Acanthamoeba is an opportunistic pathogen responsible for granulomatous amoebic encephalitis and amoebic keratitis. Despite its clinical significance, effective treatments remain challenging due to a limited understanding of its pathogenic mechanism. This study developed a genetic manipulation system in Acanthamoeba to facilitate gene function and drug screening studies. We applied the Cre/loxP system to integrate the gene encoding the tdTomato fluorescent protein into the genome of Acanthamoeba castellanii via homologous recombination. The polyubiquitin gene and its untranslated regions were identified and verified, after which the tdTomato gene was cloned between the untranslated regions of the polyubiquitin gene. The construct was then introduced into the Acanthamoeba genome using a modified pLPBLP vector containing loxP sites. Cre recombinase was utilized to remove the neomycin resistance cassette flanked by loxP sites, and genetically modified cells were selected by clonal dilution. The integration of the tdTomato gene, confirmed through PCR and fluorescence microscopy, showed stable expression in both trophozoites and cysts without the need for antibiotic selection. We demonstrated the feasibility of antibiotic-free reporter gene expression in Acanthamoeba. The system provides a valuable tool for functional genomics, allowing us to explore gene functions in Acanthamoeba and develop reliable drug screening models. Furthermore, the ability to express genes without the continuous use of selection markers opens up new possibilities for studying the pathobiology of this pathogen and advancing the development of novel therapeutic strategies against Acanthamoeba infections.
Trypanosoma cruzi is the etiological agent of Chagas disease. Epimastigote forms of T. cruzi can be readily cultured in axenic conditions. Ethanol and dimethyl sulfoxide (DMSO) are commonly used solvents employed as vehicles for hydrophobic compounds. In order to produce a reference plot of solvent dependent growth inhibition for T. cruzi research, the growth of epimastigotes was analyzed in the presence of different concentrations of ethanol (0.1-4.0%) and DMSO (0.5-7.5%). The ability of the parasites to resume growth after removal of these solvents was also examined. As expected, both ethanol and DMSO produced a dose-dependent inhibition of cellular growth. Parasites could recover normal growth after 9 days in up to 2% ethanol or 5% DMSO. Since DMSO was better tolerated than ethanol, it is thus recommended to prefer DMSO over ethanol in the case of a similar solubility of a given compound.
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