| Giardiasis is an intestinal disease caused by the protozoan parasite Giardia lambliaworldwide. Its main symptomis is diarrhea. It has been recognized as one of10major parasiticdiseases that have a high impact on public health.There is currently no effective method treatmentagainst these protozoa by reason that details of molecular and cell biology viewpoints of gairdiado not be understood. Giardia lamblia has two stages in the life cycles–trophozoite and cyst. Theresistant cyst can remain infective for long periods once released by the host. Giardial encystationhas been studied from structural genomics and identified many genes that may play importantroles during this process, but the molecular mechanism of encystation remains unknown.Observation of Giardia lamblia virus (GLV) that inhabits the cytoplasm of Giardia lamblia madeit more complicated. Studies showed that GLV high concentration infection enables change ofsubcellular structure of giardia resulted in an increase in the number of nonadhering cells andcessation of growth. The present study of GLV has focused on construction of virus-mediatedvector, and virus-mediated vector have been used successfully to determine the roles of specificgenes in giardia. There were no reports on the life cycle of GLV. The study of life cycle of GLVprovided new directions for molecular, cell biology viewpoints of gairdia, as well as virus-hostinteractions.Proteomics study is a powerful tool for the growth, development, vaccines andchemotherapies of parasites. Comparative proteomic analysis is an important strategy ofproteomics. The advanced technology of comparative proteomic can quickly and effectively beused in the study of several organism differential proteomics at different times or differentstates．So it is very important for understanding disease progression and pathogenesis. Withdeveloping of comparative proteomic technology, people arouse more interests to pursue differentstudy by using these technologies. iTRAQ (isobaric tag for relative and absolute quantitation) canbe used to simultaneously label and analyze up to four different samples and was quickly adoptedby proteomic community to give more reliable determination of sequences of peptides which canbe subsequently used for more accurate protein determination. In this study, comparative proteomeanalysis of different strains and development stages of Giardia canis (G.canis) were done byiTRAQ. Comparative proteomic analysis of trophozoites from G. canis with virus and virus-freestrains. Of total311proteins were identified by iTRAQ labelling. The expression levels of58identified proteins were drastic altered (>1.2),37of which were described as being giardiaproteins. We demonstrated that the expression of15proteins increases significantly introphozoites from G. canis with virus, on the other hand22proteins decrease. These virus-inducedproteins included cytoskeletal proteins, metabolic enzymes, and a protein involved in translation.Expression patterns of α-11giardin (Alp), kinase (Kin), and transcription elongation factor (TEF)of the identified proteins which may be involved in the cycle of virus were examined by real-timePCR, and gene expression was inhibited with hammerhead ribozymes by a G. canisvirus-mediated vector (pNEO/GDH/MCS) respectively in trophozoites from G. canis with virus.The results showed that three genes varied significantly during virus infection which is similarwith expression levels of protein. In this study, we down-regulate the Alp, Kin and TEF genesresulted in a significant decrease of G. canis virus (GCV) copy numbers (P<0.01), however thisdecrease were represented in different forms. We noticed that copy numbers of GCV had irregulardecrease when expression of Alp was inhibited; and when expression of TEF was suppressed,copy number of GCV had declined persistently; additionally the copy numbers of GCV hadsignificantly decreased (95%) two days after expression of Kin was inhibited, and kept at thelower level. Thus, those three proteins are involved in the life cycle of GCV, so we can expect thatinfection mechanism of GCV may be similar with reovirus according to the following reasons,GCV was connected with Alp (homologue of annexin) to form a complex; complex entered intocell and GCV replicated under control of Kin and TEF. We noticed also that inhibition of thosegenes cause significant slowing in trophozoites growth (P<0.05). All our results, indicated animportant role of those three proteins in the life cycle of GCV and growth of G. canistrophozoites.Comparative proteomic analysis of trophozoites versus cysts of G. canis. Totally143proteins were identified by iTRAQ labelling,63of which, described as being giardia proteins. Theexpression levels of16identified proteins were dramatically altered (>1.0) during differentiation,7of which, described as giardia proteins. We demonstrated that the expression of3proteinsincreases and4proteins decreases significantly in cyst protein. Compared with the previous study,ornithine carbamoyltransferase and β-tubulin were up-regulated in cyst, but on the other handα-tubulin was down-regulated in cyst. Arginine deaminase was expressed constitutively both in two different stages. Glyceraldehyde-3-phosphate dehydrogenase (gap1) and VSP were firstreported as differential proteins. Expression patterns of four different (ornithinecarbamoyltransferase、α-tubulin、gap1、VSP) proteins were examined by real-time PCR. Theresults showed that four genes of the previous proteins varied significantly which were similar toexpression levels of protein. gap1was knocked down with hammerhead ribozymes TRzS-G by aG. canis virus-mediated vector, then observation of trophozoites growth and encystation processwas done. The result showed that down-regulation of gap1inhibited trophozoites growth (P<0.05)and changed the morphology of trophozoites. However, gap1down-regulation did not interferewith encystation and the immuno-localization of CWP2. These data strongly suggested that gap1is a critical molecule to modulate this parasite growth and maintain the morphology oftrophozoites.In conclusion, different protein expression profiling was obtained from G. canis with virusand virus-free strains trophozoites, cyst and trophozoite. We also demonstrated4proteins whichare critical molecule to modulate this parasite growth and GCV replication. |
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