| Congresso Brasileiro de Microbiologia 2023 | Resumo: 464-1 | ||||
Resumo:INTRODUCTION. Acanthamoeba castellanii is an emerging human pathogen that causes ocular keratitis and encephalitis and is also a potential environmental host for a myriad of human pathogens. Differences were observed between environmental and clinical strains of A. castellanii upon infection, indicating the involvement of distinct virulence factors and pathogenesis mechanisms. These could be released by means of extracellular vesicles (EVs), highlighting the importance of multi-omic analysis of the EVs contents of distinct A. castellanii strains.
METHODS. EVs were isolated from two distinct A. castellanii strains, an environmental Neff (ATCC 30010) and a clinical T4 (ATCC 50370) strains. EVs dimensions of both isolates were compared morphologically by DLS, nanosite and transmission electron microscopy. EVs preparations were also processed through the MPLEx technique for the analysis of metabolites, proteins, and lipids by mass spectrometry. After obtaining the data, proteins were identified using the MaxQuant platform using the A. castellanii Uniprot database (www.uniprot.org). Venn diagrams were constructed by the online tool (https://bioinformatics.psb.ugent.be/webtools/Venn/) for the comparison between the isolates. Functional annotation of proteins and enrichment analysis were performed using DAVIDs (https://david.ncifcrf.gov). Identified lipids and metabolites were analyzed and compared using the KEGG databases (www.genome.jp/kegg/) and maps of the enriched lipids or metabolic pathways for each isolate were constructed using the Vanted software (https://www.cls.uni-konstanz.de/software/vanted/).
RESULTS. Striking differences were observed on the EVs from both isolates. The EVs dimensions of the environmental (Neff) isolate were slightly larger than the clinical (T4) isolate (198 x 177.5 nm). The protein contents of these EVs varied depending on the isolates, with 1107 common protein detected (and 242 differentially expressed), 161 proteins exclusively detected in Neff EVs and 84 proteins exclusively detected in the T4 EVs. Proteins annotation and classifications revealed distinct carbon pathways usage, and distinct response to osmotic and oxidative stress between the isolates. Regarding the lipids contents, ceramide, phosphatidic acid, diacylglyceryl-trimethylhomoserine and phosphatidylcholine were enriched in the T4 EVS; instead Neff EVs were enriched in phosphatidylserine and triacylglycerol. Comparison of the phosphatidylethanolamine (PE) content demonstrated an enrichment of a higher degree of unsaturation (20:3) for Neff, in comparison to lower unsaturation of PE in T4 EVs. Metabolites involved in the amino acid metabolism, such as L-alanine, L-glutamine and L-methionine sulfoxide and carbohydrate metabolism (dihydroxyacetone phosphate and glycerol 3-phosphate) were enriched in the Neff EVs, while in T4, disaccharides such as maltose and cellobiose were enriched.
CONCLUSION. Proportional differences of the molecules identified between the isolates were observed, suggesting distinctly active biomolecules within the T4 and Neff strains, which may alter their pathogenesis.
Palavras-chave: Extracellular vesicles (EVs), free-living amoeba, MPLEx, multi-omics analyses Agência de fomento:CAPES; CNPQ; FAPERJ | |||||