Abstract
ObjectiveRNA interference and miRNA mediated mechanisms have been proposed to explain the expression of a specific variant of VSP at a time on the surface of Giardia lamblia. Recently, epigenetic mechanisms involving histone acetylations have been proposed to explain the process of vsp gene switching in Giardia lamblia. However, due to the limited availability of specific antibodies for all the vsp variants present in the genome, it was difficult to monitor vsp gene switching. In this study, we have used an endogenous tagging method to tag specific vsp genes vsp1267 and vsp9B10A with a sequence encoding hemagglutinin (HA) epitope at the 3′end of the coding sequences without altering the 5′ upstream elements. With this method, we have monitored the expression of the tagged vsp genes in cells treated with histone deacetylase inhibitors using RT-PCR.ResultsOur results show that vsp1267-3XHA can be induced by treatment with sodium 4-phenylbutyrate, M344 and splitomicin but not by apicidin and Trichostatin A, while vsp9B10A-3XHA expression can be induced by Trichostatin A and splitomicin but not by sodium 4-phenylbutyrate, M344 and apicidin. The induced expression of these variants was not due to growth inhibition. These results support the role of histone acetylations in vsp expression.
Highlights
Giardia lamblia antigenic variation involves the expression of a single variant-specific surface protein (VSP) on the trophozoite surface from a library of approximately 200 distinct vsp genes [1]
Our results show that vsp1267-3XHA can be induced by treatment with sodium 4-phenylbutyrate, M344 and splitomicin but not by apicidin and Trichostatin A, while vsp9B10A-3XHA expression can be induced by Trichostatin A and splitomicin but not by sodium 4-phenylbutyrate, M344 and apicidin
It was demonstrated that histone deacetylase inhibitor (HDACi) Trichostatin A increases the rate of VSP switching after 5 days of treatment, while sodium butyrate and nicotinamide had minor effects on switching rates [6]
Summary
Endogenous tagging The plasmid constructs pKS-BSR-vsp1267tr-3XHA and pKS-BSR-9B10Atr-3XHA were linearized with Eco721 and transfected into Giardia cells (Fig. 1a). These amplicons may represent the intermediates of transcripts degraded by RNAi and/or miRNA mediated mechanisms [4, 5] Both in untreated control and treated cell lines, the expression of the internal control gene GleIF4A was detected (Fig. 2b, lanes, 2, 4, 6, 8, 10 and 12). There was no significant decrease in the growth of the parasites when treated with M344, NaPB, or splitomycin, when compared to the untreated controls (Fig. 3) These results suggest that the changes in the expression of vsp1267-3XHA (Fig. 2a) observed after 24 h of HDAC inhibitor treatment were not due to cell toxicity
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