Biology subjects

Ittiprasert, W.

Publications and source records attributed to Ittiprasert, W..

6 recordsLinked to original sources

bioRxiv2021

Programmed knockout mutation of liver fluke granulin, Ov-grn-1, attenuates virulence and impedes malignant transformation during chronic opisthorchiasis

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes ({Delta}Ov-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, however less hepatobiliary tract disease manifested during chronic infection with{Delta} Ov-grn-1 worms in comparison to hamsters infected with control gene-edited and non-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with{Delta} Ov-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis. Author summaryInfection with the human liver flukes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis remains a public health concern in regions where these parasites are endemic. O. viverrini is endemic in the Mekong River drainage countries of including Thailand and the Lao Peoples Democratic Republic. Infection follows the consumption of undercooked freshwater fish harboring the parasite. Liver fluke infection, opisthorchiasis, is associated with diseases of the liver and bile ducts including cancer of the biliary tract, cholangiocarcinoma, a cancer with a poor prognosis. This report characterizes for the first time experimental infection with gene-edited O. viverrini liver flukes during concurrent exposure to nitrosamine in a rodent model of liver fluke infection-associated cancer. Cancer development was slowed in hamsters infected with the parasite following CRISPR-based knock-out mutation and loss of a parasite gene known to stimulate growth of cells lining the bile ducts. These findings describe a new model for investigation of risk factors for infection-associated cholangiocarcinoma and to assess efficacy of anti-infection/anti-cancer vaccines.

cancer biology↗

bioRxiv2021

Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production

-galactosidase (-GAL) and -N-acetylgalactosaminidase (-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki-diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important -GAL and -NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansonis -GAL and -NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative -GAL/-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for -GAL/-NAGAL substrate cleavage. Both -GAL and -NAGAL enzymatic activities were higher in females compared to males (p<0.05; -NAGAL > -GAL), which was consistent with smp_089290s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited -NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in -GAL activities were observed in the same extracts. Despite this, decreases in -NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an -NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Pharmacological inhibition of SmNAGAL may lead to the development of a novel anthelmintic class of compounds. Author summarySchistosomiasis is a parasitic disease caused by infection with blood flukes, which leads to acute and chronic pathology in millions of infected individuals located in deprived tropical and subtropical regions. Elucidating the function of schistosome genes has provided a clearer view on their roles in various molecular pathways, which are critical to successful parasitism. This information is invaluable when progressing novel drug and vaccine candidates. Here, we add to the existing knowledge of the Schistosoma mansoni parasitic glycan processing and modification machinery by functionally characterising a glycosyl hydrolase (S. mansoni -N-acetylgalactosaminidase, SmNAGAL). We demonstrate that this protein is enzymatically active and important in coordinating parasite movement in adult male and female schistosomes. Additionally, we provide evidence that this protein regulates pathways associated with egg production in female schistosomes, which is responsible for inducing pathological reactions. Developing drugs that inhibit SmNAGAL enzymatic activity could provide a novel approach for controlling schistosomiasis.

molecular biology↗

bioRxiv2020

Tumor Necrosis Factor and Schistosoma mansoni egg antigen Omega-1 shape distinct aspects of the early egg-induced granulomatous response

Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg.

microbiology↗

bioRxiv2019

Exosome-mediated crosstalk stimulated by liver fluke granulin promotes a microenvironment conducive to cholangiocarcinoma

Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with fish-borne liver flukes is a major risk factor for cholangiocarcinoma (CCA). The parasite secretes a growth factor termed liver fluke granulin (Ov-GRN-1), a homologue of the human progranulin (huPGRN), which contributes significantly to biliary tract fibrosis and morbidity during infection. Here, exosome-mediated transfer of mRNAs from the human cholangiocyte cell line (H69) was investigated following exposure to Ov-GRN-1, to naive recipient H69 cells. To minimize the influence of endogenous huPGRN, the gene encoding huPGRN was inactivated using CRISPR/Cas9-based gene knock-out. Several huPGRN-depleted cell lines, termed {Delta}huPGRN-H69, were established. These lines exhibited >80% reductions in levels of huPGRN transcripts and protein, both in gene-edited cells and within exosomes released by these cells. Profiles of exosomal RNAs (exRNA) from {Delta}huPGRN-H69 for CCA-associated characteristics revealed a paucity of transcripts for estrogen- and Wnt-signaling pathways, peptidase inhibitors and tyrosine phosphatase related to cellular processes including oncogenic transformation. Several CCA-specific mRNAs including MAPK/AKT pathway members were induced by exposure to Ov-GRN-1. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 exposed to Ov-GRN-1. Of these, CCA-associated exRNAs modified the CCA microenvironment in naive recipient cells co-cultured with exosomes from {Delta}huPGRN-H69 exposed to Ov-GRN-1, and induced translation of MAPK phosphorylation related-protein in naive recipient cells comparing with control recipient cells. Exosome-mediated crosstalk in response to liver fluke granulin promoted a CCA-specific program through MAPK pathway which, in turn, established a CCA-conducive disposition.

molecular biology↗

bioRxiv2018

Programmed mutation of liver fluke granulin using CRISPR/Cas9 attenuates virulence of infection-induced hepatobiliary morbidity

Infections with several flatworm parasites represent group 1 biological carcinogens, i.e. definite causes of cancer. Infection with the food-borne liver fluke Opisthorchis viverrini causes cholangiocarcinoma (CCA). Whereas the causative agent for most cancers, including CCA in the West, remains obscure, the principal risk factor for CCA in Thailand is opisthorchiasis. We exploited this established link to explore the role of the secreted parasite growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions of the biliary tract. We targeted the Ov-grn-1 gene for programmed knockout and investigated gene-edited parasites in vitro and in experimentally infected hamsters. Both adult and juvenile stages of the liver fluke were transfected with a plasmid encoding a guide RNA sequence specific for exon 1 of Ov-grn-1 and the Cas9 nuclease. Deep sequencing of amplicon libraries from genomic DNA from gene-edited parasites exhibited programmed, Cas9-catalyzed mutations within the Ov-grn-1 locus, and tandem analyses by RT-PCR and western blot revealed rapid depletion of Ov-grn-1 transcripts and protein. Newly excysted juvenile flukes that had undergone editing of Ov-grn-1 colonized the biliary tract, grew and developed over a period of 60 days, were active and motile, and induced a clinically relevant pathophysiological tissue phenotype of attenuated biliary hyperplasia and fibrosis in comparison to infection with wild type flukes. This is the first report of gene knock-out using CRISPR/Cas9 in a parasitic flatworm, demonstrating the activity and utility of the process for functional genomics in these pathogens. The striking clinical phenotype highlights the role in virulence that liver fluke growth factors play in biliary tract morbidity during chronic opisthorchiasis.

molecular biology↗

bioRxiv2018

Functional genomics and programmed genome editing of omega-1 of the blood fluke Schistosoma mansoni

CRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 ({omega}1) of Schistosoma mansoni as a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of {omega}1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5-and 3-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within {omega}1. CRISPResso analysis of amplicons spanning the DSB revealed [~]4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding {omega}1 were reduced >80% and lysates of {omega}1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the {omega}1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of {omega}1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding {omega}1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of {omega}1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of {omega}1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

molecular biology↗