Fig 1.
TEM showed purified A. castellanii cyst walls retained endocyst and ectocyst layers and ostioles.
A, B. A mature cyst and a purified cyst wall had an outer ectocyst layer (yellow arrows), an inner endocyst layer (pink arrows), and ostioles (turquoise arrows) that connect the layers. Endocyst and ectocyst layers had the same appearance in mature cysts (C) and purified cyst walls (D). Purified cyst walls were missing amorphous material (purple arrow) between the wall and the plasma membrane of mature cysts. E. At the edge of the ostiole of a mature cyst, the endocyst layer bifurcated, and the outer branch met the ectocyst layer. In the center of the ostiole, the ectocyst layer formed a narrow cap over the inner branch of the endocyst layer. Scale bars as marked on micrographs.
Fig 2.
SIM showed purified A. castellanii cyst walls retained distinct ectocyst layer and endocyst layer, as well as ostioles.
The ectocyst layer (yellow arrows) of a mature cyst (A) and purified cyst wall (B) labeled red with GST-AcCBM49; the edges of ostioles (turquoise arrows) labeled green with WGA; and the endocyst layer (pink arrows) labeled blue with CFW. WGA also labeled less strongly the endocyst and ectocyst layers. While it appears that some ostioles overlap each other, rotation of the deconvolved images showed they are actually on opposite sides of the spherical surface. Scale bars are 2 μm.
Table 1.
Candidate cyst wall proteins of A. castellanii identified by mass spectrometry.
Fig 3.
Abundant cyst wall proteins contained two CBM49s (Luke(2) lectin), two 8-Cys domains (Leo lectin), or one CAA domain (Jonah(1) lectin).
The Luke(2) lectin had an N-terminal signal peptide (purple) and two CBM49s separated by short Ser- and Pro-rich spacers (light blue). The N-terminal CBM49 contained four Trp residues (red Ws), three of which were conserved in a C-terminal CBM49 of a tomato cellulase (larger font) and three of which were conserved in a single CBM49 of Dictyostelium cellulose-binding protein (XP_629733) (underlined). In contrast, the C-terminal CBM49 had two conserved Trp residues. The Leo lectin had a signal peptide and two unique domains (dark blue) containing eight Cys residues each (red Cs). The Jonah(1) lectin had a signal peptide, a Thr-, Lys-, and Cys-rich domain (gray), and a single CAA domain (green). Peptides used to immunize rabbits are underlined. An abundant Luke(3) lectin with three CBM49s, Leo(TKH) lectin with a Thr-, Lys-, and His-rich spacer, and an abundant Jonah(3) lectin with three CAA domains are shown in S2 Fig.
Fig 4.
During the first stage of encystation, SIM showed Jonah(1)-GFP and glycopolymers labeled by WGA and GST-CBM49 were present in dozens of distinct vesicles.
A. After 3 hr encystation, Jonah(1)-GFP (green), which was expressed under its own promoter, was present in dozens of small vesicles. WGA (red) labeled fewer but larger vesicles, which did not overlap with those containing Jonah(1)-GFP (see merge). B. After 6 hr encystation, glycopolymers labeled by GST-CBM49 (red) were present in dozens of vesicles that did not overlap with those labeled by WGA (green). C. After 9 hr encystation, glycopolymers labeled with GST-CBM49 and WGA were again very abundant in vesicles that did not overlap. CFW was not visible in vesicles of organisms encysting for 3 to 6 hr, but CFW (blue) labeled the surface of encysting protists at 9 hr. A single ostiole, which was small and circular (turquoise arrow), was present on the surface of one encysting protist, while ostioles were absent from the other organisms. When expressed under its own promoter, neither Luke(2)-GFP nor Leo-GFP was present in the first stage of encystation. A to C. Scale bars are 2 μm.
Fig 5.
During the second stage of encystation, SIM showed a primordial cyst wall was comprised of Jonah(1)-GFP and glycopolymers labeled with GST-CBM49 and WGA, each in a diffuse pattern, while small, flat ostioles were outlined by Luke(2)-GFP and labeled with CFW.
A. After 12 hr encystation, GST-CBM49 (red) diffusely labeled a thin, primordial wall, which contained small, flat ostioles (turquoise arrows) visible only with CFW (blue). WGA (green), which predominantly labeled vesicles, also labeled the thin, primordial wall. After 15 hr encystation, Jonah(1)-GFP (green), expressed under its own promoter, was homogenously distributed in the primordial wall (B), while Luke(2)-GFP (green), also expressed under its own promoter, outlined the edges of small ostioles in some cells (C). After 18 hr encystation, Luke(2)-GFP, which continued to outline the edges of small ostioles, also spread across the surface of some primordial walls (D), while Leo-GFP, expressed under its own promoter, was in a patchy distribution in primordial walls that was, for the most part, independent of ostioles (E). Also at 18 hr in some cells, there were the beginnings of an outer ectocyst layer (yellow arrow) and an inner endocyst layer (pink arrows). A to E. Scale bars are 2 μm.
Fig 6.
During the third stage of encystation, SIM showed Jonah(1)-GFP remained in the ectocyst layer, while Luke(2)-GFP and Leo-GFP moved to the endocyst layer and ostioles.
Each of the GFP-tagged lectins was expressed under its own promoter. Jonah(1)-GFP (green) was abundant in the ectocyst layer (yellow arrows) of walls of protists encysting for 24 hr (A) or 36 hr (D). Luke(2)-GFP was homogeneously distributed in the endocyst layer (pink arrows), as well as dome-shaped ostioles (turquoise arrows), at 24 hr (B) and at 36 hr (E) encystation. Leo-GFP was present in vesicles and in a somewhat patchy distribution in both the ectocyst and endocyst layers of organisms encysting for 24 hr (C). It was not until 36 hr (F) that Leo-GFP began to sharply outline ostioles. CFW (blue) consistently labeled the endocyst layer and occasionally labeled the ostioles (C) or ectocyst layer (F). WGA (red) labeled the endocyst layer (A), the ectocyst layer (C and F), or both layers (B, D, and E). A to F. Scale bars are 2 μm.
Fig 7.
SIM showed an abundant Jonah(1) lectin localized to the ectocyst layer of mature cyst walls, while abundant Luke(2), Luke(3), and Leo lectins localized to the endocyst layer and ostioles.
A. A Jonah(1) lectin with a single CAA domain, which was tagged with GFP and expressed under its own promoter in transfected A. castellanii, localized to the ectocyst layer (yellow arrows) of the wall of mature cysts. WGA labeled both ectocyst and endocyst layers, while CFW labeled the endocyst layer (pink arrows) and ostioles (turquoise arrows). B. Leo-GFP with two 8-Cys domains, which was also expressed under its own promoter, was present in the endocyst layer and sharply outlined the ostioles. Luke(2)-GFP with two CBM49s (C) and Luke(3) with three CBM49s (D), each expressed under its own promoter, were also present in the endocyst layer and outlined conical ostioles of mature cysts. When expressed under a GAPDH promoter, Jonah(1)-GFP localized to the ectocyst layer (E), while Luke(2)-GFP localized to the endocyst layer and ostioles (F). CFW (blue) consistently labeled the endocyst layer, often labeled the ostioles (A to D), and rarely labeled the ectocyst layer (A and E). In this experiment, WGA (red) consistently labeled the ectocyst layer, often labeled the endocyst layer (C, D, and F), and often labeled ostioles (C, D, and F). A to F. Scale bars are 2 μm.
Fig 8.
Western blots showed Luke(2), Leo, and Jonah(1) lectins fused to MBP or tagged with GFP bound well to microcrystalline cellulose, while binding to chitin beads was variable.
MBP-lectin fusions and MBP alone, which were made as recombinant proteins in the periplasm of bacteria, were incubated with microcrystalline cellulose (A) or chitin beads (B). Total proteins (T), bound proteins (B), and unbound proteins (U), as well as molecular weight markers (M), were run on SDS-PAGE, transferred to PVDF membranes, and detected with an anti-MBP reagent. Full-length products in total fractions are underlined in red. MBP-Leo partially bound to microcrystalline cellulose and bound weakly, if at all to chitin. MBP-Luke(2) and MBP-Jonah(1) each bound more completely to cellulose than to chitin. MBP alone (negative control) did not bind to cellulose or chitin. Luke(2)-GFP, Jonah(1)-GFP, and GFP alone, each of which was expressed under the GAPDH promoter, were released from lysed trophozoites and incubated with microcrystalline cellulose or chitin beads. Luke(2)-GFP, which included some breakdown products, bound completely to cellulose and partially to chitin. Jonah(1)-GFP, which also included some breakdown products, bound partially to microcrystalline cellulose but not at all to chitin. GFP alone (negative control) did not bind to cellulose or chitin.
Fig 9.
Widefield microscopy showed Jonah(1)-GFP and glycopolymers labeled by MBP-Jonah(1) were accessible in the ectocyst layer of mature cyst walls, while Luke(2)-GFP, Leo-GFP, and glycopolymers labeled by MBP-Luke(2) and MBP-Leo were inaccessible in the endocyst layer and ostioles.
A. Nearly 100% of mature cysts expressing Jonah(1)-GFP (green) under its own promoter were labeled with anti-GFP antibodies (red). CFW (blue) labeled the endocyst layer of cyst walls. B. Just 3% of mature cysts expressing Luke(2)-GFP under its own promoter labeled with anti-GFP antibodies. C. Just 2% of mature cysts expressing Leo-GFP under its own promoter labeled with anti-GFP antibodies. D. MBP-Jonah(1) (red) labeled 100% of mature cysts, which were also labeled with WGA (green) and CFW. In contrast, MBP-Luke(2) (E) and MBP-Leo (F) each labeled 9% of mature cysts. While it was sometimes possible to distinguish the ectocyst and endocyst layers with widefield and DIC microscopy, it was not easy. Similarly, it was very difficult to identify ostioles with widefield microscopy and impossible with DIC. A to F. Scale bars are 5 μm. SIM of mature cysts labeled with MBP-lectin fusions are shown in S7 Fig.