Abstract

During periods of stress, trophozoites of Entamoeba invadens (strain IP-1) undergo a process of differentiation (encystment) that results in a dormant cyst with a chitin-containing cyst wall. Encystment can be induced by resuspension of trophozoites from growth medium into a diluted glucose-free medium (47% LG) containing 5% adult bovine serum (ABS). ABS is thought to be a source of gal-terminated ligands that are required for high levels of encystment. After resuspension of trophozoites in 47% LG, encystment cultures were examined every 2 h for responses to the (i) addition of 10 mM free-galactose, (ii) resuspension of cells to serum-free medium, (iii) and dilution of encysting cultures to cell densities below that known to support full encystment (from 5 × 10⁵ to 1 × 10⁴ cells/ml). The role of serum components (and the gal-terminated ligand asialofetuin; ASF) adsorbed onto the surface upon which encystment proceeds, and their effect on the multi-cellular aggregation patterns formed during encystment, were also investigated. The addition of free-galactose reduced the levels of encystment (compared with the control) even when added at 10 h after resuspension of trophozoites in 47% LG. The requirement for the presence of ABS during encystment was lost within 6 h, with levels of encystment of cells washed free of serum reaching 80% of the control. The ability of cells to encyst when diluted to a cell density below that normally thought to support encystment reached over 50% by 8 h. Efficient encystment could be obtained in 47% LG in the absence of ABS or ASF using pre-treated glass culture tubes. Encystment (47% LG; 5% ABS) using ultra low attachment plates was poor, suggesting attachment of cells to a surface via gal-terminated ligands was important for efficient encystment.

The results suggest that ABS is probably not the only source of gal-terminated ligands necessary for high levels of encystment in 47% LG. While serum may provide a source of ligands which enhance the levels of encystment initially, other gal-terminated ligands possibly released by the encysting cells are still required for the completion of the encystment process and the formation of mature cysts. In addition, the gal-terminated ligands necessary for encystment efficiency may be adsorbed onto the glass surface of culture tubes and aid the initial aggregation process, as well as be involved in cell signaling during the encystment process.

Index Descriptors and Abbreviations: Trophozoite; Cyst; Encystment; Galactose lectin; Gal-terminated ligands; Adsorption

Article Outline

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Organism and culture

2.3. Encystment method

2.4. Encystment efficiency

2.5. Time point analyses of the sensitivities to the addition of free-galactose or N-acetylglucosamine (glcNAc), and the need for a specific cell density or serum

2.6. Pre-treatment of culture tubes with adult bovine serum (ABS) or asialofetuin (ASF) prior to use with encystment cultures

2.7. Comparison of encystment efficiency using either tissue culture treated plates or ultra low attachment plates

3. Results

3.1. Time point analyses of the sensitivities to the addition of free-galactose or N-acetylglucosamine (glcNAc), and the need for a specific cell density or serum

3.2. Pre-treatment of culture tubes with adult bovine serum (ABS) or asialofetuin (ASF) prior to use with encystment cultures

3.3. Comparison of encystment efficiency using either tissue culture treated plates or ultra low attachment plates

4. Discussion

References