Genomic Landscape Reveals Correlation of Endosymbiont Ralstonia With Acanthamoeba Keratitis Severity.

Purpose: To identify the basic genomic profile of Acanthamoeba, obtain information on Acanthamoeba endosymbionts, and analyze the correlation between these endosymbionts and the prognosis of Acanthamoeba keratitis (AK) patients. Methods: Whole-genome sequencing was conducted on 30 cornea-derived Acanthamoeba strains. Pan-genome analysis was performed, and endosymbionts were identified by metagenomic analysis. Gimenez staining, fluorescence in situ hybridization, and transmission electron microscopy were used to prove the existence of endosymbionts. Linear discriminant analysis effect size was used to associate endosymbiont species with AK clinical prognosis. The correlation between the endosymbiont Ralstonia and pathogenicity was experimentally validated by assessing the biological characteristics of Acanthamoeba and by performing clinical and histopathological evaluations in AK mouse models. Results: Whole genome sequencing revealed that the Acanthamoeba genome size was 37.1-105.0 Mb and GC content was 53.9%-60.5%. Pan-genomic analysis indicated an open state of the Acanthamoeba genome. Metagenomic analysis identified the presence of endosymbionts within Acanthamoeba, notably the endosymbiont Ralstonia, which was associated with poor prognosis at the genus level (P = 0.047). Acanthamoeba harboring the endosymbiont Ralstonia exhibited an increased migration area, enhanced adhesion, and had a more pronounced cytopathic effect. The size of clinical scores and corneal ulcers showed a significant increase in mouse models induced by Acanthamoeba with endosymbiont Ralstonia. Conclusions: Whole-genome sequencing highlighted the symbiotic relationship between Acanthamoeba and associated microorganisms. The presence of the endosymbiont Ralstonia influenced the biological characteristics of Acanthamoeba and was correlated with clinical poor prognosis in AK, suggesting its potential as a target for clinical intervention.