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(The FASEB Journal. 2007;21:823-835.)
© 2007 FASEB

Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases

Jennifer M. Fitzpatrick*, Yuriko Hirai{dagger}, Hirohisa Hirai{dagger} and Karl F. Hoffmann*,1

* Department of Pathology, University of Cambridge, Cambridge, UK; and

{dagger} Primate Research Institute, Kyoto University, Inuyama, Aichi, Japan

1Correspondence: Department of Pathology, University of Cambridge, Tennis Court Rd., CB2 1QP, UK. E-mail: kfh24{at}cam.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Egg production is responsible for life cycle progression and host immunopathology during schistosomiasis, with the associated parasite molecules being investigated as potential novel chemotherapeutic targets. Here, we characterize two Schistosoma mansoni products, tyrosinase 1 and tyrosinase 2 (SmTYR1/SmTYR2) and show that their diphenol oxidase enzyme activities are critical for eggshell formation and production. The genes encoding these bifunctional enzymes (monophenol and diphenol oxidases) result from a duplication event that likely occurred before speciation and exist in the parasite’s genome as multiple copies, which are linked and localized to chromosomes 4 and W. SmTYR1/SmTYR2 transcription and diphenol oxidase action are developmentally regulated with most enzyme activity localized to the eggshell–producing cells contained within the vitellaria of adult female worms. Importantly, kojic-acid mediated inhibition (IC50=0.5µM) of SmTYR1/SmTYR2’s diphenol oxidase activity during in vitro culture of sexually mature adult worms resulted in a significant decrease in the production of phenotypically normal eggs. Therefore our data suggest that SmTYR1/2 inhibition represents a novel and potentially effective strategy for combating schistosomiasis and furthermore, it may point to new methods for combinatorial control of immunopathology and egg transmission during platyhelminth infection.—Fitzpatrick J. M., Hirai Y., Hirai H., Hoffmann K. F. Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases.


Key Words: helminth • schistosoma • kojic acid • developmental regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SCHISTOSOMIASIS REMAINS A MAJOR disease of the developing world and re-evaluation of disability-linked morbidity has suggested that the World Health Organization (WHO) estimation of 0.5% disability should be raised to 2–15% as a more accurate reflection of disease burden (1) . This suggests that past and present control strategies have done little to lessen the burden associated with schistosomiasis, where current estimates indicate that 200 million human infections exist leading to chronic debilitating disease and up to 300,000 deaths per year (2) . Praziquantel remains the drug of choice to treat schistosomiasis (3) , although it does not prevent reinfection (especially in children (4) ), is minimally effective against larval stages of the parasite (5) , resistance can develop (6) , and its mechanism of action is currently unknown (7) . The search for new drug targets is therefore an important priority in developing novel strategies to combat this major pathogen of humankind (8) . As eggs produced by sexually mature, adult female worms precipitate most lethal forms of host immunopathology during experimental schistosomiasis (9) , we have focused our search for novel chemotherapeutic targets on the molecules involved in their production. Toward this end, we recently completed a series of related studies using Schistosoma mansoni and S. japonicum specific DNA microarrays to identify gender-enriched gene transcripts associated with sexually mature stages of these trematode species (10 11 12) . Here, we extend our DNA microarray findings by characterizing two female-associated transcripts sharing high-sequence similarity to the bifunctional enzyme tyrosinase (EC 1.14.18.1) and provide evidence that these enzymes are suitable novel chemotherapeutic targets.

Tyrosinases are copper-containing glycoenzymes which catalyze the hydroxylation of monophenols to o-diphenols (monophenol oxidase activity EC 1.14.18.1) and the oxidation of those o-diphenols to o-quinones (diphenol oxidase activity EC 1.10.3.1) (13) . These enzymes are widely distributed in nature, occurring in most phylogenetic taxa, ranging from bacteria to humans, in which they participate in diverse functional capacities, including melanin synthesis, light adaptation, wound healing, melanocytic encapsulation, immune responses, and protein cross-linking. As protein cross-linking is thought to be essential for helminth eggshell sclerotization (hardening) (14) , identifying the molecular components that are instrumental in such a fundamentally important biological process will be critical in developing new strategies that target the machinery used by schistosomes to produce pathogenic eggs.

We demonstrate here that the two S. mansoni tyrosinase orthologs, SmTYR1 and SmTYR2, are the products of two linked genes arising from a duplication event prior to speciation, are mapped to chromosomes 4 (autosome) and W (female-specific chromosome) and are each represented by at least two copies in the parasites genome. The transcripts for each gene are developmentally regulated with peak transcription occurring in sexually mature, egg-laying female worms. Tyrosinase diphenol oxidase activity, measured across the parasites life cycle, also strongly correlated with SmTYR1 and SmTYR2’s transcriptional developmental expression and was found highest in adult female extracts. Importantly, this enzymatic activity was specifically localized to cells within the vitellaria, an organ that produces the eggshell precursors (15) and is the major repository for the tyrosinase substrate tyrosine (14) . Kojic acid-mediated inhibition of schistosome tyrosinase diphenol oxidase activity during in vitro culturing of mated parasites significantly decreased the production of phenotypically normal eggs, and further microscopic examination of these abnormal eggs revealed the presence of severe morphological defects in the eggshell. These data strongly suggest that tyrosinase diphenol oxidase inhibition represents a potentially novel and feasible therapeutic approach to limit parasite transmission, as well as egg-induced pathology during schistosomiasis and furthermore, defines SmTYR1 and SmTYR2 as important molecular targets for rational drug design.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Parasites and antigen preparations
A Puerto Rican strain of S. mansoni was used in this study. All procedures that were performed on mice in these studies adhered to the United Kingdom Home Office Animals Scientific Procedures Act of 1986. Adult worms were perfused from percutaneously infected TO (Tuck Ordinary) mice (Harlan, Bicester, UK) challenged 3-, 4-, 5-, 6-, or 7-wk earlier with 125 cercariae (16) . Cercariae were shed from Biomphalaria glabrata intermediate snail hosts. Miracidia were hatched from S. mansoni eggs harvested from 7-wk infected mouse livers. Parasite material (miracidia, cercariae, 3-wk mixed-sex worms, 7-wk male worms and 7-wk female worms) used for diphenol oxidase measurements were solubilized in a buffer containing 0.1% Nonidet P-40/0.1 M KH2PO4 (6.7) and quantified using the bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL).

Semiquantitative and quantitative reverse transcription polymerase chain reaction analysis
Mixed-sex miracidia, cercariae, 3-wk, 4-wk, 5-wk, 6-wk, and 7-wk worms (as well as gender-separated, mature adult male and female worms) were homogenized in TRIZOL reagent (Invitrogen, Paisley, UK) using a tissue dispersing tool (IKA Labortechnik, Stauffen, Germany) and total RNA was isolated as recommended by the manufacturer. All RNA was treated with DNase I (Ambion, Huntingdon, UK) before reverse transcription (RT) to remove any potential genomic DNA contamination. RT was performed using 1 µg of parasite total RNA and oligo-dT primers as described previously (12) . The semiquantitative RT-polymerase chain reaction (RT-PCR) primers for SmTYR1, SmTYR2, and alpha-tubulin (SmAT1, M80214) were synthesized at Sigma-Genosys (Poole, UK) and are listed in Table 1 . Twenty-six cycles of PCR were used to amplify the SmTYR1 and SmAT1 fragments, whereas 33 cycles were used to amplify the SmTYR2 fragment. All amplicons were electrophoresed on a 2% agarose gel and stained with ethidium bromide. Images were captured by a digital camera and analyzed by gel documentation software (Kodak 1D 2.0 electrophoresis documentation and analysis system 120, Eastman Kodak Co., New Haven, CT). Amplification of SmAT1 served as an internal control for the amount of RNA and cDNA from each sample.


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  		Table 1. Oligonucleotide primers used for studies of SmTYR1 and SmTYR2 gene expression, genomic organization, and chromosomal copy number

The quantitative RT-PCR primers for SmTYR1, SmTYR2, and SmAT1 (Sigma-Genosys, UK) are also listed in Table 1 . PCR was performed using a MiniOpticon real-time PCR thermal cycler system (Bio-Rad, Hertfordshire, UK) and SYBR Green I, according to the manufacturer’s instructions. Briefly, real-time PCR parameters included 40 cycles, fluorescent reading after each cycle and melt curve analysis of individual products at the end of the 40 cycles. Relative expression between two genes is equal to 2–{Delta}Ct, where SmAT1 is used as the reference gene. Assuming equal (perfect doubling) efficiency between reactions, the threshold cycle (CT) is the point at which fluorescent signal intensities surpass background levels and begin to increase exponentially.

SmTYR1 and SmTYR2 cDNA cloning
Using gene-specific oligonucleotide primers, the 5' and 3' cDNA sequences encoding for the N- and C-terminal domains of SmTYR1 and SmTYR2 were isolated by 5' RACE and 3' RACE strategies (GeneRacer Kit, Invitrogen, UK) from adult female RNA. Sequencing of the cDNAs encoding SmTYR1 and SmTYR2 was performed at the Department of Genetics, University of Cambridge in both orientations using Big Dye v3.1 fluorescent chemistry and an Applied Biosystems 3100 Genetic Analyser.

Sequence analysis, multiple sequence alignment, and phylogeny
Sequence analysis was performed using DNASTAR software (Lasergene, Madison, WI, USA), SignalP (http://www.cbs.dtu.dk/services/SignalP/) (17) and TopPred (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html) (18) . Multiple-sequence alignment of SmTYR1 and SmTYR2 was performed using the ClustalW program maintained at EMBL-EBI (http://www.ebi.ac.uk/clustalw/index.html). Phylogenetic analysis was performed using only the most conserved amino acid residues (selected using default parameters in gBlocks; (19) ver. 0.91b) contained within the PFAM (http://www.sanger.ac.uk/Software/Pfam/) tyrosinase domain (PF00264) of each sequence, resulting in a total of 38 amino acid residues for analysis. Data were analyzed via Bayesian inference using MrBayes (20) (ver. 3.1.1) and the WAG protein substitution model (21) . 1.5 x 106 generations were run using default values for prior parameterization, saving 1 tree every 100 generations. A plot of the tree posterior-probabilities vs. generation was used to examine the point at which parameter values stabilized and trees saved before this point were discarded as the "burnin". A consensus of the remaining trees was created using the "sumt" command (burnin=3750, contyp=allcompat) and visualized using TreeView (22) .

Gene structure, copy number, linkage and chromosomal localization of SmTYR1 and SmTYR2
SmTYR1 and SmTYR2 gene structure was predicted by performing BLASTn analyses of full-length mRNA sequences against those sequence verified genomic entries contained at the Wellcome Trust Sanger Institute housed on the S. mansoni OmniBlast server (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/s_mansoni/omni). Noncoding 5' and 3' DNA elements, intron sequences and intron/exon boundaries (defined as GT and AG dinucleotides at either end of intron sequence) for both SmTYR genes were manually resolved via this approach and further elucidated using NCBI’s Spidey (http://www.ncbi.nlm.nih.gov/spidey/). S. mansoni genomic DNA (gDNA) was prepared from cercariae using a commercially available kit (Qiagen Dneasy tissue kit). Southern blot hybridization and fluorescent in situ hybridization (FISH) techniques were utilized to identify copy number and chromosomal localization of each SmTYR gene. SmTYR1/2 PCR fragments (oligonucleotide sequences and product size, Table 1 ) for use in Southern hybridization experiments (23) were generated from female cDNA templates and labeled with alkaline phosphatase (Amersham Biosciences, UK). Hybridization of SmTYR1/2 probes to BamHI or EcoRI digested gDNA (10 µg) and detection of signal was performed following the manufacturer’s instructions (Gene Images AlkPhos Direct Labeling and Detection System). To investigate SmTYR1 and SmTYR2 linkage, PCR amplification (oligonucleotide sequences and product size, Table 1 ) of SmTYR1 exon 1, SmTYR1 exon 3, SmTYR2 exon 1, and SmTYR2 exon 3 using 150 ng BAC clone CH103–3D13 (obtained from the Children’s Hospital Oakland Research Institute, USA) was also performed following standard methodologies. To identify chromosomal localization of SmTYR1 and SmTYR2 by FISH, a biotinylated probe was made from the BAC clone CH103–3D13. This probe was hybridized to S. mansoni chromosomes using established procedures (24) .

Tyrosinase assay
Diphenol oxidase activity was determined from parasite extracts, as well as from frozen adult worms, as described previously (12) using the modified MBTH (3-methyl-2-benzothiazoline hydrazone hydrochlroide hydrate) assay (25) . The copper chelator DETC (diethyldithiocarbamic acid) was used in parallel reactions as a generalized diphenol oxidase inhibitor.

IC50 determination
The concentration of kojic acid (KA) required to effectively inhibit 50% of schistosome diphenol oxidase activity (IC50) was determined, as described previously (26) . Increasing concentrations ([X], 0–50 µM) of kojic acid were added to constant amounts of solubilized female proteins (150 µg). Diphenol oxidase activity was measured (25) in duplicate for each inhibitor concentration used. Percent diphenol oxidase inhibition was calculated from the following formula: (Abs505 nm 0 µM KA- Abs505nm [X] µM KA)/Abs505nm 0 µM KA) x 100. Concentration of kojic acid that inhibited 50% of maximal schistosome diphenol oxidase was derived from best fit on the curve.

In vitro inhibition studies
Worms were perfused from TO mice infected 7 wk previously and cultured at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, Surrey, UK) supplemented with 10% FCS, 2 mM L-glutamine, and 100 µg/ml pen/strep for up to 48 h in an atmosphere of 5% CO2. Ten male/female worm pairs were cultured per milliliter of media in 24-well tissue culture plates, and the media were changed every 24 h. For worm pairs treated with a tyrosinase inhibitor, the culture media were supplemented with 1 mg/ml (7 µM) kojic acid (dissolved in 25% PEH; 12.5% propylene glycol, 7.5% ethanol and 5% water) (27) . Eggs laid in culture were collected after 48 h, examined/counted using a sedgewick rafter, and subjected to laser confocal microscopy (Leica TCS-SP1 confocal microscope) and scanning electron microscopy (SEM) (Philips XL30-FEG scanning electron microscope). Eggs remaining in utero were also examined by confocal microscopy at the end of each experiment.

Confocal laser microscopy
Intact, fixed (10% saline-buffered formalin for 24 h) eggs and worms were viewed with a Leica TCS SP1 confocal microscope using a x63 water immersion lens with a 1.2 numerical aperture (NA). Autofluorescence was excited with a 488-nm line from an Argon laser, and emitted light was collected between 500 and 560 nm. Images were collected at a resolution of 1024 x 1024 pixels, and a series of images in z were displayed as maximum-intensity projections.

SEM
Intact eggs collected from worm cultures were fixed in 4% glutaraldehyde/4% paraformaldehyde for 24 h at 4°C, washed in PIPES buffer (pH 7.2) for 4 h at 4°C, incubated in 1% osmium tetroxide (1 h at 4°C), rewashed in PIPES buffer, and deposited on poly-L-lysine-coated coverslips before dehydration and critical point drying. After critical point drying, egg samples were mounted onto SEM stubs, sputter coated with gold/palladium and visualized with a Philips XL 30 FEG scanning electron microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
S. mansoni encodes structurally similar tyrosinase orthologs resulting from a recent gene duplication event
Full-length cloning of SmTYR1 and SmTYR2 from adult female cDNA identified highly related (37% identity and 59% similarity) open reading frames (ORFs) sharing many structural features common to the bifunctional metalloenzyme tyrosinase superfamily (Fig. 1 A). The ORFs of both SmTYR1 (481 amino acids, predicted molecular mass=56 kDa, pI=8.69) and SmTYR2 (478 amino acids, predicted molecular mass=56 kDa, pI= 8.49) contained predicted signal peptides (italicized regions; SmTYR1, M1-G23 and SmTYR2, M1-C21), presumably to aid intracellular transport through the secretory pathway. The two ORFs also contained three highly conserved cysteine-rich domains (gray-shaded boxes, CRD1-CRD3), which likely are necessary for correct secondary structure conformation mediated by disulfide bond formation. Another conserved feature shared between SmTYR1 and SmTYR2 is related to the position, amino acid sequence and organization of the two essential copper binding domains (open boxes, CuA and CuB). On the basis of crystallographic data for hemocyanin (28) and plant catechol oxidase (29) , the six histidine residues contained in these conserved domains (three in CuA and three in CuB) coordinate the placement of two copper ions and are critical for enzyme activity. The orthologous six His residues (flanked by highly conserved canonical sequence elements) likely participate in copper binding during SmTYR1 and SmTYR2 function. Other primary amino acid features of interest included one potential N-linked glycosylation motif found for SmTYR1 (N32-T34, underlined and shaded) and two for SmTYR2 (N30-T32 and N45-S47, underlined and shaded), as well as a predicted transmembrane domain in the C-terminal tail of SmTYR1 (underlined and italicized S427-P446).


Figure 1
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  		Figure 1. Schistosoma mansoni encodes two structurally similar tyrosinase orthologs, SmTYR1 and SmTYR2, which likely arose from gene duplication within the genus. A) Multiple-sequence alignment demonstrates that S. mansoni SmTYR1 and SmTYR2 are highly similar, contain three highly conserved cysteine-rich domains (Cys-rich domains 1–3; gray-boxed residues) and possess two canonical copper binding motifs (CuA and CuB; empty boxed residues). CLUSTALW was used to align the 481 AA SmTYR1 sequence with the 478 AA SmTYR2 sequence using default parameters where an asterisk (*) indicates identical residues, a colon (:) indicates conserved substitutions, and the period (.) indicates semiconserved residues. Underlined and italicized amino acid residues at the C-terminus of SmTYR1 indicate a predicted transmembrane domain. Underlined and shaded amino acid residues in the N terminus represent putative N-linked glycosylation sites. B) Genomic representation of SmTYR1 and SmTYR2. Numbers indicate the putative start and end of each gene, as well as nucleotide position for intron/exon boundaries. Conserved phase of introns 1 and 2 are also indicated. Sm- prefix numbers indicate annotated, publically available ESTs for either SmTYR1 or SmTYR2 found in S. mansoni gene DB (http://www.genedb.org/genedb/smansoni/index.jsp). C) Phylogenetic (and not phenetic) analysis of SmTYR1 and SmTYR2 inferred by MrBayes 3.1.1 and illustrated by Treeview (unrooted tree) as described in Materials and Methods. Numbers along nodes are Bayesian posterior probability values and indicate a high degree of support for the inferred tree. GenBank accession numbers are given for each sequence used in the analysis.

The predicted genomic structure of SmTYR1 and SmTYR2 differed in overall size (~17 Kbp for SmTYR1 and ~29 Kbp for SmTYR2) but showed very similar organization (three exons and two introns) (Fig. 1B ). All three CRDs of SmTYR1/2 are contained within the first exon of each gene, and the copper binding domains are split between the first two exons (CuA – exon 1; CuB – exon 2). Conservation of intron phase is observed between both genes as well (intron 1 of both SmTYR1/SmTYR2 is phase 1; intron 2 of both SmTYR1/SmTYR2 is phase 0), and along with all other common structural features discussed above, indicates a common SmTYR ancestor. Phylogenetic analysis of SmTYR1 and SmTYR2 using a Bayesian approach (20) further suggested that these two genes likely arose from a common ancestral gene by a duplication event prior to the divergence of the Schistosoma species (Fig. 1C ). The derived tree illustrated that both S. mansoni and S. japonicum encode two forms oftyrosinase and the interspecies (SmTYR1 and SjTYR1; SmTYR2 and SjTYR2) orthologs are more similar to the intraspecies (SmTYR1 and SmTYR2; SjTYR1 and SjTYR2) paralogs.

SmTYR1 and SmTYR2 are encoded by at least two genomic copies each and are spatially linked within the genome on chromosomes 4 and W
Southern blot and FISH (fluorescent ISH) were used to investigate gene structure and chromosomal localization of SmTYR1/SmTYR2. Southern blot analysis (Fig. 2 A) demonstrated identical banding patterns for both SmTYR1 and SmTYR2 when genomic DNA was digested with either EcoRI (two bands) or BamHI (two/three bands) and probed with gene specific (exon one, lacking both EcoRI and BamHI sites) PCR products. This indicated that each gene was represented by at least two (at most three) copies within the parasite’s genome and were closely linked. Use of real-time PCR and S. mansoni gDNA (Fig. 2B ) provided additional data supporting this genome copy number estimate for SmTYR1 and SmTYR2. Utilizing primers for a single copy gene (GAPDH, M92359), a gene that was represented by two copies within the genome (Cu/Zn superoxide dismutase (SOD), L12008) and exon three of both SmTYR1 and SmTYR2 revealed SmTYR1, SmTYR2, and Cu/Zn SOD products had Ct (cycle threshold) values consistently 1 or 2 cycles below that of GAPDH (Fig. 2B ). This indicated that SmTYR1 and SmTYR2 were represented in the schistosome’s genome by more than one copy and provided further support for the conclusions revealed by Southern blot analysis.


Figure 2
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  		Figure 2. SmTYR1 and SmTYR2 are encoded by multiple copy, genetically linked genes and are mapped to chromosomes 4 and W. A) Genomic DNA isolated from cercariae was digested with either BamHI or EcoRI and probed with SmTYR1 or SmTYR2 PCR products (spanning exon 1, lacking BamHI and EcoRI sites) during Southern blot hybridizations. B) SmTYR1 and SmTYR2 fragments (spanning exon 3) were PCR amplified from gDNA along with GAPDH (M92359, single copy gene) and Cu/Zn SOD (L12008, two copy gene) using a real-time MiniOpticon system. C) Genomic linkage of SmTYR1 and SmTYR2 was established by PCR amplification of exons 1 and 3 from a BAC clone CH103–3D13. Extracellular SOD (Ex-SOD, M27529) was not contained on this BAC clone and was only amplified from adult female worm cDNA. D) FISH analysis indicates positive signal (arrows) for CH103–3D13 on chromosomes 4 (autosome) and W (female sex chromosome). Scale Bar indicates 10 µM.

As Southern blot hybridization also suggested that SmTYR1 and SmTYR2 shared similar spatial organization within the parasite’s genome, additional evidence of linkage, utilizing BAC PCR amplification was sought. Here, a S. mansoni GeneDB (http://www.genedb.org/genedb/smansoni/index.jsp) verified SmTYR1 containing BAC clone (CHORI103–3D13) was used as a template for PCR amplification of exons 1 and 3 from SmTYR1, as well as exons 1 and 3 from SmTYR2 (Fig. 2C ). All four PCR reactions yielded a sequence and size-verified product, strongly suggesting that at least one copy of SmTYR1 and SmTYR2 are located on the same piece of chromosomal DNA and are closely linked within the genome (supporting the Southern blot results). Amplification of extracellular SOD (M27529) from this same BAC clone did not yield an amplicon and therefore, the SmTYR1 and SmTYR2 results are not due to experimental PCR artifacts. When CHORI103–3D13 was used as a probe for FISH analysis, positive staining was observed for chromosomes 4 and W (Fig. 2D ).

SmTYR1 and SmTYR2 transcription and diphenol oxidase activity are developmentally regulated in the sexually mature female worm with high enzyme activity found in eggshell-producing cells of the vitellaria
To determine when SmTYR1 and SmTYR2 were expressed throughout the life cycle of S. mansoni, we measured mRNA abundance by semiquantitative and quantitative RT-PCR, as well as enzymatic activity by a diphenol oxidase assay (Fig. 3 A–D). Semiquantitative RT-PCR results indicated that both transcripts were not appreciably expressed in mixed-sex miracidia (infective to the intermediate snail host), cercariae (infective to the definitive host) or 3-wk-old sexually immature adults (resident in definitive host) and were only observed in 7-wk-old sexually mature, adult female worms (Fig. 3A ). Further developmental expression studies, including life stages that span the timeframe associated with the initiation of egg production (~4- to 5-wk-old worms) were performed (Fig. 3B ). Here, the earliest expression of SmTYR1 and SmTYR2 was observed in mixed-sex 5-wk-old worms (presumably originating mostly from female RNA) with an up-regulation occurring in 6-wk-old worms. This increased rate of transcription was maintained in 7-wk-old worms. Quantitative RT-PCR analysis of SmTYR1 and SmTYR2 expression in sexually mature male and female worms indicated that both transcripts were ~4000x more abundant in females when compared to males (Fig. 3C ). Within the female worm, SmTYR1 is 8 x more abundant than SmTYR2 (Fig. 3C ).


Figure 3
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  		Figure 3. SmTYR1/SmTYR2 expression and diphenol oxidase activity are developmentally regulated with peak activity localized to the vitellaria of egg-laying mature female worms. A) Semiquantitative RT-PCR analysis of SmTYR1 and SmTYR2 transcription in mixed-sex parasite stages (M, miracidia; C, cercariae; 3-wk-old worms harvested from mice infected with S. mansoni for 3-wk-old, 7-wk-old worms harvested from mice infected with S. mansoni for 7 wk), as well as sex-separated adult male and female worms (7 wk). B) Additional semiquantitative RT-PCR analysis of SmTYR1 and SmTYR2 transcription in mixed-sex parasite life stages spanning the development of sexual maturity (3-wk-old worms through 7-wk-old worms). C) Quantitative RT-PCR analysis of SmTYR1 and SmTYR2 expression in adult male and female worms (collected at 7 wk). Amplification of SmAT1 served as a constitutively expressed gene during all PCR experiments. D) Determination of diphenol oxidase activity from parasite life stages used in (A). Bar graph depicts mean diphenol oxidase activity + SD measured in 40 µg of parasite extract from two independent experiments after correction for background absorbance at 505 nm (40 µg parasite extract+2 mM DETC). E) In situ diphenol oxidase activity localized within adult female worms. Fast Red B staining (first panel) indicates presence of concentrated phenolic tyrosine in the vitelline glands (VG) surrounding the worm gut. Diphenol oxidase activity (second panel) colocalizes to the same cells within the tyrosine-rich vitelline glands. DETC inhibits the specific diphenol oxidase-mediated staining (third panel). Scale Bar equals 0.5 mm.

Diphenol oxidase activity was measured across the parasite’s life cycle (Fig. 3D ) and found to generally agree with the SmTYR1/SmTYR2 transcriptional results (Fig. 3A ). Minimal activity was observed in extracts derived from mixed-sex miracidia, cercariae, 3-wk-old sexually immature worms and sexually mature male worms. An approximate 4x increase in diphenol oxidase activity was consistently observed in sexually mature adult female worms.

The localization of diphenol oxidase activity within whole frozen worms was examined by a modified in situ enzyme assay (25) (Fig. 3E ). Under these conditions and verifying previous observations in S. japonicum (12) , proteins containing diphenol oxidase activity were concentrated in female vitellaria surrounding the gut (easily identifiable by condensed, black hemozoin). Specific and concentrated diphenol oxidase staining was not observed in adult male worms (data not shown). The vitellaria also stained brightly with Fast Red B, a dye that specifically binds phenolic compounds (specifically the tyrosinase substrate tyrosine). This diphenol oxidase activity disappears from the vitellaria when worm samples were incubated with a specific copper chelator (diethyldithiocarbamic acid, DETC) during the enzyme assay.

Kojic acid inhibits S. mansoni diphenol oxidase activity in a dose-dependent manner and prevents the in vitro production of phenotypically normal eggs
As both SmTYR1 and SmTYR2 demonstrated developmentally regulated and female biased expression (Fig. 3) and contained sequence similarity to proteins involved in cross-linking (Fig. 1) , we investigated whether inhibition of these enzymes’ activity would lead to defects in phenotypically normal eggshell formation and production. The diphenol oxidase inhibitor utilized was kojic acid, a 5-hydroxy-2-hydroxymethyl-4-pyranone fungal metabolite (Beelik 1956) with potent and specific activity against melanocyte tyrosinase (Mishima Hatta Inazu 1988). During titration experiments, kojic acid inhibited diphenol oxidase activity in soluble adult female worm extracts dramatically (Fig. 4 A). The concentration of kojic acid sufficient to inhibit 50% of maximal schistosome diphenol oxidase activity (IC50), when L-DOPA was utilized as a substrate, was found to be ~0.5 µM.


Figure 4
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  		Figure 4. Kojic acid inhibits S. mansoni diphenol oxidase activity and significantly reduces phenotypically normal egg production during in vitro worm culture. A) Adult female worm extracts were generated as described in Materials and Methods. Increasing concentrations (0–50 µM) of kojic acid were added to constant amounts of solubilized female proteins (150 µg). Diphenol oxidase activity was measured and % inhibition and IC50 levels calculated as described in Materials and Methods. An asterisk denotes kojic acid concentration (7 µM) used during in vitro culture experiments. B) Ten worm pairs/well were cultured in the presence/absence of 7 µM of kojic acid (1 mg/ml) for 48 h. Eggs (oval with lateral spine) were counted from each well at the end of the experiment. Bar graph represents mean egg counts ± SD from two separate wells. Each experiment was performed at least twice.

When adult male and female worm pairs were cocultured in the presence of kojic acid (7 µM), a significant decrease in the number of phenotypically normal eggs laid was observed after 48 h (Fig. 4B ). Although the concentration of kojic acid used here was greater than the IC50 value on schistosome diphenol oxidase activity, there was no detrimental effect on worm viability or survival during the 48 h period (data not shown). Confocal microscopic examination of eggs collected from cultured worm pairs in control wells demonstrated a morphological normal appearance consisting of a fully formed lateral spine with continuous circumoval autofluorescence (Fig. 5 A, E). These eggs also appeared normal when examined in situ (inside the uterus, Fig. 5G ), suggesting that the culture conditions did not dramatically affect eggshell formation or production. This was in direct contrast to the phenotypes of eggs collected from worm pairs cultured in the presence of kojic acid (Fig. 5C , 5I , and 5K ). Here, confocal microscopic examination of "egg-like entities" revealed many kojic acid-mediated morphological defects, including an overall decrease in size of the egg (Fig. 5C ), irregularities/inconsistencies with circumoval autofluorescence (Fig. 5C ), a complete absence of autofluorescence (arrows, Fig. 5C ), pinching of the eggshell (Fig. 5I ), loss of the lateral spine and severe invaginations in the surface of the produced egg (Fig. 5K ). Some of these drug-mediated effects were also observed in utero (Fig. 5M ). Together, these data suggested that inhibition of schistosome diphenol oxidase activity had a profoundly negative impact on eggshell sclerotization during in vitro culture.


Figure 5
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  		Figure 5. Kojic acid inhibition of schistosome diphenol oxidase activity leads to morphological and structural defects in eggshell architecture as determined by laser confocal microscopy. Eggs and "egg-like entities" collected from in vitro cultured male/female schistosome worm pairs were processed for confocal microscopy, as described in Materials and Methods. A, B, E, F) Eggs collected from control wells (worms cultured without kojic acid). C, D, I–L) Eggs collected from treated wells (worms cultured with 7 µM kojic acid). White arrows in C) represent eggs (clearly seen in D) that completely lack spines and surface autofluorescence. G–H) In utero egg morphology in females cultured without kojic acid. M–N) In utero egg morphology in females cultured with kojic acid. A, C, E, G, I, K, and M represent confocal images, whereas B, D, F, H, J, and L represent phase contrast images. H and N represent confocal images superimposed on corresponding phase contrast images.

A more thorough examination of kojic acid-associated egg aberrations was performed by use of SEM (Fig. 6 ). In contrast to the smooth coat of microspines observed on the eggs collected from control wells (absence of kojic acid, Fig. 6A, B ), this structural coating was severely interrupted by clefts on eggs collected from worm pairs cultured in the presence of kojic acid (Fig. 6C, D ). In fact, these invaginations/clefts originally observed by confocal microscopy (Fig. 5K ) ultimately led to breaches in the integrity of the eggshell (Fig. 6E-G ). This resulted in leakage of the cellular contents of the egg (presumably vitelline cells based on size and appearance (30) , white boxed area in Fig. 6E-G ) into the surrounding culture media.


Figure 6
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  		Figure 6. Kojic acid inhibition of schistosome diphenol oxidase activity leads to invaginations and breaches in eggshell integrity as determined by scanning electron microscopy (SEM). Eggs and "egg-like entities" collected from in vitro cultured male/female schistosome worm pairs were processed for SEM, as described in Materials and Methods. A) Eggs collected from control wells (worms cultured without kojic acid) are phenotypically normal (oval with a lateral spine) and B) display evenly distributed surface microspines (enlarged area from white box (A)). C) Eggs collected from treated wells demonstrate severe and gross morphological defects. D) Enlargement of white box in (C) illustrates cleft formation and invaginations of eggshell material. E–G) Successive series of increasing magnification for one particular egg collected from a kojic acid-treated well. White box represents the subject of each successive image.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Most chemotherapeutic-based programs attempting to eradicate schistosomiasis in the developing world rely on the effectiveness of a single drug, praziquantel. Although this drug has favorable toxicological properties and is efficient at killing adult schistosomes and their eggs, it does not prevent reinfection/rebound morbidity (31) and has limited effect against larval stages of the parasite. In addition to praziquantel’s mechanism of action being poorly understood, the development of resistance is a concern and has been observed in the laboratory setting (5) . Therefore, as in other diseases that solely rely on a single drug for chemotherapeutic control, there exists an urgent need to identify new parasite targets and effective antischistosome compounds.

Our efforts in identifying novel chemotherapeutic targets have involved DNA microarray profiling of adult male and female worms with the goal of detecting those transcripts associated with the egg-laying machinery of sexually mature females. These studies identified several hundred, differentially expressed, gender-associated gene products (11) . Two of these products, SmTYR1 and SmTYR2, were found preferentially expressed in adult female worms compared to adult male worms, and this discovery was independent of the species or strain examined (12) . In the current study, we further characterized these two transcripts, believing they would have an important role in female egg-laying biology, and thus could serve as attractive novel chemotherapeutic targets.

On the basis of the proposed hardening mechanism (sclerotization) of schistosome eggshell formation, tyrosinase and the o-quinone products generated by its diphenol oxidase actvity are thought to be essential (14 , 32 33 34 35) . It has been hypothesized that the abundant tyrosine residues found among eggshell proteins (36) are converted to o-quinones by tyrosinase and these o-quinones subsequently serve as substrates for nucleophilic attack by neighboring lysine and histidine residues also contained in the same or neighboring eggshell proteins. It is thought that the resultant chain of events, additionally governed by optimal calcium and pH conditions (35) , leads to a series of crosslinks within a single protein and between neighboring protein molecules and ultimately leads to the highly rigid, hardened, schistosome eggshell. Our data greatly expand on these ideas and provide a link between tyrosinase gene expression and enzyme activity, with the formation of phenotypically normal eggs.

The major findings reported here are that SmTYR1 and SmTYR2 play critical (possibly overlapping) roles in S. mansoni egg biology, as neither transcript or diphenol oxidase activity is appreciably detected in either mixed-sex immature larval or mature male life-stages (Fig. 3A-D ) and kojic acid mediated diphenol oxidase inhibition results in severe egg-laying defects (Fig. 4) , as well as morphological abnormalities (Fig. 5 and 6) . Low SmTYR1/2 expression and diphenol oxidase activity (Fig. 3A-D ) observed in these life stages likely is related to the developmental regulation of each gene (Fig. 3 A,B ) and/or the localization and expression of one copy of each gene to chromosome 4 (Fig. 2C ). In this scenario, mixed-sex immature parasites have yet to receive the appropriate signals for maximal SmTYR1/2 expression, and adult male worms limit SmTYR1/2 expression to basal levels originating from chromosome 4. This is in contrast to adult female worms where appropriate environmental signals (currently unknown but initiated in 5-wk-old females, Fig. 3B ) lead to the high-level expression of SmTYR1/2 from both chromosome 4 and W (Fig. 2D ). Interestingly, this environmental signal is not related to the presence of adult male worms as demonstrated by a recent study exploring gene expression profiles between adult females obtained from single-sex cercariae infected mice (sexually immature due to the absence of males) to those adult females harvested from mixed-sex cercariae-infected mice (sexually mature) (37) . Although the expression of most vitellaria-localized transcripts was, not surprisingly (e.g., 38), linked to the developmental status of the female in this study, expression of SmTYR1 and SmTYR2 was not. This suggests that female expression of both SmTYR1 and SmTYR2 is regulated in a developmentally independent manner, once a certain point in the parasite’s life cycle is reached (at least 5-wk-old females, Fig. 3B ), and this serves to ready the female worm for the initiation of male-stimulated egg-laying behavior. Therefore, SmTYR1/SmTYR2 are always expressed at levels in the adult female worm sufficient to catalyze the sclerotization of newly formed eggshells, and this trait is surprisingly not dependent on male interaction. The reported discrepancy between gender-associated SmTYR transcript expression and diphenol oxidase activity (4000x more SmTYR transcript expressed and 4x more diphenol oxidase activity measured in adult females compared to adult males) observed here may simply be due to sensitivity differences of the employed assays or some, as of yet unknown, mechanism of transcript or enzyme regulation.

Why there are two distinct, but genetically linked, multiple-copy (2 or 3) tyrosinase genes present in the schistosome genome (Figs. 1 and 2) is presently unclear, although duplicated isoforms have been found in other organisms (39) , suggesting that this is not a unique phenomenon. It is clear, however, that SmTYR1 and SmTYR2 are bona fide members of the tyrosinase superfamily and not related to the highly similar tyrosinase-related proteins (TRP) because they, like all eukaryote tyrosinases, contain a HH amino acid pair at the C-terminal end of their CuB binding site (Fig. 1A ) (compared to LH for TRPs). As female schistosomes produce several hundred eggs every day (40) , they may need the cooperation of both SmTYR1 and SmTYR2 isoforms for the demanding biochemical task of sclerotization prior to oviposition. SmTYR1 and SmTYR2 presumably share paralogous functions within the female, although heterologous expression studies of each polypeptide (in bacteria, yeast, or vertebrate cells) have yet to produce successful results despite several attempts. However, as catechol oxidase (the only other copper dependent enzyme capable of diphenol oxidase activity (41) ) has not been detected in the schistosome genome, the diphenol oxidase activity measured in this study almost certainly is due to the function of both SmTYR1 and SmTYR2. One possible explanation for the evolution (gene duplication from an ancestral gene) of two different schistosome tyrosinases is that they display slightly different, but equally important, biophysical properties and this is related to localization and abundance. SmTYR1 may be associated with membranous structures inside the vitelline cells (Fig. 3E ) due to its C-terminal transmembrane domain (Fig. 1A ), whereas SmTYR2 may have a more predominant cytosolic localization (Fig. 3E ). Diphenol oxidase fractionation experiments in S. mansoni yielding two different active components (predominant membrane bound and minor cytosolic) (42) , as well the identification of multiple diphenol oxidase-containing protein isoforms in both S. mansoni (43) and S. japonicum (44) supports this interpretation. Different subcellular localization, together with the 8-fold greater abundance of SmTYR1 compared to SmTYR2 (either due to increased SmTYR1 expression or decreased SmTYR2 stability, Fig. 3C ), may affect the contribution of each enzyme’s activity toward eggshell sclerotization. Expression studies of recombinant SmTYR1 and SmTYR2 for functional and structural investigations are ongoing, and it is anticipated that these will shed light on this interesting hypothesis.

Clearly, inhibiting the schistosome’s ability to produce morphologically normal numbers of eggs by targeting the activity of SmTYR1 and SmTYR2 provides a rationale for the continued investigation of this approach toward blocking oviposition and preventing immunopathology during schistosomiasis. As DETC also prevents sclerotization of Fasciola hepatica eggs (45) , this therapeutic strategy may be applicable to all trematodes within the phylum Platyhelminthes. However, it is presently unclear whether these abnormal ‘eggs’, leaking cellular components (Fig. 6E-G ), are capable of inducing classical type-2 immunological responses (46) or are capable of producing infective-stage miracidia. Additional studies are under way to elucidate these questions, as well as to translate these in vitro findings into an in vivo setting, where schistosome-infected mice will be treated with kojic acid derivatives.

Importantly, this approach may not be limited to the phylum Platyhelminthes, as evidence also suggests that disulfiram (parent compound of DETC and general copper chelator) treatment of Trichuris muris (phylum Nematoda) infected mice leads to the production of malformed eggs incapable of infecting naive mice (47) . Although disulfiram can affect other copper-dependent enzymes, the conclusion from this study was that diphenol oxidase inhibition was responsible for the production of malformed eggs and implicated tyrosinase as the molecular target. In another investigation, kojic acid supplemented diets also affected the egg-laying ability of Lygus hesperus (phylum Insecta), although the authors of this study did not measure the specific effect kojic acid had on the insect’s tyrosinase activity (48) . Extrapolating from our studies, it is likely that this observed effect on egg production was directly due to tyrosinase inhibition. Therefore, utilization of kojic acid and derivatives (or other active compounds) to block the diphenol oxidase activity of tyrosinases may be considered a novel therapeutic aimed at inhibiting eggshell formation/laying, which is effective across phyla. Furthermore, zoonotic schistosomes (e.g., S. japonicum) that infect cattle or other livestock may be particularly amenable to tyrosinase inhibition as drugs could theoretically be incorporated into feed or drinking water, and thereby lengthen the healthy life span (and use) of infected animals by preventing the build-up of egg-induced inflammatory reactions. Together, with the continued use of current antihelminthics, this approach may offer a new combinatorial strategy to combat the dispersal of eggs and the pathology of disease associated with a variety of biomedically important pathogens.


   ACKNOWLEDGMENTS
 
We thank Susan Arnold and Frances Jones for excellent technical help with S. mansoni life cycle maintenance, Dr. Peter Olson (Department of Zoology, Natural History Museum, London, UK) and Mr. Iain Chalmers (Department of Pathology, University of Cambridge) for assistance with phylogenetic analyses. We also thank Dr. Peter Ashton (Department of Biology, University of York, UK) for assistance with gene structure analysis, Dr. Jeremy Skepper and Mr. Tony Burgess (Multi-imaging Center, Department of Anatomy, University of Cambridge) for assistance with confocal and scanning electron microscopy and Prof. David Dunne (Department of Pathology, University of Cambridge) for critically reviewing the manuscript. This work was supported by means of a Wellcome Trust Career Development Grant (RG35177) awarded to KFH and a Biodiversity Research of the 21st century COE (A14) Grant awarded to H.H. Protein sequence data reported in this paper is available in the GenBank, EMBL and DDBJ databases under the accession numbers AY266330 and AY675348.

Received for publication September 11, 2006. Accepted for publication September 29, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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