Signal peptide variation in cyst lectins as a potential marker for pathogenic Acanthamoeba spp

Acanthamoeba species are free-living protists widely distributed in natural and artificial environments, including freshwater, soil, and water-associated facilities. Some isolates can cause opportunistic human infections, such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. The life cycle of Acanthamoeba includes an active trophozoite stage and a dormant cyst stage. The cyst is surrounded by a complex double-layered wall composed of cellulose and multiple structural proteins, including cyst wall lectins. While the architecture and molecular composition of the cyst wall have been investigated, inter-isolate sequence variation of cyst wall components remains poorly characterized.

We performed a genome-based comparative analysis of 31 previously characterized cyst wall lectins, analyzing the homologous sequence of each lectin across 31 publicly available Acanthamoeba genomes. Isolates were classified as clinical or environmental on the basis of their reported sources. Sequence similarity-based clustering was conducted to identify lectins associated with clinical isolates. For selected genes, sequence features at the 5' region were further examined and experimentally validated by polymerase chain reaction using long-term axenic clinical isolates maintained at National Cheng Kung University Hospital.

Similarity-based clustering identified eight cyst wall lectins that formed clusters enriched for clinical isolates. Among these, three lectins showed pronounced sequence variation at the 5' region in multiple clinical isolate genomes. Polymerase chain reaction (PCR) validation confirmed that these variations were present in clinical isolates and were not attributable to genome assembly artifacts. Sequence analysis suggested that these variations may affect signal peptide features at the amino terminus of the proteins.

Our findings reveal previously underexplored sequence diversity in cyst wall lectins across Acanthamoeba isolates. This diversity may contribute to differences in cyst wall architecture between clinical and environmental isolates. These results provide new insight into cyst-associated molecular features that may be linked to pathogenic potential and offer a foundation for future studies on cyst-mediated adaptation and infection.