTGCCGTCTTTCAACGTCATCAATGGCGG ||||||||||||||| |||||||||| || |||||||||||||||||||||||||||||| TGCGGGAAATAAAGA-GGTTATAATG-CC-GGTGCCGTCTTTCAACGTCATCAATGGCGG
417
TAGTCACGCTTGGGAAACAAACTAGCGATGCAGGAGTTTTATGATAATGCCAACGGGTGC |||||||||| |||||||||||||||||||||||| ||||||||||||||||||||||| TAGTCACGCT--GGAAACAAACTAGCGATGCAGGAG-TTTATGATAATGCCAACGGGTGC
477
TAGTTCTNTTCACTGAAGCTATGGAAGATCGGAAGTGAGGTTTACCATAACCTCAGAGCT ||||||| |||||||||||||| ||||||||||||||||||||||||||||||||||||| TAGTTCT-TTCACTGAAGCTAT-GAAGATCGGAAGTGAGGTTTACCATAACCTCAGAGCT
537
GTTATCAAAAGCAAGTACGGTTTGGACGCCTGCAATGTGGGTGACGAGGGTGGTTTCNCC |||||||||||||||||||||||||||||||||||||||||||||||||||||||| | GTTATCAAAAGCAAGTACGGTTTGGACGCCTGCAATGTGGGTGACGAGGGTGGTTT--CG
597
CTCCAAGTATTCAAGATAATTTGGAAAAGGACTTGAGCTGCTTCGTACCGCAATAGACAA |||||||||||||||||||||||| |||||||||||||||||||||||||||||||||| CTCCAAGTATTCAAGATAATTTGG--AAGGACTTGAGCTGCTTCGTACCGCAATAGACAA
657
AGCTGGATATACGGGAAAAGTCAAGATTGCCATGGATTGCGCTGCCTCGGGAATTTTACA ||||||||||||||||||||||||||||||||||||||||||||| || ||||||||||| AGCTGGATATACGGGAAAAGTCAAGATTGCCATGGATTGCGCTGCTTC-GGAATTTTACA
717
AGGAAGGGAAATACGATTTGGATTTCAAAAACCCCAAGTCTCAGGCCAGTTCTTGGATCA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AGGAAGGGAAATACGATTTGGATTTCAAAAACCCCAAGTCTCAGGCCAGTTCTTGGATCA
777
CTTCGGATGCCATGGCTGATGTCTACAAGAAAATGATGTCTACTTACCCGATCGTTAGCA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTTCGGATGCCATGGCTGATGTCTACAAGAAAATGATGTCTACTTACCCGATCGTTAGCA
837
TCGAGGACCCGTTTCGATCAAGACGATTGGCCGGCTTGGACTAAACTGACGGGCGAATGT ||||||||||| ||||| |||||||||||||| |||||||||||||||||||| |||||| TCGAGGACCCG-TTCGACCAAGACGATTGGCCAGCTTGGACTAAACTGACGGGTGAATGT
897
AAAATCCAGATCGTTGGTGATGACTTAANCTGTCACCAANCCCCTCTTCGTGTGCAAAAA |||||||||||||||||||||||||||| |||||||||| |||| ||||||||||||||| AAAATCCAGATCGTTGGTGATGACTTAA-CTGTCACCAA-CCCC-CTTCGTGTGCAAAAA
957
GCTATCGACCAAAAGGCATGCCATTGCCTGTTGCTAAAAGTCAACCAGATCGGTTCGGGG ||||||||||||||||||||| |||||||||||||||||||||||||||| ||||||| | GCTATCGACCAAAAGGCATGCAATTGCCTGTTGCTAAAAGTCAACCAGATTGGTTCGGTG
1017
TCANANTCCATTNAGGCTTGCAAGATGGCCCAGGAAGCCGGTTGGGGTGTGATGGTATCA ||| | |||||| ||||||||||||||||||| ||||| ||||||||||||||||||||| TCAGAGTCCATTAAGGCTTGCAAGATGGCCCAAGAAGCTGGTTGGGGTGTGATGGTATCA
1077
464
521
579
637
695
754
814
874
933
990
1050
1110
CATCGTNCAGGATANACCGAAGATAANTTCATCGCTGATTTNGTGGNNGGNCTNNGNANC |||||| ||||| | || |||||||| |||||||||||||| |||| || || | | CATCGTTCAGGAGAAACTGAAGATAACTTCATCGCTGATTTGGTGGTTGGACTGCGTACT
1137
GGCCNGATTAAAACAGGTNCNCCTTGTCGTTCAGANCNTCTTNCGAAATACGAACCGAGT |||| ||||||||||||| | |||||||||||||| | |||| |||||||| |||| ||| GGCCAGATTAAAACAGGTGCACCTTGTCGTTCAGAACGTCTTGCGAAATAC-AACC-AGT
1197
TGCNGCGTNTTNAAG ||| |||| || ||| TGCTGCGTATTGAAG
1170
1228
1212 1243
Fig. 4A.
Fg
2
EDDVTTAX-VIPCGVPSGASTGVHEALELRDGPPGYMGKGVLKAVANVNSQIAPNLIKSG
60
Fh
21
Fg
61
Fh
81
Fg
121
Fh
141
Fg
181
Fh
201
E DVTTA + VPSGASTGVHEALELRDGPPGYMGKGVLKAVANVNSQIAPNLIKSG EVDVTTAKGLFRAAVPSGASTGVHEALELRDGPPGYMGKGVLKAVANVNSQIAPNLIKSG
80
INVTDQAAVDKFMLDLDGTPNKEKLGANAILGVSLAVCKAGAAEKGLPLYKYIATLAGNK INVTDQAAVDKFMLDLDGTPNKEKLGANAILGVSLA CKAGAAEKGLPLYKYIATLAGNK INVTDQAAVDKFMLDLDGTPNKEKLGANAILGVSLAXCKAGAAEKGLPLYKYIATLAGNK
120
EVIMPVPSFNVINGGSHAGNKLAMQEFMIMPTGASSFTEAMKIGSEVYHNLRAVIKSKYG EVIMPVPSFNVINGGSHAGNKLAMQEFMIMPTGASSFTEAMKIGSEVYHNLRAVIKSKYG EVIMPVPSFNVINGGSHAGNKLAMQEFMIMPTGASSFTEAMKIGSEVYHNLRAVIKSKYG
180
LDACNVGDEGGF LDACNVGDEGGF LDACNVGDEGGF
140
200
192 212
Fig. 4B.
Fig. 4C.
Table 1 Effect of sheep vaccination with TSMTP species of 60, 32, and 28 kDa on protection against challenge Fasciola gigantica. _____________________________________________________________________ Fluke burden P value a
Mean + SD
Percent reduction b
Sheep group _____________________________________________________________________ Adjuvant controls
74.5 + 16.6
60 kDa
43.7 + 10.6
0.020
32 kDa
64.0 + 7.8
NS
28 kDa
60.2 + 12.8
NS
41.3
_____________________________________________________________________ Sheep (4 per group) administered with immunogen-free adjuvant (adjuvant controls) or adjuvanted TSMTP species of 60, 32, or 28 kDa were orally challenged 5 weeks after the last, adjuvant-free injection with 150 viable F. gigantica metacercariae, and assessed for fluke burden 12 weeks later. a
Two-tailed Student's t test.
23
b
Percentage protection was calculated as C – T/C x 100 where C = mean worm count
in adjuvant control sheep and T = mean worm count in vaccinated sheep. Table 2 Fractionation of TSMTP of 60 kDa by amino acid microsequencing. _____________________________________________________________________ Peptides identity
Number
percentage
_____________________________________________________________________ Enolase (gi|3023708)
10
32.2
Actin (gi|113230)
8
25.8
Actin (gi|3328)
3
9.6
Tubulin beta chain (gi|109431)
6
19.3
Alpha tubulin (gi|1223784)
2
6.4
Thiol-specific anti-oxidant protein (gi|3549894) 2
6.4
_____________________________________________________________________
24