Figure 1.
Analysis of S-palmitoylated proteins displays a different pattern in Giardia growing and encysting parasites.
(A) Giardia trophozoites (T) or encysting trophozoites (ET) were labeled with [3H]-palmitic acid and loaded onto SDS-PAGE. The gel was treated with (hyd+) or without (hyd−) the thioester cleavage reagent hydroxylamine. Samples were then analyzed by autoradiography. (B) Western blotting performed on palmitoylated proteins purified by ABE from hcncp-V5 transgenic trophozoites (HCNCp T) or hcncp-V5 transgenic encysting trophozoites (HCNCp ET). (C) Western blotting performed on palmitoylated proteins purified by ABE from wild-type trophozoites (T) or encysting parasites (ET). The approximate sizes are indicated on the right in kDa.
Figure 2.
Inhibition of protein palmitoylation yields a low amount of Giardia cysts.
(A) Growth curves displaying optimal concentrations of 2-BP (left panel) or 2-FP (right panel) that do not affect Giardia growth. Giardia trophozoites were cultured with different concentrations of 2-BP (10, 20, 40, 50, 75 or 100 µM), 2-FP (100, 150 or 200 µM), or DMSO (control) for 48 h. The parasites were then analyzed by staining them with Trypan blue to distinguish live from dead cells and by counting them in a Neubauer chamber. The graph displays the number (mean ± SEM) of parasites counted in three independent experiments. (B) Percentage of encysting parasites and cysts after inhibition of protein palmitoylation. Giardia trophozoites were induced to encyst and 2-BP (10, 20 or 40 µM), 2-FP (100 uM) or DMSO (Control) added to the encysting media. After 48 h, the encysting parasites were stained with anti-CWP1 mAb and analyzed by fluorescence microscopy. One representative cell of each encystation state (encysting I, encysting II, cyst) is shown in the upper panel. The graph in the lower panel represents the percentage (mean + SEM) of the cells counted in each state in three independent experiments. The asterisks indicate significant difference compared with the control (Student's t test: * p<0.05; **p<0.01; ***p<0.001). (C) Number of nuclei in encysting II parasites treated with palmitoylation inhibitors. Trophozoites were induced to encyst and 2-BP (20 or 40 µM), 2-FP (100 µM) or DMSO (Control) added to the encystation media as described above. After 48 h, the encysting parasites were stained with anti-CWP1 mAb and DAPI, and analyzed by fluorescence microscopy. One representative encysting II cell is shown. Scale bars = 5 µm.
Figure 3.
Sequence alignment and schematic drawing of Giardia DHHC proteins.
(A) Multiple Sequence alignment of DHHC proteins shows conserved regions. The amino acid sequences of the total set of Giardia DHHC proteins, Erf2 (Yeast), ZDHHC4 (Human), and PF11_0167 (Plasmodium falciparum) were aligned using T-Coffee software [104]. The conserved DHHC-CRD domain and the DPG and TTxE motifs are indicated in bold. Positions exhibiting absolute identity are shown in pink, and high and lower amino acid similarities in green and yellow, respectively. (B) Schematic representation of the primary structure of Giardia DHHC proteins. The domains were searched using SMART (http://smart.embl-heidelberg.de) [105], [106]. Transmembrane domains were predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM) [107] and TMPred (http://www.ch.embnet.org/software/TMPRED_form.html) with default settings. Signal peptides were predicted with signalP (http://www.cbs.dtu.dk/services/SignalP) [108].
Table 1.
Collection of DHHC proteins in Giardia lamblia.
Figure 4.
(A) Phylogenetic relationships between DHHC proteins from Giardia and several other species. Phylogenetic tree of DHHC proteins inferred from ML analyses is depicted in the left panel. Symbols correspond to aLRT values >0.7. Sequence taxonomic identity is displayed with colors (outer circle around the tree), as shown in the upper right panel. MCs are labeled as A, B, C, D, E and F. Giardia DHHC proteins are colored in red and indicated in black in the inner circle around the tree. Each Giardia DHHC protein position in the tree (MC) is indicated in the table (lower right panel). (B) Trichomonas duplicated DHHC sequences accumulate mutations. Giardia DHHC proteins are indicated in light blue, and Trichomonas DHHC proteins in yellow. Variations in the HC, C, and DHHC portions of the DHHC-CRD domain were mapped in the tree using a green-to-black-to-red color code. Full conservation is depicted in light green, while lack of conservation is shown in red. A clade of highly mutated Trichomonas sequences is displayed in red.
Figure 5.
Orthology relationships between Giardia isolates WB, GS and P15 (Assemblages A, B and E, respectively).
Phylogenetic tree of Giardia DHHC sequences from the three isolates inferred from ML analyses is depicted. Each isolate is indicated with a different color.
Figure 6.
Differential expression of Giardia dhhc genes in growing and encysting parasites.
Expression of gla_8619, gla_1908, gla_8711, EAA36893, gla_9529, gla_16928, gla_6733, gla_96562, gla_2116 transcripts from 48 h encysting parasites (white bars) relative to the expression in growing parasites (black bars). The data are the means and SEM of three separate experiments, and each experiment was carried out in triplicate. The qRT-PCR analysis of dhhc genes was performed as described in Methods. The asterisks indicate that there was significant difference compared with growing parasites (Student's t test: * p<0.05; **p<0.01; ***p<0.001).
Figure 7.
Expression of DHHC-HA proteins in Giardia trophozoites.
Western blotting performed on total protein extracts from dhhc-ha transgenic trophozoites. Expected sizes are indicated in brackets. Relative molecular weights of protein standards (kDa) are indicated on the left.
Figure 8.
Localization of DHHC-HA proteins in trophozoites and effect of DHHC-HA overexpression in encystation.
Subcellular localization of gla_1908-HA (A), gla_2116-HA (B), gla_16928-HA (C), or gla_8711-HA (D) in trophozoites or encysting parasites. For trophozoites, gla_1908-HA, gla_2116-HA or gla_16928-HA were stained with anti-BiP (ER) mAb, anti-HA mAb and DAPI; gla_8711-HA was stained with anti-AP2 (PVs) mAb, anti-HA mAb and DAPI. For encysting parasites, after 48 h of encystation dhhc-ha transgenic parasites were stained with anti-HA mAb, anti-CWP1 mAb and DAPI. The cells were analyzed by fluorescence microscopy. One representative cell from each stage is shown. Yellow areas in trophozoites indicate co-localization between DHHC-HA and ER (gla_1908-HA, gla_2116-HA or gla_16928-HA), or between DHHC-HA and PVs (gla_8711-HA). Yellow areas in encysting parasites indicate co-localization between DHHC-HA and CWP1. The inset in C (gla_16928 transgenic encysting II parasites) corresponds to the zoomed area indicated by the lined box. Scale bars = 5 µm.
Figure 9.
The expression of cyst wall protein transcripts and the amount of cysts are different among dhhc transgenic encysting parasites.
(A) qRT-PCR analysis of cwp1, cwp2, and cwp3 transcripts expression in dhhc transgenic parasites after 48 h of encystation (white bars), relative to the expression in wild-type encysting cells (control) (black bars). The data are the means and SEM of three separate experiments, and each experiment was carried out in triplicate. (B) Percentage of water-resistant cysts in dhhc transgenic parasites determined by flow cytometry after 48 h of encystation. The results are presented as the percentage (mean ± SEM) of cysts in three independent experiments. The asterisks indicate that there was significant difference compared with the control (Student's t test: * p<0.05; **p<0.01; ***p<0.001).
Table 2.
Main features of the Giardia DHHC proteins analyzed in this paper.
Figure 10.
Analysis of palmitoylated proteins in dhhc transgenic growing and encysting parasites displays a similar pattern to wild type parasites.
Giardia trophozoites (T) or encysting trophozoites (ET) were labeled with [3H]-palmitic acid and loaded onto SDS-PAGE. Samples were then analyzed by autoradiography. The approximate sizes are indicated on the right in kDa.