Figure 1.
Proteome profile of G. lamblia trophozoites analyzed at 0, 2, 4, 6, 8, 10, 12 and 14hpie.
(A) Upper row: Wide-field immunofluorescence microscopy analysis of CWP1 (red) localization in representative G. lamblia trophozoites induced to encyst over a 14 hr time period using the 2-step encystation method [29]. Between 2 and 4hpie, CWP1 is mainly localized to the ER. From 6 to 8hpie, ESVs emerge and develop, reaching the partitioning phase for CWPs between 10 and 12hpie. At 14hpie, cyst production is already under way within a population of late-encysting cells. Lower row and far right: corresponding bright-field images. Condensed-core ESVs become visible at 8hpie (white arrow). hpie: hours post induction of encystation. Scale bars: 1 µm. (B) Regulation of protein abundance within 2-hour transitions during the 14 hour encystation time-course experiment. Based on relative quantitative information by nSpC for each identified protein (further information in Table S1), protein abundance across each transition was either up-regulated (up), down-regulated (down) or did not change. The total number of proteins for each dataset is indicated above each bar. Transitions between 0-2-4 and 4-6-8hpie showed a trend for increased and decreased protein abundances respectively while the last 4 hours of encystation were marked by a slight tendency of increased abundances. The associated table reports the exact number of proteins in each category. hpie: hours post induction of encystation.
Table 1.
Encystation markers identified at 0, 2, 4, 6, 8, 10, 12 and 14hpie.
Figure 2.
Venn diagrams depicting proteome profiles of G. lamblia trophozoites analyzed at 0, 4, 8 and 12hpie.
(A) The proteomic analysis done in triplicates of G. lamblia trophozoites after 4, 8 and 12hpie yielded a total of 960 proteins with at least two unique peptides (further information in Table S2). Although the total number of proteins identified in each of the 4 time-points (in bold) was comparable, mean Spearman rank correlation coefficients (in italics) indicated that the 4hpie time-point differed the most with respect to the other 3 time-points. hpie: hours post induction of encystation. (B) In this study, we identified a total of 1063 proteins, 316 of which were annotated as “hypothetical” in the Giardia Genome Database. Based on its current release (GiardiaDB 3.0; 11th of March 2013), the G. lamblia assemblage A WB strain genome was assigned 5237 protein-encoding genes, with 3557 genes awaiting annotation.
Figure 3.
Protein abundance and predicted subcellular distribution of G. lamblia trophozoites analyzed at 0, 4, 8 and 12hpie.
(A) Comparison of changes in protein abundance between 0 and 4hpie (0–4), 4 and 8hpie (4–8) and 8 and 12hpie (8–12). Based on relative quantitative information by nSpC for each identified protein (further information in Table S2), protein abundance across each 4 hour interval was either up-regulated (up), down-regulated (down) or did not change. The total number of proteins for each dataset is indicated above each bar. In total, the abundance of 342 and 303 proteins was significantly increased or decreased between two different time-points. The 4hpie time-point presented the highest number of both detected and significantly regulated proteins, while only few changes were recorded at 12hpie compared to 8hpie. The associated table reports the exact number of proteins in each category. hpie: hours post induction of encystation. (B) Target P and NucPred predictions for the subcellular distribution of all proteins detected at each time-point. The predicted contribution of mitochondrion (mitosome), nucleus and cytosol localized proteins was similar across time-points. In contrast, the number of proteins predicted to traffic to or through the ER was reduced by ca. 50% at the 4hpie time-point (black arrow). The number of proteins which satisfied both algorithm thresholds for reliability is indicated beneath the respective pie-chart; in brackets, the overall number of proteins for each dataset is indicated. hpie: hours post induction of encystation. (C) Target P and NucPred predictions for the subcellular distribution of significantly regulated proteins between 0 and 4 and 4 and 8hpie. Proteins more abundant at 4hpie compared to 0 and 8hpie (4>0hpie and 4>8hpie) were significantly depleted for predicted ER-targeted hits and enriched for putative nuclear targeted proteins. This view was almost reversed for proteins with higher abundance at 0 and 8hpie compared to 4hpie (0>4hpie and 8>4hpie). The number of proteins which satisfied both algorithm thresholds for reliability is indicated beneath the respective pie-chart; in brackets, the overall number of proteins for each dataset is indicated. hpie: hours post induction of encystation.
Figure 4.
Functional annotation for the proteome of G. lamblia trophozoites at 0, 4, 8 and 12hpie.
(A) Proteins identified at 0, 4, 8 or 12hpie were assigned to a total of 17 functional annotation clusters using DAVID. Only clusters with enrichment scores of at least 1 were included in the graph and were further discussed. The number of proteins in each cluster dataset is indicated, followed by the total number of proteins submitted to DAVID (in brackets). Functional clusters that were up-regulated at 0hpie (up 0hpie) and that were either up- or down-regulated at 4hpie (up 4hpie and down 4hpie, respectively) are indicated on the graph. hpie: hours post induction of encystation. (B) Significantly regulated proteins between 0 and 4hpie (0>4hpie and 4>0hpie) and 4 and 8hpie (4>8hpie and 8>4hpie) were assigned to functional annotation clusters using DAVID. The number of proteins in each cluster dataset is indicated, followed by the total number of proteins submitted to DAVID (in brackets). Only clusters with enrichment scores of at least 1 were included in the graph and were further discussed. hpie: hours post induction of encystation.