Molecular and biochemical characterizations of a Fasciola gigantica retinoid X receptor-α isoform A (FgRXRα-A).

Fascioliasis is a parasitic infection in animals and humans caused by the parasitic flatworm genus Fasciola, which has two major species, F. hepatica and F. gigantica. A major concern regarding this disease is drug resistance, which is increasingly reported worldwide. Hence, the discovery of a novel drug as well as drug targets is crucially required. Therefore, this study aims to characterize the novel drug target in the adult F. gigantica. In the beginning, we hypothesized that the parasite might interact with some host molecules when it lives inside the liver parenchyma or bile ducts, specifically hormones and hormone-like molecules, through the specific receptors, primarily nuclear receptors (NRs), which are recognized as a major drug target in various diseases. The retinoid X receptor (RXR) is a member of subfamily 2 NRs that plays multitudinous roles in organisms by forming homodimers or heterodimers with other NRs. We obtained the full-length amino acid sequences of F. gigantica retinoid X receptor-alpha (FgRXRα-A) from the transcriptome of F. gigantica that existed in the NCBI database. The FgRXRα-A were computationally predicted for the basic properties, multiple aligned, phylogeny analyzed, and generated of 2D and 3D models. Moreover, FgRXRα-A was molecular cloned and expressed as a recombinant protein (rFgRXRα-A), then used for immunization for specific polyclonal antibodies. The native FgRXRα-A was detected in the parasite extracts and tissues, and the function was investigated by in vitro binding assay. The results demonstrated the conservation of FgRXRα-A to the other RXRs, especially RXRs from the trematodes. Interestingly, the native FgRXRα-A could be detected in the testes of the parasite, where the sex hormones are accumulated. Moreover, the binding assay revealed the interaction of 9-cis retinoic acid and FgRXRα-A, suggesting the function of FgRXRα-A. Our findings suggested that FgRXRα-A will be involved with the sexual reproduction of the parasite by forming heterodimers with other NRs, and it could be the potential target for further drug development of fascioliasis.

Production and immunological characterization of the novel single-chain variable fragment (scFv) antibodies against the epitopes on Opisthorchis viverrini cathepsin F (OvCatF).

BACKGROUND: Opisthorchis viverrini infection is a significant health problem in several countries, especially Southeast Asia. The infection causes acute gastro-hepatic symptoms and also long-term infection leading to carcinogenesis of an aggressive bile duct cancer (cholangiocarcinoma; CCA). Hence, the early diagnosis of O. viverrini infection could be the way out of this situation. Still, stool examination by microscopic-based methods, the current diagnostic procedure is restricted by low parasite egg numbers in the specimen and unprofessional laboratorians. The immunological procedure provides a better chance for diagnosis of the infection. Hence, this study aims to produce single-chain variable fragment (scFv) antibodies for use as a diagnostic tool for O. viverrini infection. METHODS: This study uses phage display technologies to develop the scFv antibodies against O. viverrini cathepsin F (OvCatF). The OvCatF-deduced amino acid sequence was analyzed and predicted for B-cell epitopes used for short peptide synthesis. The synthetic peptides were used to screen the phage library simultaneously with OvCatF recombinant protein (rOvCatF). The potentiated phages were collected, rescued, and reassembled in XL1-blue Escherichia coli (E. coli) as a propagative host. The positive clones of phagemids were isolated, and the single-chain variable (scFv) fragments were sequenced, computationally predicted, and molecular docked. The complete scFv fragments were digested from the phagemid, subcloned into the pOPE101 expression vector, and expressed in XL1-blue E. coli. Indirect ELISA and Western analysis were used to verify the detection efficiency. RESULTS: The scFv phages specific to OvCatF were successfully isolated, subcloned, and produced as a recombinant protein. The recombinant scFv antibodies were purified and refolded to make functional scFv. The evaluation of specific recognition of the particular epitopes and detection limit results by both computational and laboratory performances demonstrated that all three recombinant scFv antibodies against OvCatF could bind specifically to rOvCatF, and the lowest detection concentration in this study was only one hundred nanograms. CONCLUSION: Our produced scFv antibodies will be the potential candidates for developing a practical diagnostic procedure for O. viverrini infection in humans in the future.

Current prevalence and geographic distribution of helminth infections in the parasitic endemic areas of rural Northeastern Thailand.

BACKGROUND: Helminth infection is a global health issue that not only causes acute helminthiasis but long-term infection may lead to complicated symptoms as well as severe complications. The World Health Organization cooperated with the Ministry of Public Health in many countries, particularly where high prevalence, spending a lot of resources for limiting the infection. In Thailand, the incidence of parasitic helminth infections was continuously declined in the last few decades according to several campaigns for parasitic elimination. However, the rural community in the northeast of Thailand where the highest prevalence of the country still needs to be monitored. This present study aims to report the current prevalence of parasitic helminth infections in Nakhon Ratchasima and Chaiyaphum provinces where sharing a huge area of the northeastern region of Thailand but only a few studies have been published. METHODS: The stool specimens were collected from 11,196 volunteers and processed by modified Kato-Katz thick smear, PBS-ethyl acetate concentration techniques, and PCR. The epidemiological data were collected, analyzed, and used for generating of parasitic hotspots. RESULTS: The results indicated that O. viverrini remains the major parasite in this area with a total prevalence of 5.05% followed by Taenia spp., Hookworms, T. trichiura, and Echinostoma spp., respectively. Mueang district of Chaiyaphum province has the highest prevalence especially O. viverrini with a prevalence of 7.15% that higher than the latest national surveillance. Interestingly, the prevalence of O. viverrini was hugely reported (more than 10%) in five subdistricts. The geographic localization of O. viverrini infections revealed that a lot of water reservoirs such as the lakes or branches of the river in the two-most prevalent subdistricts. Our finding indicated that gender and age were insignificantly different. CONCLUSION: This finding suggested that the parasitic helminth infection in the rural areas of northeast of Thailand remains high and the housing location is a major contributing factor for the parasitic infection.

Molecular Cloning and Characterization of a Fasciola gigantica Nuclear Receptor Subfamily 1 (FgNR1).

Fasciola gigantica, a giant liver fluke, causes tremendous loss to the livestock economy in several regions throughout the world. The situation of drug resistance has been emerging increasingly; therefore, novel drugs and drug targets need to be discovered. The adult F. gigantica inhabits the major bile ducts where bile salts accumulate­these are steroid-like molecules that mediate several physiological processes in organisms through interacting with their specific nuclear receptors. However, the molecular mechanism of the interaction in the parasitic organisms have not been clearly understood. In this study, putative nuclear receptor subfamily 1 of F. gigantica (FgNR1) was identified. Nucleotide and amino acid sequences of the FgNR1 homolog were obtained from the transcriptome of F. gigantica and predicted for properties and functions using bioinformatics. The full-length cDNA was cloned and expressed in the bacterial expression system and then used for immunization. Western analysis and immunolocalization suggested that FgNR1 could be detected in the crude worm antigens and was highly expressed in the caeca and testes of the adult parasite. Moreover, the bile could significantly activate the expression of FgNR1 in cultured parasites. Our results indicated that FgNR1 has high potential for the development of a novel anthelminthic drug in the future.

Evaluation of indirect-ELISA using eluted antigens from Trichinella spiralis muscle larvae for diagnosis of swine trichinellosis.

Trichinellosis is caused by Trichinella spiralis muscle larvae (TsML), which is transmitted to human when they eat infected raw or undercooked meat. T. spiralis infection is detected by an enzyme-linked immunosorbent assay (ELISA) using excretory-secretory antigens (ESAg); however, the preparation of ESAg is challenging, and yields are low, which hampers screening efforts. In this study, crude somatic antigens (CSAg) of TsML with molecular weights (MWs) of 43, 79 and 101 kDa have been identified in swine trichinellosis sera with less cross-reaction with uninfected sera and other parasitic infected sera. After that, the CSAg at MWs of 43, 79 and 101 kDa (TsCSAg-43, TsCSAg-79, and TsCSAg-101, respectively) were isolated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The eluted antigens were analyzed by IgG-ELISA for sensitivity and specificity, and specific antigens from the three regions were identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The sensitivity of IgG-ELISA using the three eluted antigens was 100% with specificities of 97.77%, 95.54%, 90.63% and for TsCSAg-43, TsCSAg-79, and TsCSAg-101, respectively. The LC-MS-MS results of immunomics showed that 18/20 spots of the antigens with MWs of 43, 79, and 101 kDa represent 11 different proteins identified. TsCSAg-43 showed the highest specificity, indicating that the specific proteins identified, including 45 kDa antigen-trichina [fragment], DNA topoisomerase 2-alpha antigen targeted by protective antibodies, and a conserved hypothetical protein (gi339234223), should be developed and produced in large volumes for further immunodiagnostic studies.

Molecular cloning and characterization of serine protease inhibitor from food-borne nematode, Gnathostoma spinigerum.

Gnathostoma spinigerum is a causative agent of human gnathostomiasis and infects people residing in endemic areas as well as travelers. Cutaneous and visceral larval migrants cause clinical manifestations, resulting in severe morbidity and mortality. To survive in hosts, these parasites have evolved various immune evasion mechanisms, including the release of regulatory molecules. Serine protease inhibitors (serpins) that are present in many parasitic helminths are proteins suspected of suppressing host serine protease-related digestion and immune responses. In this study, the serpin secreted by G. spinigerum (GsSerp) was characterized using bioinformatics and molecular biology techniques. The bioinformatics revealed that GsSerp contains 9 helices, 3 ß-sheets, and a reactive central loop, which are conserved structures of the serpin superfamily. Recombinant GsSerp (rGsSerp) was expressed in E. coli (molecular weight, 39 kDa) and could inhibit chymotrypsin. Mouse polyclonal antibody against GsSerp could detect the native GsSerp in crude worm antigen but not the excretory-secretory product (ES) of infective-stage larva (aL3Gs). Moreover, the expression of GsSerp in the aL3Gs tissue was located in the hemolymph and intestinal tissue, indicating its role in parasite homeostasis. Our findings may help develop effective strategies for preventing and controlling gnathostomiasis.

Molecular characterization and functional analysis of the Schistosoma mekongi Ca2+-dependent cysteine protease (calpain).

BACKGROUND: Schistosoma mekongi, which causes schistosomiasis in humans, is an important public health issue in Southeast Asia. Treatment with praziquantel is the primary method of control but emergence of praziquantel resistance requires the development of alternative drugs and vaccines. Calcium-dependent cysteine protease (calpain) is a novel vaccine candidate that has been studied in S. mansoni, S. japonicum, and protozoans including malaria, leishmania and trypanosomes. However, limited information is available on the properties and functions of calpain in other Schistosoma spp., including S. mekongi. In this study, we functionally characterized calpain 1 of S. mekongi (SmeCalp1). RESULTS: Calpain 1 of S. mekongi was obtained from transcriptomic analysis of S. mekongi; it had the highest expression level of all isoforms tested and was predominantly expressed in the adult male. SmeCalp1 cDNA is 2274 bp long and encodes 758 amino acids, with 85% to 90% homology with calpains in other Schistosoma species. Recombinant SmeCalp1 (rSmeCalp1), with a molecular weight of approximately 86.7 kDa, was expressed in bacteria and stimulated a marked antibody response in mice. Native SmeCalp1 was detected in crude worm extract and excretory-secretory product, and it was mainly localized in the tegument of the adult male; less signal was detected in the adult female worm. Thus, SmeCalp1 may play a role in surface membrane synthesis or host-parasite interaction. We assessed the protease activity of rSmeCalp1 and demonstrated that rSmeCalp1 could cleave the calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, that was inhibited by calpain inhibitors (MDL28170 and E64c). Additionally, rSmeCalp1 could degrade the biological substrates fibronectin (blood clotting protein) and human complement C3, indicating important roles in the intravascular system and in host immune evasion. CONCLUSIONS: SmeCalp1 is expressed on the tegumental surface of the parasite and can cleave host defense molecules; thus, it might participate in growth, development and survival during the entire life-cycle of S. mekongi. Information on the properties and functions of SmeCalp1 reported herein will be advantageous in the development of effective drugs and vaccines against S. mekongi and other schistosomes.

Transcriptomic analysis of male and female Schistosoma mekongi adult worms.

BACKGROUND: Schistosoma mekongi is one of five major causative agents of human schistosomiasis and is endemic to communities along the Mekong River in southern Lao People's Democratic Republic (Laos) and northern Cambodia. Sporadic cases of schistosomiasis have been reported in travelers and immigrants who have visited endemic areas. Schistosoma mekongi biology and molecular biology is poorly understood, and few S. mekongi gene and transcript sequences are available in public databases. RESULTS: Transcriptome sequencing (RNA-Seq) of male and female S. mekongi adult worms (a total of three biological replicates for each sex) were analyzed and the results demonstrated that approximately 304.9 and 363.3 million high-quality clean reads with quality Q30 (> 90%) were obtained from male and female adult worms, respectively. A total of 119,604 contigs were assembled with an average length of 1273 nt and an N50 of 2017 nt. From the contigs, 20,798 annotated protein sequences and 48,256 annotated transcript sequences were obtained using BLASTP and BLASTX searches against the UniProt Trematoda database. A total of 4658 and 3509 transcripts were predominantly expressed in male and female worms, respectively. Male-biased transcripts were mostly involved in structural organization while female-biased transcripts were typically involved in cell differentiation and egg production. Interestingly, pathway enrichment analysis suggested that genes involved in the phosphatidylinositol signaling pathway may play important roles in the cellular processes and reproductive systems of S. mekongi worms. CONCLUSIONS: We present comparative transcriptomic analyses of male and female S. mekongi adult worms, which provide a global view of the S. mekongi transcriptome as well as insights into differentially-expressed genes associated with each sex. This work provides valuable information and sequence resources for future studies of gene function and for ongoing whole genome sequencing efforts in S. mekongi.