Maria E. Morales

piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni

The transposon piggyBac from the genome of the cabbage looper moth Trichoplusia ni has been observed in the laboratory to jump into the genomes of key model and pathogenic eukaryote organisms including mosquitoes, planarians, human and other mammalian cells, and the malaria parasite Plasmodium falciparum. Introduction of exogenous transposons into schistosomes has not been reported but transposon‐mediated transgenesis of schistosomes might supersede current methods for functional genomics of this important human pathogen. In the present study we examined whether the piggyBac trans‐poson could deliver reporter transgenes into the genome of Schistosoma mansoni parasites. A piggyBac donor plasmid modified to encode firefly luciferase under control of schistosome gene promoters was introduced along with 7‐methylguanosine capped RNAs encoding piggyBac transposase into cultured schistosomula by square wave electroporation. The activity of the helper transposase mRNA was confirmed by Southern hybridization analysis of genomic DNA from the transformed schistosomes, and hybridization signals indicated that the piggyBac transposon had integrated into numerous sites within the parasite chromosomes. piggyBac integrations were recovered by retrotransposon‐anchored PCR, revealing characteristic piggyBac TTAA footprints in the vicinity of the endogenous schistosome retro‐transposons Boudicca, sri, and sr2. This is the first report of chromosomal integration of a transgene and somatic transgenesis of this important human pathogen, in this instance accomplished by mobilization of the piggyBac transposon.—Morales, M. E., Mann, V. H., Kines, K.J., Gobert, G. N., Fraser, M. J.,Jr., Kalinna, B. H., Correnti, J. M., Pearce, E. J., Brindley, P. J. piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni. FASEB J. 21, 3479–3489 (2007)

RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs

Schistosoma mansoni leucine aminopeptidase (LAP) is thought to play a central role in hatching of the miracidium from the schistosome egg. We identified two discrete LAPs genes in the S. mansoni genome, and their orthologs in S. japonicum. The similarities in sequence and exon/intron structure of the two genes, LAP1 and LAP2, suggest that they arose by gene duplication and that this occurred before separation of the mansoni and japonicum lineages. The SmLAP1 and SmLAP2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP2 expression was higher in free living larval (miracidia) and in parasitic intra-snail (sporocysts) stages. We investigated the role of each enzyme in hatching of schistosome eggs and the early stages of schistosome development by RNA interference (RNAi). Using RNAi, we observed marked and specific reduction of mRNAs, along with a loss of exopeptidase activity in soluble parasite extracts against the diagnostic substrate l-leucine-7-amido-4-methylcoumarin hydroxide. Strikingly, knockdown of either SmLAP1 or SmLAP2, or both together, was accompanied by >or=80% inhibition of hatching of schistosome eggs showing that both enzymes are important to the escape of miracidia from the egg. The methods employed here refine the utility of RNAi for functional genomics studies in helminth parasites and confirm these can be used to identify potential drug targets, in this case schistosome aminopeptidases.

Development of Functional Genomic Tools in Trematodes: RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica

The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite–host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi) reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC). We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA) specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP), and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth parasites. These could facilitate the study of gene function and the identification of relevant targets for intervention in organisms that are by other means intractable. More specifically, these results open new perspectives for functional genomics of F. hepatica, which hopefully can lead to the development of new interventions for fascioliasis.

Integration of reporter transgenes into Schistosoma mansoni chromosomes mediated by pseudotyped murine leukemia virus

The recent release of draft genome sequences of two of the major human schistosomes has underscored the pressing need to develop functional genomics approaches for these significant pathogens. The sequence information also makes feasible genomescale investigation of transgene integration into schistosome chromosomes. Retrovirus‐mediated transduction offers a means to establish transgenic lines of schistosomes, to elucidate schistosome gene function and expression, and to advance functional genomics approaches for these parasites. We investigated the utility of the Moloney murine leukemia retrovirus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) for the transduction of Schistosoma mansoni and delivery of reporter transgenes into schistosome chromosomes. Schistosomula were exposed to virions of VSVG‐pseudotyped MLV, after which genomic DNA was extracted from the transduced schistosomes. Southern hybridization analysis indicated the presence of proviral MLV retrovirus in the transduced schistosomes. Fragments of the MLV transgene and flanking schistosome sequences recovered using an anchored PCR‐based approach demonstrated definitively that somatic transgenesis of schistosome chromosomes had taken place and, moreover, revealed widespread retrovirus integration into schistosome chromosomes. More specifically, MLV transgenes had inserted in the vicinity of genes encoding immunophilin, zinc finger protein Sma‐Zic, and others, as well as near the endogenous schistosome retrotransposons, the fugitive and SR1. Proviral integration of the MLV transgene appeared to exhibit primary sequence site specificity, targeting a gGATcc‐like motif. Reporter luciferase transgene activity driven by the schistosome actin gene promoter was expressed in the tissues of transduced schistosomula and adult schistosomes. Luciferase activity appeared to be developmentally expressed in schistosomula with increased activity observed after 1 to 2 wk in culture. These findings indicate the utility of VSVG‐pseudotyped MLV for transgenesis of S. mansoni, herald a tractable pathway forward toward germline transgenesis and functional genomics of parasitic helminths, and provide the basis for comparative molecular pathogenesis studies of chromosomal lesions arising from retroviral integration into human compared with schistosome chromosomes.—Kines, K. J., Morales, M. E., Mann, V. H., Gobert, G. N., Brindley, P. J. Integration of reporter transgenes into Schistosoma mansoni chromosomes mediated by pseudotyped murine leukemia virus. FASEB J. 22, 2936–2948 (2008)

Culture for genetic manipulation of developmental stages of Schistosoma mansoni.

Genomes of the major human helminth parasites, and indeed many others of agricultural significance, are now the research focus of intensive genome sequencing and annotation. A draft genome sequence of the filarial parasite Brugia malayi was reported in 2007 and draft genomes of two of the human schistosomes, Schistosoma japonicum and S. mansoni reported in 2009. These genome data provide the basis for a comprehensive understanding of the molecular mechanisms involved in schistosome nutrition and metabolism, host-dependent development and maturation, immune evasion and invertebrate evolution. In addition, new potential vaccine candidates and drug targets will likely be predicted. However, testing these predictions is often not straightforward with schistosomes because of the difficulty and expense in maintenance of the developmental cycle. To facilitate this goal, several developmental stages can be maintained in vitro for shorter or longer intervals of time, and these are amenable to manipulation. Our research interests focus on experimental studies of schistosome gene functions, and more recently have focused on development of transgenesis and RNA interference with the longer term aim of heritable gene manipulation. Here we review methods to isolate and culture developmental stages of Schistosoma mansoni, including eggs, sporocysts, schistosomules and adults, in particular as these procedures relate to approaches for gene manipulation. We also discuss recent advances in genetic manipulation of schistosomes including the deployment of square wave electroporation to introduce reporter genes into cultured schistosomes.

Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini

Background The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities. Methodology/Principal Findings Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt) encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa), prosegment (95 aa), and mature protease (213 aa). BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%), Paragonimus westermani (58%), Schistosoma mansoni and S. japonicum (52%), and with vertebrate cathepsin F (51%). Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of ∼3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and activate to a mature enzyme, trans-processing by Fasciola hepatica cathepsin L cleaved the prosegment of Ov-CF-1, releasing a mature cathepsin F with activity against the peptide Z-Phe-Arg-NHMec >50 times that of the zymogen. Immunocytochemistry using antibodies raised against the recombinant enzyme showed that Ov-CF-1 is expressed in the gut of the mature hermaphroditic fluke and also in the reproductive structures, including vitelline glands, egg, and testis. Ov-CF-1 was detected in bile duct epithelial cells surrounding the flukes several weeks after infection of hamsters with O. viverrini and, in addition, had accumulated in the secondary (small) bile ducts where flukes cannot reach due to their large size. Conclusions/Significance A cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level. Secretion of this protease may contribute to the hepatobiliary abnormalities, including cholangiocarcinogenesis, observed in individuals infected with this parasite.

Electroporation Facilitates Introduction of Reporter Transgenes and Virions into Schistosome Eggs

Background The schistosome egg represents an attractive developmental stage at which to target transgenes because of the high ratio of germ to somatic cells, because the transgene might be propagated and amplified by infecting snails with the miracidia hatched from treated eggs, and because eggs can be readily obtained from experimentally infected rodents. Methods/Findings We investigated the utility of square wave electroporation to deliver transgenes and other macromolecules including fluorescent (Cy3) short interference (si) RNA molecules, messenger RNAs, and virions into eggs of Schistosoma mansoni. First, eggs were incubated in Cy3-labeled siRNA with and without square wave electroporation. Cy3-signals were detected by fluorescence microscopy in eggs and miracidia hatched from treated eggs. Second, electroporation was employed to introduce mRNA encoding firefly luciferase into eggs. Luciferase activity was detected three hours later, whereas luciferase was not evident in eggs soaked in the mRNA. Third, schistosome eggs were exposed to Moloney murine leukemia virus virions (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG). Proviral transgenes were detected by PCR in genomic DNA from miracidia hatched from virion-exposed eggs, indicating the presence of transgenes in larval schistosomes that had been either soaked or electroporated. However, quantitative PCR (qPCR) analysis determined that electroporation of virions resulted in 2–3 times as many copies of provirus in these schistosomes compared to soaking alone. In addition, relative qPCR indicated a copy number for the proviral luciferase transgene of ∼20 copies for 100 copies of a representative single copy endogenous gene (encoding cathepsin D). Conclusions Square wave electroporation facilitates introduction of transgenes into the schistosome egg. Electroporation was more effective for the transduction of eggs with pseudotyped MLV than simply soaking the eggs in virions. These findings underscore the potential of targeting the schistosome egg for germ line transgenesis.

The Contribution of Alu Elements to Mutagenic DNA Double-Strand Break Repair

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

Transgenesis of schistosomes: approaches employing mobile genetic elements.

Draft genome sequences for Schistosoma mansoni and Schistosoma japonicum are now available. However, the identity and importance of most schistosome genes have yet to be determined. Recently, progress has been made towards the genetic manipulation and transgenesis of schistosomes. Both loss-of-function and gain-of-function approaches appear to be feasible in schistosomes based on findings described in the past 5 years. This review focuses on reports of schistosome transgenesis, specifically those dealing with the transformation of schistosomes with exogenous mobile genetic elements and/or their endogenous relatives for the genetic manipulation of schistosomes. Transgenesis mediated by mobile genetic elements offers a potentially tractable route to introduce foreign genes to schistosomes, a means to determine the importance of schistosome genes, including those that could be targeted in novel interventions and the potential to undertake large-scale forward genetics by insertional mutagenesis.

Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes

Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs) as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl2 favored resolution of DSBs through homologous recombination (HR) and single strand annealing (SSA), which were inhibited by higher NiCl2 doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the “error prone” non-homologous end joining (alt-NHEJ) while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair.