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.     

Genome-wide RNAi screening in Caenorhabditis elegans

In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.     

Molecular Characterization of a Tetraspanin from the Human Liver Fluke, Opisthorchis viverrini

Background

The human liver fluke, Opisthorchis viverrini, is designated as a group 1 carcinogen, and is the major risk factor for cholangiocarcinoma in endemic countries throughout Southeast Asia. Proteins in the excretory-secretory products and tegumental surface membranes of the fluke have been proposed to play pivotal roles in parasite survival in the host, and subsequent pathogenesis. These macromolecules are therefore valid targets for the development of vaccines and new drugs to control the infection. Tetraspanins (TSP) are prominent components of the tegument of blood flukes where they are essential for tegument formation, are directly exposed to the immune system, and are major targets for a schistosomiasis vaccine. We propose that similar molecules in the surface membranes of O. viverrini are integral to tegument biogenesis and will be efficacious vaccine antigens.

Methodology/Principal Findings

The cDNA sequence encoding O. viverrini tetraspanin-1 (Ov-TSP-1) was identified and cloned. The Ov-tsp-1gene was isolated from a cDNA library. Ov-tsp-1 mRNA was expressed most highly in metacercariae and eggs, and to a lesser extent in juvenile and adult worms. Immunolocalization with adult flukes confirmed that Ov-TSP-1 was expressed in the tegument and eggs in utero. Western blot analysis of rOv-TSP-1 probed with sera from O. viverrini-infected humans and hamsters indicated that both hosts raise antibody responses against the native TSP. Using RNA interference we silenced the expression level of Ov-tsp-1 mRNA in adult flukes by up to 72% by 10 days after delivery of dsRNA. Ultrastructural morphology of adult worms treated with Ov-tsp-1 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls.

Conclusions/Significance

This is the first report of a tetraspanin from the tegument of a liver fluke. Our data imply that tetraspanins play important structural roles in the development of the tegument in the adult fluke. Potential uses of O. viverrini tetraspanins as novel interventions are discussed.     

Comparative histopathology of Opisthorchis felineus and Opisthorchis viverrini in a hamster model: an implication of high pathogenicity of the European liver fluke

European liver fluke (Opisthorchis felineus) and Asian liver fluke (Opisthorchis viverrini) are similar in morphology but comparative pathology of the infections has not been described. We therefore did comparative histopathology of both parasites in an experimental animal model. The study was conducted in 3 groups of 105 Syrian golden hamsters; the first and second groups fed with 50 metacercariae of O. felineus (OF) or O. viverrini (OV) and the last group was uninfected controls. Five hamsters in each group were euthanized on weeks 1, 2, 4, 8, 12 and 24 post-infection. The liver tissue was fixed and processed for routine histopathology and immunohistochemistry for proliferation markers (BrdU or PCNA). Overall, the liver histopathology of O. felineus and O. viverrini infection was generally similar. However, various histopathogical features including intense inflammation, fibrosis, biliary and goblet cell hyperplasia and dysplasia occurred earlier in the OF group. In addition, the existence of precancerous lesions such as cholangiofibrosis in a long-term infection was observed only in this group. O. felineus is larger in size than O. viverrini which, together with its excreted and secreted antigens, likely is crucial in the induction of liver fluke induced disease. The differences in nature and timing of the histopathological profile indicate that opisthorchiasis caused by the European liver fluke O. felineus is more pathogenic than its Asian relative O. viverrini.     

Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis

The efficiency of RNA interference (RNAi) delivery to L1 through L3 stage worms of the sheep parasitic nematode Trichostrongylus colubriformis was investigated using several techniques. These were: (i) feeding of Escherichia coli expressing double stranded RNA (dsRNA); (ii) soaking of short interfering (synthetic) RNA oligonucleotides (siRNA) or in vitro transcribed dsRNA molecules; and (iii) electroporation of siRNA or in vitro transcribed dsRNA molecules. Ubiquitin and tropomyosin were used as a target gene because they are well conserved genes whose DNA sequences are available for several nematode parasite species. Ubiquitin siRNA or dsRNA delivered by soaking or electroporation inhibited development in T. colubriformis but with feeding as a delivery method, RNAi of ubiquitin was not successful. Feeding was, however, successful with tropomyosin as a target, suggesting that mode of delivery is an important parameter of RNAi. Electroporation is a particularly efficient means of inducing RNA in nematodes with either short dsRNA oligonucleotides or with long in vitro transcribed dsRNA molecules. These methods permit routine delivery of dsRNA for RNAi in T. colubriformis larval stage parasites and should be applicable to moderate to high-throughput screening.     

Transport of orally delivered dsRNA in southern green stink bug,Nezara viridula

The southern green stink bug (SGSB, Nezara viridula) is an emerging polyphagous pest in many regions of the world. RNA interference (RNAi) is a valuable method for understanding gene function and holds great potential for pest management. However, RNAi efficiency is variable among insects and the differences in transport of double-stranded RNA (dsRNA) are one of the major factors that contribute to this variability. In this study, Cy3 labeled dsRNA was used to track the transport of dsRNA in SGSB tissues. Cy3_dsRNA was detected in the hemocytes, fat body (FB), epidermis, and midgut tissues at 24–72 hr after injection. Orally delivered Cy3_dsRNA or Cypher-5E labeled dsRNA was mostly detected in the midgut and a few signals were detected in parts of the FB and epidermis. Both injected and fed Cy3_dsRNA showed stronger signals in SGSB tissues when compared to Cy3_siRNA (small interfering RNA) or Cy3_shRNA (short hairpin RNA). dsRNA targeting the gene for a vacuolar-sorting protein, SNF7, induced higher knockdown of the target gene and greater SGSB mortality compared to siRNA or shRNA targeting this gene. ³²P-labeled dsRNA injected into SGSB was processed into siRNA, but fed ³²P-labeled dsRNA was not efficiently processed into siRNA. These data suggest that transport of orally delivered dsRNA across the midgut epithelium is not efficient in SGSB which may contribute to variable RNAi efficiency. Targeting genes expressed in the midgut rather than other tissues and using dsRNA instead of siRNA or shRNA would be more effective for RNAi-mediated control of this pest.     

The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini

Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products and, utilizing selective labeling and sequential solubilization, from the host‐exposed tegument. The excretory/secretory included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti‐apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno‐modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin‐2. Novel roles in pathogenesis were suggested for the tegument–host interface since more than ten surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke.     

Organization of the nervous system in Opisthorchis viverrini investigated by histochemical and immunohistochemical study

The structure and organization of the nervous system has been documented for various helminth parasites. However, the neuroanatomy of the carcinogenic liver fluke, Opisthorchis viverrini has not been described. This study therefore investigated the organization of the nervous system of this fluke using cholinesterase activity, aminergic and peptidergic (FMRFamide-like peptides) immunostaining to tag major neural elements. The nervous system, as detected by acetylcholinesterase (AchE) reaction, was similar in newly excysted metacercariae, migrating juveniles and adult parasites. In these stages, there were three pairs (dorsal, ventral and lateral) of bilaterally symmetrical longitudinal nerve cords and two cerebral ganglia. The ventral nerve cords and the cerebral ganglia were well-developed and exhibited strong AchE reactivity, as well as aminergic and FMRFamide-like immunoreactivity. Numerous immunoreactive nerve cell bodies were observed around the inner surface of the ventral sucker. Fine FMRFamide-like peptides immunopositive nerve fiber was rarely observed. Overall, the organization of the nervous system of O. viverrini is similar to other trematodes.     

Secreted cysteine proteases of the carcinogenic liver fluke,Opisthorchis viverrini: regulation of cathepsin F activation by autocatalysis and trans‐processing by cathepsin B

Opisthorchis viverrini is an important helminth pathogen of humans that is endemic in Thailand and Laos. Adult flukes reside within host bile ducts and feed on epithelial tissue and blood cells. Chronic opisthorchiasis is associated with severe hepatobiliary diseases such as cholangiocarcinoma. Here we report that adult O. viverrini secrete two major cysteine proteases: cathepsin F (Ov‐CF‐1) and cathepsin B1 (Ov‐CB‐1). Ov‐CF‐1 is secreted as an inactive zymogen that autocatalytically processes and activates to a mature enzyme at pH 4.5 via an intermolecular cleavage at the prosegment–mature domain junction. Ov‐CB‐1 is also secreted as a zymogen but, in contrast to Ov‐CF‐1, is fully active against peptide and macromolecular substrates despite retaining the N‐terminal prosegment. The active Ov‐CB‐1 zymogen was capable of trans‐activating Ov‐CF‐1 by proteolytic removal of its prosegment at pH 5.5, a pH at which the Ov‐CF‐1 zymogen cannot autocatalytically activate. Both cathepsins hydrolyse human haemoglobin but their combined action more efficiently degrades haemoglobin to smaller peptides than each enzyme alone. Ov‐CF‐1 degraded extracellular matrix proteins more effectively than Ov‐CB‐1 at physiological pH. We propose that Ov‐CB‐1 regulates Ov‐CF‐1 activity and that both enzymes work together to degrade host tissue contributing to the development of liver fluke‐associated cholangiocarcinoma.     

Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans

Background  

In Caenorhabditis elegans, injection of double-stranded RNA (dsRNA) results in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). It has previously been shown that RNAi can also be achieved by feeding worms Escherichia coli expressing dsRNA corresponding to a specific gene; this mode of dsRNA introduction is conventionally considered to be less efficient than direct injection, however, and has therefore seen limited use, even though it is considerably less labor-intensive.     

Functional Analysis of the Unique Cytochrome P450 of the Liver Fluke Opisthorchis felineus

The basic metabolic cytochrome P450 (CYP) system is essential for biotransformation of sterols and xenobiotics including drugs, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms have numerous paralogues of the CYP gene encoding heme monooxygenases with specific substrate range. Notably, by contrast, the parasitic flatworms have only one CYP gene. The role of this enzyme in the physiology and biochemistry of helminths is not known. The flukes and tapeworms are the etiologic agents of major neglected tropical diseases of humanity. Three helminth infections (Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium) are considered by the International Agency for Research on Cancer (IARC) as definite causes of cancer. We focused our research on the human liver fluke Opisthorchis felineus, an emerging source of biliary tract disease including bile duct cancer in Russia and central Europe. The aims of this study were (i) to determine the significance of the CYP activity for the morphology and survival of the liver fluke, (ii) to assess CYP ability to metabolize xenobiotics, and (iii) to localize the CYP activity in O. felineus tissues. We observed high constitutive expression of CYP mRNA (Real-time PCR) in O. felineus. This enzyme metabolized xenobiotics selective for mammalian CYP2E1, CYP2B, CYP3A, but not CYP1A, as determined by liquid chromatography and imaging analyses. Tissue localization studies revealed the CYP activity in excretory channels, while suppression of CYP mRNA by RNA interference was accompanied by morphological changes of the excretory system and increased mortality rates of the worms. These results suggest that the CYP function is linked to worm metabolism and detoxification. The findings also suggest that the CYP enzyme is involved in vitally important processes in the organism of parasites and is a potential drug target.     

Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria

Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites.     

Fasciola gigantica: Production and characterization of a monoclonal antibody against recombinant cathepsin B3

A number of monoclonal antibodies (MoAbs) against a recombinant cathepsin B3 (rCatB3) of Fasciola gigantica were produced in BALB/c mice. Reactivity and specificity of these MoAbs were assessed by indirect ELISA and immunoblotting techniques. Six stable clones, namely 1C4, 1E9, 2E5, 2F9, 5B4, 5D7 were obtained. All MoAbs reacted with rCatB3 at molecular weight (MW) 37 kDa as well as the glycosylated peptide at 55–75 kDa and with the native CatB3 at MW 37 kDa in WB extracts of metacercariae (Met) and newly excysted juveniles (NEJ). It was found to be IgG₁ and λ light chain isotypes. Immunolocalization of CatB3 in metacercariae, NEJ, 4-week-old juvenile and adult F. gigantica performed by immunoperoxidase technique by using these MoAbs as probes indicated that CatB3 was present in high concentration in the caecal epithelium and caecal lumen of the Met and NEJ, but not in the 4-week-old juvenile and adult fluke. The MoAbs show no cross-reactions with antigens of other parasites including Gigantocotyl explanatum, Eurytrema pancreaticum, Paramphistomum cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-papillosa. Thus, it is possible that these MoAbs could be a good candidate for immunodiagnosis of fasciolosis.