ABSTRACT
Screening of vaccine or drug target in parasitic helminth is hindered by lack of robust tool for functional studies of parasite protein which account for the availability of only a few anti-helminthic vaccines, diagnostic assay and slower pace of development of an anthelmintic drug. With the piling up of parasite transcriptomic and genomic data, in silico screening for possible vaccine/drug target could be validated by functional characterization of proteins by RNA interference or CRISPR/Cas9. These reverse genetic engineering tools have opened up a better avenue and opportunity for screening parasitic proteins in vitro as well as in vivo. RNA interference provides a technique for silencing targeted mRNA transcript for understanding a gene function in helminth as evidence by work in Caenorhabditis elegans. Recent genetic engineering tool, CRISPR/Cas9 allows knock-out/deletion of the desired gene in parasitic helminths and the other provision it provides in terms of gene knock-in/insertion in parasite genome is still to be explored in future. This manuscript discussed the work that has been carried out on RNAi and CRISPR/Cas9 for functional studies of helminth parasitic proteins.
ABSTRACT
Fasciola gigantica faces a series of threats from various free radicals produced by the host immune system during its invasion through the abdominal cavity and establishment in the bile duct of ruminants, limiting the fluke viability. The role of the superoxide radical produced by Muzaffarnagari sheep immune effector cells against F. gigantica newly excysted juveniles (NEJs) is highlighted in this study, as is the critical role of superoxide dismutase enzyme (SOD) in dismutation of superoxide radicals derived from host immune effector cells in vitro. Three concentrations of the ovine immune effector cells viz. 2.5, 5, and 10 × 106 cells were tested for their ability to induced cytotoxic killing of the parasite. All the three cell concentrations caused significant (p < 0.01) cytotoxic killing of NEJs in comparison to the control groups. Also, reduction of the immune effector cell concentration directly correlates with the NEJs killing. Attachment of immune effector cells to the parasite tegument in the presence of anti-F. gigantica antibodies was found to be critical in inducing NEJs killing via antibody-dependent cell-mediated cytotoxicity (ADCC). However, the addition of SOD greatly inhibits cytotoxic killing of NEJs, demonstrating the importance of SOD enzyme in fluke survival and parasite evasion of the host immunity. Thus, F. gigantica SOD warrants a promising candidate for immunoprophylactic studies in ruminants against the tropical liver fluke.
