Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths

Abstract

Background: The thioredoxin and/or glutathione pathways occur in all organisms. They provide electrons for deoxyribonucleotide synthesis, function as antioxidant defenses, in detoxification, Fe/S biogenesis and participate in a variety of cellular processes. In contrast to their mammalian hosts, platyhelminth (flatworm) parasites studied so far, lack conventional thioredoxin and glutathione systems. Instead, they possess a linked thioredoxin-glutathione system with the selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR) as the single redox hub that controls the overall redox homeostasis. TGR has been recently validated as a drug target for schistosomiasis and new drug leads targeting TGR have recently been identified for these platyhelminth infections that affect more than 200 million people and for which a single drug is currently available. Little is known regarding the genomic structure of flatworm TGRs, the expression of TGR variants and whether the absence of conventional thioredoxin and glutathione systems is a signature of the entire platyhelminth phylum.

Results: We examine platyhelminth genomes and transcriptomes and find that all platyhelminth parasites (from classes Cestoda and Trematoda) conform to a biochemical scenario involving, exclusively, a selenium-dependent linked thioredoxin-glutathione system having TGR as a central redox hub. In contrast, the free-living platyhelminth Schmidtea mediterranea (Class Turbellaria) possesses conventional and linked thioredoxin and glutathione systems. We identify TGR variants in Schistosoma spp. derived from a single gene, and demonstrate their expression. We also provide experimental evidence that alternative initiation of transcription and alternative transcript processing contribute to the generation of TGR variants in platyhelminth parasites.

Conclusions: Our results indicate that thioredoxin and glutathione pathways differ in parasitic and free-living flatworms and that canonical enzymes were specifically lost in the parasitic lineage. Platyhelminth parasites possess a unique and simplified redox system for diverse essential processes, and thus TGR is an excellent drug target for platyhelminth infections. Inhibition of the central redox wire hub would lead to overall disruption of redox homeostasis and disable DNA synthesis.

Figure 1

Amino acid sequence alignment of TR, GR and TGRs of platyhelminths. Sec is indicated by U. The position of the redox active residues in the sequences is indicated by a star. Conserved residues in all proteins are highlighted in dark grey, conserved residues in the Grx domain of TGRs are highlighted in light grey. Location of the Grx domain is indicated above the sequence. ORFs for TGR, TR and GR from S. mediterranea (SCHME) genome (assembly 31) were predicted in the contigs 000676, 000203 and 001663, respectively. ORF for E. multilocularis (ECHMU) TGR was assembled from contigs 0007357 and 0007358. Full-length TGR sequences of S. mansoni (SCHMA),S. japonicum (SCHJA), E. granulosus (ECHGR), and F. hepatica (FASHE) were retrieved from Genebank (gb|AAK85233.1|AF395822_1, gb|AAW25951.1, emb|CAM96615.1, and gb|AAN63052.1, respectively). T. solium (TAESO) TGR partial sequences were retrieved from the EST repository at Genebank (gb|EL757065.1 and gb|EL743442.1). The putative mitochondrial leader peptide of S. mediterranea TGR is not included in the sequence, neither leader peptide variants of Schistosoma and Echinococcus TGRs. Sequences were aligned with Clustal W2 [29], with final manual adjustment after inspection.

Figure 2

Structures and nucleotide sequence alignment of SECIS elements of TR and TGRs of platyhelminths. The SECIS elements were predicted using the SECISearch program [27]. Functionally important nucleotides in the apical loop and the quartet (SECIS core) are shown in bold in the structure and in bold and underlined in the alignment. ECHGR: E. granulosus, ECHMU: E. multilocularis, SCHMA: S. mansoni, SCHJA: S. japonicum, SMED: S. mediterranea, FASHE: F. hepatica.

Figure 3

Phylogenetic relationships of GRs, TRs and TGRs of platyhelminths and mammals. TRs, GRs and TGRs from platyhelminths and mammals were aligned with Clustal W2 [29]. Human dihydrolipoamide dehydrogenase (DLDH) was used as outgroup. A Neighbor-Joining tree was constructed using MEGA4 [30] with pairwise deletion and default parameters. A condensed tree is shown, and bootstrap values of reliable nodes (above 50) are indicated. The polytomy displayed at the TGR node denotes that the evolutionary relationships within the node can not be resolved with at least 50% of bootstrap support. In other words, nodes with less than 50% of bootstrap support were collapsed and are displayed as polytomies. The results indicate that the TR and GR genes present in the planarian lineage were lost in the neodermata lineage. Very similar topology and statistical support were obtained using different phylogenetic reconstruction methods (i.e. Maximum Parsimony, UPMGA and Minimum Evolution). ECHGR: E. granulosus, ECHMU: E. multilocularis, TAESO: T. Solium, SCHMA: S. mansoni, SCHJA: S. japonicum, SMED: S. mediterranea, FASHE: F. hepatica.

Figure 4

TGR variants in Schistosoma spp. Amino acid sequence alignment of S. japonicum (denoted as SCHJA) and S. mansoni (denoted as SCHMA) TGR variants. Variant 1 (v1) encodes a TGR with a mitochondrial signal peptide, variant 2 (v2) encodes a TGR with shorter leader peptide with no topology prediction, and variant 3 (v3) encodes a cytosolic TGR. B. Nucleotide sequence alignment of ESTs encoding S. japonicum and S. mansoni TGR variants; the sequence of SCHMA_v1 was deduced from the corresponding TGR gene. C. Nucleotide genomic sequence of S. mansoni and S. japonicum TGRs. The sequence corresponds to the end of the first exon, the first intron (indicated by italics) and the beginning of the second exon. GT and AG donor and acceptor splice sites of intron I, whose splicing generates variant 1, are shown underlined in lower case. Underlined in capital letters and in italics is shown a presumptive leaky GT donor splice site present in exon I, that, if spliced, gives rise to variant 2. Sequences of variant 3 were retrieved from translated full-cDNAs deposited in Genebank (accession Numbers gb|AAK85233.1|AF395822_1, gb|AAW25951.1), sequences of variants 1 and 2 correspond to ESTs deposited in Genebank (gb|BU801474.1 for Sja variant 1, gb|BU791993.1 and gb|CV688441.1 for Sja2, and gb|CD202891.1 for Sma variant 2). D. Proposed model of how Schistosoma mRNA variants would be generated. From TGR gene, two primary transcripts would be synthesized from alternative transcription initiation sites: core promoter and a putative intron I promoter. The transcript derived form the core promoter would give rise to two different mRNAs by alternative transcript processing.

Figure 5

Expression of S. mansoni variants detected by RT-PCR. A. Schematic representation of the three mRNA variants of S. mansoni TGR, the variant 1 and variant 2 specific primers (F1 and F2) and the primer corresponding to the 5'-end of the cytosolic variant (F3) used in RT-PCRs in combination with a reverse primer derived from exon 3 sequence. B. Electrophoresis of PCR products from S. mansoni cercariae: a band of the expected size was observed in the PCRs with F1, F2 and F3 primers and the reverse primer (lanes 2 to 4, respectively). MWM lane corresponds to 100 bp plus ladder (Fermentas).

Figure 6

Analysis of alternative initiation of transcription from E. granulosus TGR intron 1. A. The sequence of E. granulosus TGR gene starting from intron 1 ending at exon 3. Exons 2 and 3 are shaded in grey. The putative TATA box present in intron 1 is highlighted in bold and italics. Sequences of the primers used in the PCR experiments are shown underlined and in italics: forward primers 1, 2 and 3 span intron 1 from 5' to 3'; the reverse primer derives from exon 3 sequence. B. Schematic representation of the primers used in PCR reactions, localized in the TGR sequence. C. Gel-electrophoresis of PCR reaction carried out from genomic DNA (lanes 2 to 4) and from E. granulosus cDNA (lanes 5 to 7) using a combination of the reverse primer and forward primers 1, 2 and 3 (lanes 2 and 5, lanes 3 and 6, lanes 4 and 7, respectively). Lane 1 corresponds to 100 bp plus ladder (Fermentas).