METHODS: The role of ion transporters in the encystation and excystation of Acanthamoeba remains unclear. Here, we investigated the role of sodium, potassium and calcium ion transporters as well as proton pump inhibitors on A. castellanii encystation and excystation and their effects on trophozoites.
RESULTS: Remarkably 3',4'-dichlorobenzamil hydrochloride a sodium-calcium exchange inhibitor, completely abolished excystation of Acanthamoeba. Furthermore, lanthanum oxide and stevioside hydrate, both potassium transport inhibitors, resulted in the partial inhibition of Acanthamoeba excystation. Conversely, none of the ion transport inhibitors affected encystation or had any effects on Acanthamoeba trophozoites viability.
CONCLUSIONS: The present study indicates that ion transporters are involved in sensory perception of A. castellanii suggesting their value as potential therapeutic targets to block cellular differentiation that presents a significant challenge in the successful prognosis of Acanthamoeba infections.
METHODS: Anti-amoebic effects of phosphanegold(I) thiolates were tested against clinical isolate of A. castellanii belonging to the T4 genotype by employing viability assays, growth inhibition assays, encystation assays, excystation assays, and zymographic assays.
RESULTS: The treatment of A. castellanii with the phosphanegold(I) thiolates tested (i) had no effect on the viability of A. castellanii as determined by Trypan blue exclusion test, (ii) did not affect amoebae growth using PYG growth medium, (iii) did not inhibit cellular differentiation, and (iv) had no effect on the extracellular proteolytic activities of A. castellanii.
CONCLUSION: Being free-living amoeba, A. castellanii is a versatile respirator and possesses respiratory mechanisms that adapt to various aerobic and anaerobic environments to avoid toxic threats and adverse conditions. For the first time, our findings showed that A. castellanii exhibits resistance to the toxic effects of gold compounds and could prove to be an attractive model to study mechanisms of metal resistance in eukaryotic cells.
ANIMALS: A convention on international trade in endangered species (CITES) of wild fauna and flora registered crocodile farm, provided a healthy male saltwater crocodile, Crocodylus porosus for this study.
PROCEDURES: Three samples were taken from the oral cavity, 3 samples from the proximal region of the small intestine (jejunum), and 3 samples from the distal part of the large intestine of the gastrointestinal tract of C. porosus were obtained using sterile cotton swabs. Next, swabs were placed in 15 mL sterile centrifuge tubes, individually, and kept on ice for immediate transportation to the laboratory. This was followed by 16S rRNA gene analysis using specific primers (341F-CCTAYGGGRBGCASCAG, and 806R-GGACTACNNGGGTATCTAAT). Amplicons were sequenced on Illumina paired-end platform, and bacterial gastrointestinal communities, the relative abundance of taxa, and principal component and coordinate analysis were performed.
RESULTS: The findings revealed that bacterial community structures from differing regions exhibited several differences. The number of observed bacterial operational taxonomic units (OTUs) was 153 in the oral cavity, 239 in the small intestine, and 119 in the large intestine of C. porosus. The small intestine reflects the highest richness. In contrast, the large intestine exhibited the least richness of microbial communities. Relative abundance of taxa showed that Proteobacteria, Bacteroidetes, and Firmicutes were dominant in all 3 sample sites. Pseudomonas differed in the oral cavity and the large intestine, with the latter exhibiting less distribution of Pseudomonas. Stenotrophomonas and Castellaniella were higher in the oral cavity, while the relative abundance of Comamonas and Salmonella was higher in the small intestine. Conversely, the relative abundance of Salmonella and Pannonibacter was augmented in the large intestine.
CLINICAL RELEVANCE: For the first time, this study demonstrates the bacterial diversity along the segments of the gastrointestinal tract of C. porosus. Bacterial flora varies throughout the gastrointestinal tract. Although further studies using large cohorts are warranted; however, our findings suggest that microbiome composition may have the potential as a biomarker in determining the overall health and well-being of C. porosus.