RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro.

BACKGROUND: Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST). METHODOLOGY/PRINCIPAL FINDINGS: Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200-320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively. CONCLUSIONS/SIGNIFICANCE: In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.

First insight into CD59-like molecules of adult Fasciola hepatica.

The present study focussed on investigating CD59-like molecules of Fasciola hepatica. A cDNA encoding a CD59-like protein (termed FhCD59-1) identified previously in the membrane fraction of the F. hepatica tegument was isolated. This homologue was shown to encode a predicted open reading frame (ORF) of 122 amino acids (aa) orthologous to human CD59 with a 25 aa signal peptide, a mature protein containing 10 cysteines and a conserved CD59/Ly-6 family motif "CCXXXXCN". An analysis of cDNAs from two different adult specimens of F. hepatica revealed seven variable types of FhCD59-1 sequences, designated FhCD59-1.1 to FhCD59-1.7, which had 94.3-99.7% amino acid sequence identity upon pairwise comparison. Molecular modeling of FhCD59-1.1 with human CD59 confirmed the presence of the three-finger protein domain found in the CD59 family and predicted three disulphide bonds in the F. hepatica sequence. The interrogation of F. hepatica databases identified two additional sequences, designated FhCD59-2 and FhCD59-3, which had only 23.4-29.5% amino acid identity to FhCD59-1.1. Orthologues of the inferred CD59 protein sequences of F. hepatica were also identified in other flatworms, including Fasciola gigantica, Fascioloides magna, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Clonorchis sinensis, Opisthorchis viverrini, Taenia solium, Echinococcus granulosus and the free living Schmidtea mediterannea. The results revealed a considerable degree of sequence complexity in the CD59-like sequence families in F. hepatica and flatworms. Phylogenetic analysis of CD59-like aa sequences from F. hepatica and flatworms showed that FhCD59-2 clustered with the known surface-associated protein SmCD59-2 of S. mansoni. Relatively well-supported clades specific to schistosomes, fasciolids and opisthorchiids were identified. The qPCR analysis of gene transcription showed that the relative expression of these 3 FhCD59-like sequences varied by 11-47-fold during fluke maturation, from the newly excysted juvenile (NEJ) to the adult stage. These findings suggest that different FhCD59-like sequences play distinct roles during the development of F. hepatica.

Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1-P4 specificity reveals an unusual preference.

Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S(2) subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola. An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P(2) proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P(2) proline, further highlighting the importance of Y69 for FhCatL2 P(2) proline acceptance. Finally, the P(1)-P(4) specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P(2,) which is unique amongst Fasciola cathepsins examined to date.

Cathepsin B proteases of flukes: the key to facilitating parasite control?

Cysteine proteases are important virulence factors for parasites. This review will focus on the cathepsin B proteases of trematodes (also known as flukes) which are abundant in juvenile and immature flukes. Recent research, primarily in Fasciola, using inhibitors, RNA interference (RNAi) and vaccination studies indicates that cathepsin Bs play a key role in the biology of trematodes. As these proteases are largely expressed by infective parasite stages, their inactivation by chemotherapy or vaccination will greatly reduce the damage wrought by flukes as they invade host tissues. This validates cathepsin Bs as key strategic targets for fluke control.

A novel achiral seco-cyclopropylpyrido[e]indolone (CPyI) analog of CC-1065 and the duocarmycins: synthesis, DNA interactions, in vivo anticancer and anti-parasitic evaluation.

The synthesis of an achiral seco-hydroxy-aza-CBI-TMI analog (8) of the duocarmycins is reported. Its specificity for the DNA minor groove of AT-rich sequences and covalent bonding to adenine-N3 was ascertained by a thermal cleavage assay. Compound 8 was found to be cytotoxic in the nanomolar range against murine and human cancer cells. It was further demonstrated that compound 8 was active against murine melanoma (B16-F0) grown in C57BL/6 mice. Compound 8 was also shown to inhibit the growth of the protozoan parasites Leishmania donovani, Leishmania mexicana, Trypanosoma brucei, and Plasmodium falciparum in culture.

Identification, expression and immunolocalization of cathepsin B3, a stage-specific antigen expressed by juvenile Fasciola gigantica.

To identify antigens that could potentially be developed as vaccines against Fasciola gigantica, somatic antigens were analyzed by immunoprecipitation using pooled sera from rats infected with F. gigantica metacercariae. A prominent antigen of the newly excysted juveniles (NEJ), cathepsin B3 protease (FgCatB3), was identified by N-terminal sequencing and PCR screening of a cDNA library. Recombinant FgCatB3 (rFgCatB3) was expressed in Pichia pastoris, and shown to catalyse the digestion of gelatin, the fluorometric substrate Z-Phe-Arg-AMC and native fibronectin, suggesting that this enzyme may be involved in digesting host connective tissues during the fluke's penetration and migration in the host. Rabbit polyclonal sera against rFgCatB3 was produced and used to determine the distribution of the native cathepsin B3 protease in various developmental stages of F. gigantica. By Western blotting, cathepsin B3 was detected in the whole body (WB) extract of metacercariae and NEJ but not in 4-week-old juveniles or adult parasites which confirmed the stage-specific characteristics of cathepsin B3. Immunolocalization of cathepsin B3 protease in each parasite stage showed that high levels of FgCatB3 were present in the caecal epithelium of the metacercariae and NEJ. The differential distribution of FgCatB3 in the different life cycle stages suggests that this protease is functionally important for the juvenile stage of F. gigantica.

A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites.

The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.

Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens.

Liver flukes produce cathepsin B and cathepsin L in their excretory-secretory material. These proteases are proposed to be key virulence factors for parasite infection, and are therefore targets for vaccination. Cathepsin B is predominately released in the juvenile stage of the life cycle, while different cathepsin L's are released throughout the cycle. Three proteases (cathepsin L5, cathepsin L1g and cathepsin B) were expressed in yeast from cDNA clones isolated from adult, metacercariae and newly excysted juvenile flukes respectively. Each was used singly or in combination to vaccinate rats that were subsequently challenged with Fasciola hepatica metercercariae. Each protein induced an immune response, and all groups vaccinated with recombinant protein yielded significantly fewer and smaller flukes than the control group. Maximal protection of 83% was seen in the group vaccinated with cathepsin B and cathepsin L5 in combination.

Chemically attenuated Plasmodium sporozoites induce specific immune responses, sterile immunity and cross-protection against heterologous challenge.

Vaccination with Plasmodium sporozoites attenuated by irradiation or genetic manipulation induces a protective immune response in rodent malaria models. Recently, vaccination with chemically attenuated P. berghei sporozoites (CAS) has also been shown to elicit sterile immunity in mice. Here we show that vaccination with CAS of P. yoelii also protects against homologous infection and that a P. berghei CAS vaccine cross protects against heterologous challenge with P. yoelii sporozoites. Vaccination with P. yoelii or P. berghei CAS induced parasite-specific antibodies and IFN-gamma-producing CD8(+) T cells at levels not significantly different from radiation-attenuated sporozoites. Our findings provide an initial characterization of the immune response generated by CAS vaccination and suggest that this attenuation process could be used in the production of an effective cross-protective liver stage vaccine for malaria.

Chemical attenuation of Plasmodium berghei sporozoites induces sterile immunity in mice.

Radiation and genetic attenuation of Plasmodium sporozoites are two approaches for whole-organism vaccines that protect against malaria. We evaluated chemical attenuation of sporozoites as an alternative vaccine strategy. Sporozoites were treated with the DNA sequence-specific alkylating agent centanamycin, a compound that significantly affects blood stage parasitemia and transmission of murine malaria and also inhibits Plasmodium falciparum growth in vitro. Here we show that treatment of Plasmodium berghei sporozoites with centanamycin impaired parasite function both in vitro and in vivo. The infection of hepatocytes by sporozoites in vitro was significantly reduced, and treated parasites showed arrested liver stage development. Inoculation of mice with sporozoites that were treated in vitro with centanamycin failed to produce blood stage infections. Furthermore, BALB/c and C57BL/6 mice vaccinated with treated sporozoites were protected against subsequent challenge with wild-type sporozoites. Our findings demonstrate that chemically attenuated sporozoites could be a viable alternative for the production of an effective liver stage vaccine for malaria.

The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration.

Probing protein function in parasitic flatworms is hampered by the difficulties associated with the development of transgenic approaches. Although RNA interference (RNAi) in schistosomes shows much promise, it has not been reported in other trematodes. Here, we show the successful silencing of the cysteine proteases cathepsin B and L in the infective stage of Fasciola hepatica newly excysted juveniles (NEJs). Silencing resulted in marked reductions in target transcript levels and significant diminution in the encoded proteins in the gut. RNAi of either enzyme in NEJs induced transient, abnormal locomotory phenotypes, and significantly reduced penetration of the rat intestinal wall.

Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite.

The liver fluke, Fasciola hepatica, apparently uses a number of cysteine proteases during its life cycle, most likely for feeding, immune evasion and invasion of tissues. A cathepsin B-like enzyme (herein referred to as FhcatB1) appears to be a major enzyme secreted by the invasive, newly excysted juvenile flukes of this parasite. To examine the processing mechanisms for this enzyme, a recombinant form was expressed in Pichia pastoris and purified to yield a homogenous pool of the enzyme. The purified enzyme could be autoactivated at low pH via a bi-molecular mechanism, a process that was greatly accelerated by the presence of large, negatively charged molecules such as dextran sulfate. The enzyme could also apparently be processed to the correct size by an asparaginyl endopeptidase via cleavage in an unusual insertion N-terminal to the normal cleavage site used to yield the active form of the enzyme. Thus, there appear to be a number of ways in which this enzyme can be processed to its optimally active form prior to secretion by F. hepatica.

DNA vaccines in sheep: CTLA-4 mediated targeting and CpG motifs enhance immunogenicity in a DNA prime/protein boost strategy.

DNA vaccines have proven to be an efficient means of inducing immune responses in small laboratory animals; however, their efficacy in large out-bred animal models has been much less promising. In addressing this issue, we have investigated the ability of ovine cytotoxic lymphocyte antigen 4 (CTLA-4) mediated targeting and ruminant specific CpG optimised plasmids, both alone and in combination, to enhance immune responses in sheep to the pro cathepsin B (FhCatB) antigen from Fasciola hepatica. In this study, CTLA-4 mediated targeting enhanced the speed and magnitude of the primary antibody response and effectively primed for a potent memory response compared to conventional DNA vaccination alone, which failed to induce a detectable immune response. While the CpG-augmentation of the CTLA-4 targeted construct did not further enhance the magnitude or isotype profile of the CTLA-4 induced antibody titres, it did result in the induction of significant antigen-specific, lymphocyte-proliferative responses that were not observed in any other treatment group, showing for the first time that significant cellular responses can be induced in sheep following DNA vaccination. In contrast, CpG-augmentation in the absence of CTLA-4 mediated targeting failed to induce a detectable immune response. This is the first study to explore the potential adjuvant effects of ruminant specific CpG motifs on DNA vaccine induced immune responses in sheep. The ability of CpG-augmented CTLA-4 mediated targeting to induce both humoral and cellular immune responses in this study suggests that this may be an effective approach for enhancing the efficacy of DNA vaccines in large out-bred animal models.

The evolution of enzyme specificity in Fasciola spp.

Fasciola spp., commonly known as liver fluke, are significant trematode parasites of livestock and humans. They secrete several cathepsin L-like cysteine proteases, some of which differ in enzymatic properties and timing of expression in the parasite's life cycle. A detailed sequence and evolutionary analysis is presented, based on 18 cathepsin L-like enzymes isolated from Fasciola spp. (including a novel clone identified in this study). The enzymes form a monophyletic group which has experienced several gene duplication events over the last approximately 135 million years, giving rise to the present-day enzymatic repertoire of the parasite. This timing of these duplications appears to correlate with important points in the evolution of the mammalian hosts. Furthermore, the dates suggest that Fasciola hepatica and Fasciola gigantica diverged around 19 million years ago. A novel analysis, based on the pattern of amino acid diversity, was used to identify sites in the enzyme that are predicted to be subject to positive adaptive evolution. Many of these sites occur within the active site cleft of the enzymes, and hence would be expected to lead to differences in substrate specificity. Using homology modeling, with reference to previously obtained biochemical data, we are able to predict S2 subsite specificity for these enzymes: specifically those that can accommodate bulky hydrophobic residues in the P2 position and those that cannot. A number of other positions subject to evolutionary pressure and potentially significant for enzyme function are also identified, including sites anticipated to diminish cystatin binding affinity.

Fasciola hepatica cathepsin L suppresses sheep lymphocyte proliferation in vitro and modulates surface CD4 expression on human and ovine T cells.

Fasciola hepatica infection has been shown to suppress sheep lymphocyte proliferation in vitro and this is at least partially attributable to excretory/secretory products (ES) released by F. hepatica parasites. We identified a suppressive component in ES by analysing the effect of ES fractions, separated by gel filtration, on the proliferation of sheep T cells in vitro. A major proportion of the suppressive activity in ES was shown to coelute with the cathepsin L proteases: E64, a cysteine protease inhibitor, blocked the suppressive activity of cathepsin L. In order to identify possible mechanisms by which cathepsin L could suppress T cell proliferation, the effect of ES and F. hepatica recombinant cathepsin L (rFhCatL) on the expression of 22 different sheep T cell surface markers was analysed by flow cytometry. Incubation of sheep T cells with ES or two rFhCatL significantly reduced surface CD4 expression and this effect was prevented in the presence of E64. In similar experiments with human T lymphocytes, ES and rFhCatL were shown to down regulate surface CD4 expression. These results show that F. hepatica cathepsin L both suppresses sheep T cell proliferation and reduces surface CD4 expression on both human and ovine T cells.