Endogenous tagging
The plasmid constructs pKS-BSR-vsp1267tr-3XHA and pKS-BSR-9B10Atr-3XHA were linearized with Eco721 and transfected into Giardia cells (Figure 1A). To confirm the integration of the truncated and tagged version of the vsp gene, primers encompassing the entire coding sequence starting from the initiation codon to the stop codon located after the triple HA tag were used (Figure 1B). Amplification of full length HA tagged vsp1267 of size 1.7Kb (Figure 1C lane 2) and full length HA tagged vsp9B10 of size 2.2 Kb (Figure 1D, lane 2), indicated proper integration of the 3XHA tagged gene into the chromosome.
HDACi induce transcription of endogenously tagged vsp genes
To test the hypothesis that inhibition of histone deacetylase activity leads to the expression of vsp genes, trophozoites were incubated with 2µM concentrations of HDAC inhibitors for 24 hours. Total RNA was extracted from Giardia cell lines Glvsp1267-3HA and Glvsp9B10A-3XHA after 24 h post-treatment. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect for HA-tagged vsp gene expression using a full-length vsp primer and a 3XHA primer (Figure 1B). Additionally, Giardia lamblia eukaryotic translation initiation factor 4A (GleIF4A) was used as an internal control. A full length amplicon of size 1.7Kb was detected in Glvsp1267-3XHA cell lines that were treated with M344, NaPB and splitomicin but not in the untreated control (Figure 2A, lanes 5, 7, 11). For M344 and NaPB treatments, full length amplicon of vsp1267-3XHA was detected in 2 out of 3 separate experiments performed on different days. In splitomicin treated cell lines, 8 out of 13 independent experiments detected full length amplicon. In the experiments where the full length amplicons were not detected when treated with M344, NaPB or splitomicin, a smear of amplicons ranging from 0.5 to 1Kb were observed (data not shown). These partial amplicons could represent the intermediates of degraded vsp1267 transcripts. Interestingly, no full length amplicons were detected when the cells were treated with apicidin or TSA (Figure 2A, lanes 3 and 9) and the results were consistent in 7 independent treatments performed on different days. These results agree with the previous reports that showed a decrease in the expression of vsp1267 upon treatment with TSA [4] or no change in expression when treated with apicidin [9]. Both untreated control and HDAC inhibitor treated cells showed a full-length GleIF4A amplicon of 1.2 kb (Figure 2A, lanes, 2,4, 6, 8, 10 and 12).
In contrast to vsp1267-3XHA, full length vsp9B10A-3XHA amplicon of 2.2 Kb was detected in Glvsp9B10A cell lines that were treated with splitomycin or trichostatin A (Figure 2B, lanes 9 and 11, respectively) but not in cell lines treated with apicidin, M344 or NaPB (Figure 2B, lanes 3, 5, and 7, respectively). However, a smear of amplicons ranging from 0.5 to 1.5 Kb was observed in untreated control and in Apicidin, M344 or NaPB treated cells but not in cells treated with splitomicin or TSA. 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 (Figure 2B, lanes, 2, 4, 6, 8, 10 and 12).
Effect of HDACi on Parasite Growth
Replicating trophozoites of Glvsp1267-3XHA cell lines were treated with 2 µM of HDAC inhibitors for up to 48 hours. After 24 hours, no significant growth inhibition was observed in the cell lines treated with all the inhibitors when compared to control (Figure 3). However, after 48 hours of treatment, Apicidin inhibited the parasite growth by 87% (P< 0.001%), while Trichostatin A inhibited the growth by 82.2% (P< 0.001%) when compared to the untreated controls. However, there was no significant decrease in the growth of the parasites when treated with M344, NaPB, or splitomycin, when compared to the untreated controls (Figure 3). These results suggest that the changes in the expression of vsp1267-3XHA (Figure 2A) observed after 24 hours of HDAC inhibitor treatment were not due to cell toxicity.