Effects Of Giardia Cathepsin B-like Gene Interference On Parasite Growth And Giardia Virus MRNA

Giardia lamblia, the cause of zoonotic giardiasis worldwide, is aflagellated unicellular eukaryotic microorganism. Giardia parasites can infect avariety of hosts, including humans, mammals, birds and amphibians. In recent years,Giardia is considered to be an opportunistic pathogenic protozoa. Giardia lambliavirus (GLV) is a dsRNA virus that infects Giardia lamblia. Viral vector system basedon GLV has been successfully used in the field of gene expression and gene structureand function analysis. Miniature ribozyme molecule is easy-to-design and synthesis,and can be applied both in vitro and in vivo for target RNA degradation. Comparativeproteomic analysis in our group by iTRAQ between trophozoites of G. canis withvirus and trophozoites without virus has identified Cathepsin B-like protein as thedifferentially regulated protein.In the present study, the effects of Cathepsin B-likegene interference on Giardia canis growth and Giardia virus mRNA expression wereinvestigated.The secondary structure of Cathepsin B-like Protease mRNA was analyzed withthe RNA draw software and the Ribozyme cleavage points were chosen. The specificsequence for the antisense-miniature ribozyme was synthesized and the ribozyme wascloned into Giardia canis virus vector with long and short Cathepsin B-like Proteaseantisense RNA sequences flanked with each arm.To test the in vitro cleavage efficiency, the recombinant vector was linearized todetect the cleavage of Cathepsin B-like Protease mRNA. The cleavage activity of invitro transcribed miniature ribozyme against Cathepsin B-like Protease was analyzedby real-time quantitative RT-PCR. The in vitro cleavage activities of the two ribozymeCRzS or CRzL on Cathepsin B-like Protease mRNA were75.42%and83.14% respectively as detected by real-time quantitative RT-PCR. On contrast, PCB revealedan activity of23.0%and GRzL of0.02%. The miniature ribozyme showed a highcleavage activity against the in vitro transcripts of Cathepsin B-like Protease.To examine the in vivo efficiency, the two ribozymes CRzS and CRzL wereintroduced into GCV-infected G. canis trophozoites by electroporation. CathepsinB-like mRNA in the transfected cells were decreased by73.5%with CRzS and88.5%with CRzL as detected by relative real-time quantitative RT-PCR.To test the effects of the two ribozymes CRzS and CRzL on the growth ofparasites and the mRNA expression of the virus, ribozymes were introduced intoGCV-infected G. canis trophozoites by electroporation. The results showed that theRDRP gene mRNA of Giardia canis virus in CRzL and CRzS treated cells declinedup to91%and82%respectively, and the capsid protein gene mRNA of Giardia canisvirus declined up to94%and82%respectively. The inhibition of Cathepsin B-likegene mRNA also caused a significant slowing down in trophozoites growth (P<0.05).So Cathepsin B-like Protease is a factor relatived to parasite growth and Giardia virusreplication.