Comprehensive characterization of extracellular vesicles produced by environmental (Neff) and clinical (T4) strains of
Acanthamoeba castellanii
Authors: Medeiros Elisa Gonçalves , Valente Michele Ramos , Honorato Leandro , Ferreira Marina da Silva , Mendoza Susana Ruiz , Gonçalves Diego de Souza , Martins Alcântara Lucas , Gomes Kamilla Xavier , Pinto Marcia Ribeiro , Nakayasu Ernesto S. , Clair Geremy , da Rocha Isadora Filipaki Munhoz , dos Reis Flavia C. G. , Rodrigues Marcio L. , Alves Lysangela R. , Nimrichter Leonardo , Casadevall Arturo , Guimarães Allan Jefferson 
Published: May 2024

We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two
Acanthamoeba castellanii
strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of
A. castellanii
EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for
Acanthamoeba
-related diseases.

IMPORTANCE

A comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two
Acanthamoeba castellanii
strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of
A. castellanii
EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of
Acanthamoeba
-related diseases.