Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia

Abstract

Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1-3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this "minimal Golgi" hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting functions.

Figure 1. Processing of epitope-tagged CWP2 variants and CWM distribution in differentiating cells.

Western blot analysis of Flag-CWP2-HA expression and processing between 0 to 12 h p.i. (A). Products (pro-form: asterisk, mature form: arrowhead) were detected in separated total lysates using anti-Flag (left panel) or anti-HA (right panel) antibodies. B) A schematic rendering of the Flag-CWP2-HA expression construct: bent arrow, CWP1 promoter; solid line, CWP1 flanking regions; boxes: signal sequence (black), epitope tags (white), CWP2 N-terminal (dark gray) and C-terminal (light gray) domains. Deleted regions in ΔPS or ΔPS3 variants are indicated. Western blots of separated crude lysates of transgenic parasites (12 h p.i.) expressing full-length Flag-CWP2-HA, or deletion products ΔPS or ΔPS3 were probed with anti-HA antibody (right panel) which labels pro-forms only while the anti-Flag antibody (left panel) labels also the large N-terminal mature forms. Note the absence of a processed form of ΔPS3. To avoid any cross-reactions two separate blots were made. C) Fluorescence and transmission EM micrographs of parasites 12 h p.i. Typical “ring-like” distribution of CWP1 in ESVs (left panel, arrows) detected with a specific monoclonal antibody. Accumulation of electron dense material in ESVs (arrows) suggests condensation of cargo (right panel, arrows). N, nucleus; scale bars 2 µm. Inset: bright field differential interference contrast (DIC) image.

Figure 2. Confocal microscopy of Flag-CWP2-HA and HA-CWP3 reporters in representative cells during early stages of encystation.

CWP1 (red) is used as a counter stain throughout. Insets: DIC images; scatter plots show the results of colocalization analyses of the respective markers in the entire image stacks. A) Signals from unprocessed Flag-CWP2-HA (green) overlaps with CWP1 (red) in emerging ESVs at 6 h post-induction. B, C) Partitioning of processed Flag-CWP2-HA at 12 h p.i.: The N-terminal Flag-N overlaps with CWP1 in a “ring-like” distribution, whilst the C-terminal ΔC-HA fragment is concentrated in the center of ESVs resulting in dramatically reduced overlap with CWP1. D) Like ΔC-HA, HA-tagged CWP3 (HA-CWP3) partitions in the central region of ESVs. Nuclear DNA is stained with DAPI (blue). Scale bars: 3 µm.

Figure 3. Fluorescence recovery after photobleaching (FRAP) analysis reveals condensation of CWP3::GFP during ESV maturation.

Quantitative analysis of cargo motility: Photobleaching of a single ESV (region of interest 1 (ROI 1) arrow) in a living cell at 6 h p.i. (A) results in recovery (green line in graphs) of fluorescence (see also Figure S3A) with similar kinetics as a CWP1::GFP reporter . Purple and amber lines represent unbleached control organelles (ROIs 2 and 3). Fluorescence micrographs from the image series at the start (0 sec) of the experiment, during bleaching, and at the beginning of the recovery phase (19 sec) are shown. In cells at 12 h p.i. (B) fluorescence in a bleached ESV (ROI 1, arrow) does not recover (see also Figure S3B) which is consistent with immobilization of CWP3::GFP in a condensed core. Arbitrary units of fluorescence are indicated [I]. Unbleached control organelles (ROIs 2 and 3) are indicated. Broken lines connect pre- and post bleaching values in the graph.

Figure 4. Confocal microscopy of the Flag-CWP2-HA and HA-CWP3 reporters in representative cells during late stages of encystation.

In transgenic cells at 14 h post-induction, CWP1 (red) appears to be sorted away from ΔC-HA (A) or HA-CWP3 (B) to compartments at the cell periphery. In cysts emerging between 16–24 h p.i (C), CWP1 appears quantitatively secreted to the cyst wall when morphological transformation takes place whilst all ΔC-HA (green) remains in internal compartments. ΔC-HA is secreted at a later time (D, E) and eventually fully incorporated into the cyst wall. Images show representative cysts (24 and 26 h p.i., respectively) at different stages of maturation. Insets: DIC images; scatter plots show the results of colocalization analyses of the respective markers in the entire image stacks. Nuclear DNA is stained with DAPI (blue). Scale bars: 3 µm.

Figure 5. Inhibition of CWP2 and Flag-CWP2-HA processing by E64 treatment leads to mis-targeting and formation of cysts which are significantly less water resistant.

(A) Western blots of separated crude lysates of transgenic parasites (12 h p.i.) expressing Flag-CWP2-HA in the presence (+) or absence (−, DMSO alone) of E64. The drug significantly abolishes processing of the Flag-CWP2-HA pro-form as well as the endogenous pro-CWP2 (B). Immunofluorescence analysis of early cysts derived from cells expressing Flag-CWP2-HA (C) shows mis-targeting of the unprocessed reporter and extensive co-localization of the Flag (red) and HA epitopes (green) in internal compartments containing CWMco after treatment with E64 (left panels). CWP1 is sorted and secreted with CWMfl, however (upper panel). In untreated control cells (right panel) the mature N-Flag fragment of the reporter is almost completely sorted away from ΔC-HA and incorporated into the CW. Nuclear DNA is stained with DAPI (blue). Scale bars: 3 µm. Cyst derived from E64-treated cells (D) have a considerably reduced survival rate compared to untreated controls.

Figure 6. Model for the construction of the cyst wall from a single-type cargo secreted in two stages.

The open circular graph depicts identifiable stages (hours post induction) of encystation. Sorting pathways are indicated by grey arrows. First appearance of CWPs in the ER (A) at about 2 h p.i. is followed by sorting of CWM from constitutively secreted cargo at transitional ER (tER) sites (B). ESVs increase in size (C) until all CWM is exported from the ER. Appearance of condensed cores in ESVs (D) coincides with processing of CWP2 and leads to partitioning of the CWM into two fractions. Maturing ESVs exchange soluble CWP1 (E) via dynamic membrane tubules. Cargo partitioning inside ESVs is followed by a sorting process separating the two CWM fractions is distinct compartments (F). CWMfl is secreted rapidly and establishes an outer cyst wall (G). Subsequent slow secretion of the CWMco fraction (H) over several hours likely requires decondensation of this material. Maturation must be completed before cysts become water resistant.