Conference Agenda

Entamoeba histolytica is an enteric tissue-invasion protozoan parasite that rapidly kills host cells within about 5 minutes of contact with host cells. In this study, Entamoeba-induced host cell death was comparatively investigated using two cell death measurement methods; LDH release assay and CellTiter Glo. The widely used LDH release assay measures cell death by detecting lactate dehydrogenase, which is released through cell membrane disruption. CellTiter Glo measures cell viability by detecting ATP in living cells. First, Entamoebae were pretreated with various inhibitors such as D-galactose (a competitive inhibitor of amebic Gal-lectin), E-64 (a cysteine protease inhibitor) and Ca²⁺ chelators (EGTA or EDTA), respectively. Pretreated Entamoeba was co-cultured with Caco-2 cells, and Entamoeba-induced cell death was measured using two methods. The results of these methods commonly explained that Entamoeba treated with D-galactose, E-64, or Ca²⁺ chelators lost their ability to induce host cell killing compared to untreated amoeba. Interestingly, CellTiter Glo was able to detect Entamoeba-induced cell death after only 5 minutes of co-incubation. These results show that CellTiter Glo is more suitable and sensitive method for analyzing rapid cell death caused by Entamoeba than LDH assay.

Acanthamoeba, a free-living amoeba, causes granulomatous amebic encephalitis (GAE) and Acanthamoeba keratitis (AK), an eye infection primarily affecting contact lens users due to extended wear, improper maintenance, and corneal trauma. In vitro AK studies, especially for therapeutic drug development, require in vivo confirmation. Previously, we established an AK mouse model by loading 2 mm contact lens fragments with 5 x 10⁴ A. castellanii cells, inserting them into scratched mouse eyes under anesthesia, and suturing the eyelids. Daily observations from 1 to 14 days post-infection showed AK lesion progression, confirmed by PCR detection of Acanthamoeba DNA. This study aimed to develop an effective cultivation method to validate PCR findings in the AK mouse model. Eyeball samples collected at 1, 3, 7, and 14 days post-inoculation were homogenized and successfully cultivated on non-nutrient agar with an Escherichia coli lawn, then scaled up in PYG medium. PCR analysis confirmed the cultivated amoeba's genetic information matched the inoculated strain (> 99%). This cultivation and PCR approach provides a robust method for confirming AK development in the AK mouse model.

Anaerobic protists, although often overlooked, play a crucial role in marine ecosystems, contributing to nutrient cycling, organic matter degradation, and overall ecosystem health. Despite their importance, the species diversity and ecological significance of anaerobic protists in Korean marine environments remain largely understudied. In this study, we applied environmental DNA (eDNA) metabarcoding as an effected tool for analyzing the diversity of protist in marine water and sediments. As the result, 20 phyla of Protist are detected including Apicomplexa, Cerozoa, Telonemia, Perkinsozoa and so on. The current state of marine protist research in Korea, focusing on species diversity and ecological roles of aerobic environments. Until now, 18 phyla over 3,000 marine protist species have been reported in Korea based on morphological description. This finding underscores the need for further research to comprehensively understand the diversity and ecological functions of anaerobic protists in Korean marine ecosystems. This work was supported by the National Marine Biodiversity Institute Research Program (2024M00200).