Article Figures & Data
Figures
- FIG. 1.
Gelatin zymogram analysis comparing the proteinase activities during encystation. The patterns of total proteolytic activity within culture media (CM), trophozoite (T), and cyst cell lysates (Day 1 [1D], 2D, 3D, and 4D) were compared by gelatin zymogram analysis (A). Arrows indicate new proteolytic bands that appear during encystation. Almost all proteolytic activity, induced by encystations, was inhibited by the serine protease inhibitor PMSF (B).
- FIG. 2.
The effect of proteinase activity on encystation. E64 (cysteine proteinase inhibitor) did not affect the formation of mature cyst (A). PMSF (serine proteinase inhibitor) reduced the number of mature cysts in a dose-dependent manner (B). The experiments were repeated three times, and the average values are presented with error bars representing standard deviations. *, means are significantly different at a P value of <0.05 by Student's t test.
- FIG. 3.
Proteinase mRNA expression levels as measured by RT-PCR. The cysteine proteinase, serine-type endopeptidase, peptidase M28, and aspartyl aminopeptidase-like protein all showed similar or lowered mRNA expression levels during encystation (A to D). The serine proteinases of A. castellanii and A. healyi were highly expressed during encystation (E and F). *, means are significantly different at a P value of <0.05 by Student's t test.
- FIG. 4.
Western blot analysis of the polyclonal antibody prepared against a recombinant PMSF protein. The mature form of EMSP was detected as a 33-kDa band during encystation (Day 1 [1D], 2D, 3D, and 4D).
- FIG. 5.
RT-PCR after transfection with EMSP siRNA. After transfection, cells were transferred to encystment media and gathered during encystation from 0 to 3 days. RT-PCR analysis demonstrates that EMSP levels of the trophozoite and cyst forms were similar. The means are not significantly different at a P value of <0.05 by Student's t test.
- FIG. 6.
Inhibition of encystation by EMSP siRNA. Cells transfected with SuperFect transfection reagent, nonspecific negative siRNA, or EMSP siRNA were incubated in encystment media for 4 days, and the numbers of mature cysts were scored. The formation of mature cysts was almost completely inhibited in EMSP siRNA-transfected cells.
- FIG. 7.
Transfection of pUbEMSPg for the intracellular localization of EMSP. The localization of the EMSP-EGFP fusion protein showed fluorescent vesicle-like structures distributed within the cytoplasm (A). Expressed EMSP-EGFP fusion proteins (green signal) completely overlapped with lysosomes stained with LysoTracker red DND-99 (red signal) in A. castellanii (panel B, top) and A. healyi (panel B, bottom). Amoebae with the fluorescence of EGFP alone showed dispersed distribution in the cytoplasm (C). Bar = 10 μm.
- FIG. 8.
Localization of EMSP during encystation. After transfection of plasmid encoding the EMSP-EGFP fusion protein, cells were transferred to encystment media and incubated for 24 to 48 h. Small fluorescent vesicle-like structures gathered and formed ball-like structures (A). These ball-like structures (green signal) colocalized with LysoTracker red DND-99 staining in A. castellanii (panel B, top) and A. healyi (panel B, bottom). Amoebae with the fluorescence of EGFP alone showed dispersed distribution in the cytoplasm during encystation (C). Bar = 10 μm.
- FIG. 9.
The morphology of the autophagosome after transfection of EMSP specific siRNA. Control cells and cells transfected with EMSP siRNA were fixed after 65 h of incubation in encystment media. As shown in panel A, autophagosome in mature cyst digested almost all of the cytoplasmic contents. siRNA-transfected cells showed undigested cytoplasmic materials and organelles within the autophagosome (B). Arrows, autophagosome; bar, 10 μm.
Tables
- TABLE 1.
Primer sequences for RT-PCR of proteinases
Organism Proteinase used for identificationa Primer sequence Sense Antisense A. castellanii Cysteine proteinase 5′-CCGAGCAGAACCTCATCGATT 5′-CCATGCCCTTGTTGTTGATGAT Serine-type endopeptidase 5′-GAAGGCGCTCACCGAATACAT 5′-GGTTCGTCATCTGCTGATAGCC Peptidase M28 5′-TCGGTATCGTGACGGACTACGT 5′-TAGCCGCACTTGGTGTTCTTG Aspartyl aminopeptidase-like protein 5′-ACCATCGACATCGGTTTGC 5′-TGAAGTCGGAGTAGAAGGCCTT Subtilisin-like serine proteinase 5′-CAACAAGATGCTCTACACGCC 5′-CGTAATCGGAGCCATCCAACA 18S rDNA 5′-TCCAAT TTTCTGCCACCGAA 5′-ATCATTACCCTAGTCCTCGCGC A. healyi Subtilisin-like serine proteinase 5′-TCAAGGTGCTCGGATGCAAT 5′-ATGTTAGCCACAGACTGCGTC ↵a rDNA, ribosomal DNA.
