{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T00:40:38Z","timestamp":1788482438197,"version":"build-2803163510"},"reference-count":42,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,5,6]],"date-time":"2025-05-06T00:00:00Z","timestamp":1746489600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p><jats:italic>Naegleria fowleri<\/jats:italic> is the etiologic agent of primary acute meningoencephalitis (PAM). Although this amoeba is commonly found in water and soil, reports of infections are rare; problems with diagnosis probably contribute to underestimation. Moreover, information regarding the pathogenicity of this species is still lacking. Costa Rica reported the first three cases of PAM during 2020; from these, two <jats:italic>N. fowleri<\/jats:italic> isolates were recovered. The aim of this study was to characterize and compare these isolates, focusing in excretion\/secretion products.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Analyses of protein profiles by silver staining and protease activity assays were performed to characterize whole protein extracts and conditioned media from isolates. Proteomic analyses of excretion\/secretion products, including extracellular vesicles (EVs), were performed, and cytopathic effect and drug susceptibility tests were also compared between isolates.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Results obtained were similar for both isolates. Patterns of multiple bands were observed in each isolate after silver staining. Proteomics analyses revealed a total of 88 and 62 non-redundant proteins as part of the cargo of EVs secreted by trophozoites of <jats:italic>N. fowleri<\/jats:italic> Guanacaste and <jats:italic>N. fowleri<\/jats:italic> Limo\u0301n, while conditioned media results revealed 34 and 17 non-redundant proteins, respectively;  hydrolase activity and actin filament binding were part of the most represented gene ontology terms in EVs and conditioned media. Regarding drug susceptibility assays, no statistically significant differences were identified. On the other hand, although protease activity resulted very similar with substrate buffer at pH 8.5, only <jats:italic>N. fowleri<\/jats:italic> Limo\u0301n showed activity between 40 and 70 kDa at pH 5.0; in contrast, a more rapid cytopathic effect was observed when incubating Vero cells with <jats:italic>N. fowleri<\/jats:italic> Guanacaste.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>Comparative analyses of different <jats:italic>N. fowleri<\/jats:italic> isolates, supported by their remarkable genomic heterogeneity that could be reflected in different metabolic repertoires, are key to understanding virulence and pathogenicity of this amoeba, and could help to explain whether different isolates differ in the severity or course of PAM.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fcimb.2025.1585448","type":"journal-article","created":{"date-parts":[[2025,5,6]],"date-time":"2025-05-06T05:26:01Z","timestamp":1746509161000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":4,"title":["Characterization of Naegleria fowleri from two human cases: insights into its excretion\/secretion products"],"prefix":"10.3389","volume":"15","author":[{"given":"Natalia","family":"Chac\u00f3n Camacho","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda Fernanda","family":"Steller Espinoza","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Johan","family":"Alvarado-Ocampo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonio","family":"Osuna","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lissette","family":"Retana Moreira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth","family":"Abrahams Sand\u00ed","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,5,6]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"382","DOI":"10.3201\/eid2102.141576","article-title":"Fatal meningoencephalitis in child and isolation of Naegleria fowleri from hot springs in Costa Rica","volume":"21","author":"Abrahams Sand\u00ed","year":"2015","journal-title":"Emerg. Infect. Dis."},{"key":"B2","doi-asserted-by":"publisher","first-page":"150","DOI":"10.1016\/j.jinf.2004.09.001","article-title":"Extracellular proteases of Acanthamoeba castellanii (encephalitis isolate belonging to T1 genotype) contribute to increased permeability in an in vitro model of the human blood-brain barrier","volume":"51","author":"Alsam","year":"2005","journal-title":"J. Infect."},{"key":"B3","doi-asserted-by":"publisher","first-page":"Appendix 4E","DOI":"10.1002\/9780471729259.mca04es11","article-title":"Growth and maintenance of Vero cell lines","author":"Ammerman","year":"2008","journal-title":"Curr. Protoc. Microbiol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"618","DOI":"10.3390\/genes10080618","article-title":"Host invasion by pathogenic amoebae: epithelial disruption by parasite proteins","volume":"10","author":"Betanzos","year":"2019","journal-title":"Genes"},{"key":"B5","first-page":"e1888","volume-title":"Naegleria fowleri","author":"Cabello-V\u00edlchez","year":"2023"},{"key":"B6","doi-asserted-by":"publisher","first-page":"e36","DOI":"10.1093\/cid\/civ017","article-title":"The first association of a primary amebic meningoencephalitis death with culturable Naegleria fowleri in tap water from a US treated public drinking water system","volume":"60","author":"Cope","year":"2015","journal-title":"Clin. Infect. Dis."},{"key":"B7","doi-asserted-by":"publisher","first-page":"1060","DOI":"10.1002\/cbin.11551","article-title":"Distinct immunomodulatory properties of extracellular vesicles released by different strains of Acanthamoeba","volume":"45","author":"Costa","year":"2021","journal-title":"Cell Biol. Int."},{"key":"B8","doi-asserted-by":"publisher","first-page":"360","DOI":"10.1645\/22-86","article-title":"The 72-kDa protein of Naegleria fowleri plays an important role in the adhesion of trophozoites to BALB\/C mice nasal epithelium","volume":"110","author":"Flores-Su\u00e1rez","year":"2024","journal-title":"J. Parasitol."},{"key":"B9","doi-asserted-by":"publisher","first-page":"125640","DOI":"10.1016\/j.ejop","article-title":"A comparative study of the membrane proteins from Naegleria species: A 23-kDa protein participates in the virulence of Naegleria fowleri","volume":"72","author":"Flores-Huerta","year":"2019","journal-title":"Eur. J. Protistol."},{"key":"B10","doi-asserted-by":"publisher","first-page":"740","DOI":"10.1136\/bmj.2.5464.734-a","article-title":"Acute pyogenic meningitis probably due to Acanthamoeba sp.: a preliminary report","volume":"2","author":"Fowler","year":"1965","journal-title":"Br. Med. J."},{"key":"B11","doi-asserted-by":"publisher","first-page":"13","DOI":"10.3390\/bioengineering6010013","article-title":"Extracellular vesicles from the protozoa Acanthamoeba castellanii: their role in pathogenesis, environmental adaptation and potential applications","volume":"6","author":"Gon\u00e7alves","year":"2019","journal-title":"Bioengineering"},{"key":"B12","doi-asserted-by":"publisher","first-page":"818","DOI":"10.1080\/21505594.2018.1451184","article-title":"Extracellular vesicles and vesicle-free secretome of the protozoa Acanthamoeba castellanii under homeostasis and nutritional stress and their damaging potential to host cells","volume":"9","author":"Gon\u00e7alves","year":"2018","journal-title":"Virulence"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1155\/2014\/256310","article-title":"Morphological features and in vitro cytopathic effect of Acanthamoeba griffini trophozoites isolated from a clinical case","volume":"2014","author":"Gonz\u00e1lez Robles","year":"2014","journal-title":"J. Parasitol. Res."},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1111\/pim.12715","article-title":"Identification of differential protein recognition pattern between Naegleria fowleri and Naegleria lovaniensis","volume":"42","author":"Guti\u00e9rrez-S\u00e1nchez","year":"2020","journal-title":"Parasite Immunol."},{"key":"B15","first-page":"197","article-title":"Proteolytic enzymes of pathogenic and non-pathogenic strains of Acanthamoeba spp","volume":"44","author":"Hadas","year":"1993","journal-title":"Tropical Medicine and Parasitology"},{"key":"B16","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1002\/elps.1150090106","article-title":"Improved silver staining procedure for fast staining in PhastSystem Development Unit I. Staining of sodium dodecyl sulfate gels","volume":"9","author":"Heukeshoven","year":"1988","journal-title":"Electrophoresis"},{"key":"B17","doi-asserted-by":"publisher","first-page":"2814","DOI":"10.1016\/S0021-9258(17)35860-X","article-title":"Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors","volume":"261","author":"Herron","year":"1986","journal-title":"J. Biol. Chem."},{"key":"B18","doi-asserted-by":"publisher","first-page":"2858","DOI":"10.1128\/JCM.38.8.2858-2861.2000","article-title":"Proteases as markers for differentiation of pathogenic and nonpatogenic species of Acanthamoeba","volume":"38","author":"Khan","year":"2000","journal-title":"J. Clin. Microbiol."},{"key":"B19","doi-asserted-by":"publisher","first-page":"4010","DOI":"10.1128\/AAC.00197-08","article-title":"Effect of therapeutic chemical agents in vitro and on experimental meningoencephalitis due to Naegleria fowleri","volume":"52","author":"Kim","year":"2008","journal-title":"Antimicrob. Agents Chemother."},{"key":"B20","doi-asserted-by":"publisher","first-page":"1436","DOI":"10.1099\/mic.0.000537","article-title":"Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system","volume":"163","author":"Lam","year":"2017","journal-title":"Microbiology (Reading)"},{"key":"B21","doi-asserted-by":"publisher","first-page":"467","DOI":"10.1186\/s13071-019-3725-z","article-title":"Quantitative proteomic analysis and functional characterization of Acanthamoeba castellanii exosome-like vesicles","volume":"12","author":"Lin","year":"2019","journal-title":"Parasitol. Vectors"},{"key":"B22","doi-asserted-by":"publisher","first-page":"204","DOI":"10.1016\/j.ijpddr.2021.10.003","article-title":"Repurposing in vitro approaches for screening anti-parasitic drugs against the brain-eating amoeba Naegleria fowleri","volume":"17","author":"Mart\u00edn Escolano","year":"2021","journal-title":"Int. J. Parasitol. Drugs Drug Resist."},{"key":"B23","doi-asserted-by":"publisher","first-page":"461329","DOI":"10.1155\/2013\/461329","article-title":"Reevaluating the role of Acanthamoeba proteases in tissue invasion: observation of cytopathogenic mechanisms on MDCK cell monolayers and hamster corneal cells","volume":"2013","author":"Oma\u00f1a Molina","year":"2013","journal-title":"BioMed. Res. Int."},{"key":"B24","doi-asserted-by":"publisher","first-page":"113","DOI":"10.1016\/s0020-7519(83)80059-9","article-title":"Comparative protein patterns of three thermophilic nonpathogenic Naegleria isolates and two Naegleria fowleri strains by isoelectric focusing","volume":"13","author":"Pernin","year":"1983","journal-title":"Int. J. Parasitol."},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2024.1346021","article-title":"Providing an in vitro depiction of microglial cells challenged with immunostimulatory extracellular vesicles of Naegleria fowleri","volume":"15","author":"Retana Moreira","year":"2024","journal-title":"Front. Microbiol."},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.3390\/biology11070983","article-title":"Characterization of extracellular vesicles secreted by a clinical isolate of Naegleria fowleri and identification of immunogenic components within their protein cargo","volume":"11","author":"Retana Moreira","year":"2022","journal-title":"Biology"},{"key":"B27","doi-asserted-by":"publisher","first-page":"629","DOI":"10.3390\/pathogens9080629","article-title":"Primary amebic meningoencephalitis related to groundwater in Costa Rica: diagnostic confirmation of three cases and environmental investigation","volume":"9","author":"Retana Moreira","year":"2020","journal-title":"Pathogens"},{"key":"B28","doi-asserted-by":"publisher","first-page":"122","DOI":"10.3390\/pathogens8030122","article-title":"In Vitro Activity of Statins against Naegleria fowleri","volume":"8","author":"Rizo-Liendo","year":"2019","journal-title":"Pathogens"},{"key":"B29","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1007\/s00436-024-08378-9","article-title":"Analysis of virulence factors in extracellular vesicles secreted by Naegleria fowleri","volume":"123","author":"Rodr\u00edguez Mera","year":"2024","journal-title":"Parasitol. Res."},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2023.1264348","article-title":"Characterization of the extracellular vesicles, ultrastructural morphology, and intercellular interactions of multiple clinical isolates of the brain-eating amoeba, Naegleria fowleri","volume":"27","author":"Russell","year":"2023","journal-title":"Front. Microbiol."},{"key":"B31","doi-asserted-by":"publisher","first-page":"e01899","DOI":"10.1128\/spectrum.01899-21","article-title":"Differential growth rates and in vitro drug susceptibility to currently used drugs for multiple isolates of Naegleria fowleri","volume":"10","author":"Russell","year":"2022","journal-title":"Microbiol. Spect."},{"key":"B32","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1111\/j.1550-7408.2005.00082","article-title":"In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri","volume":"53","author":"Schuster","year":"2006","journal-title":"J. Eukaryot. Microbiol."},{"key":"B33","doi-asserted-by":"publisher","first-page":"411","DOI":"10.1111\/j.1550-7408.2007.00280.x","article-title":"Biochemical comparison of proteases from pathogenic Naegleria fowleri and non-pathogenic Naegleria gruberi","volume":"54","author":"Serrano Luna","year":"2007","journal-title":"J. Eukaryot. Microbiol."},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2011.00026","article-title":"Leishmania exosomes deliver preemptive strikes to create an environment permissive for early infection","volume":"1","author":"Silverman","year":"2012","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"B35","doi-asserted-by":"publisher","first-page":"2094","DOI":"10.3390\/molecules22122094","article-title":"The eukaryotic elongation factor 1 alpha (eEF1\u03b1) from the parasite Leishmania infantum is modified with the immunomodulatory substituent phosphorylcholine (PC)","volume":"22","author":"Timm","year":"2017","journal-title":"Molecules"},{"key":"B36","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1111\/j.1574-695X.2007.00232.x","article-title":"Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea","volume":"50","author":"Visvesvara","year":"2007","journal-title":"FEMS Immunol. Med. Microbiol."},{"key":"B37","doi-asserted-by":"publisher","first-page":"25S","DOI":"10.1111\/j.1550-7408","article-title":"Epidemiology of free-living ameba infections","volume":"37","author":"Visvesvara","year":"1990","journal-title":"J. Protozool."},{"key":"B38","volume-title":"Identification of peptidases in highly pathogenic versus weakly pathogenic","author":"Vyas","year":"2014"},{"key":"B39","doi-asserted-by":"publisher","first-page":"51","DOI":"10.1111\/jeu.2015.62.issue-1","article-title":"Identification of peptidases in highly pathogenic vs. weakly pathogenic Naegleria fowleri amebae","volume":"62","author":"Vyas","year":"2015","journal-title":"J. Eukaryot. Microbiol."},{"key":"B40","doi-asserted-by":"publisher","first-page":"e12404","DOI":"10.1002\/jev2.12404","article-title":"Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches","volume":"13","author":"Welsh","year":"2024","journal-title":"J. Extracell. Vesicles"},{"key":"B41","doi-asserted-by":"publisher","first-page":"61","DOI":"10.1016\/j.exppara.2015.05.004","article-title":"In vitro growth, cytopathic effects and clearance of monolayers by clinical isolates of Balamuthia mandrillaris in human skin cell cultures","volume":"156","author":"Yera","year":"2015","journal-title":"Exp. Parasitol."},{"key":"B42","doi-asserted-by":"publisher","first-page":"968","DOI":"10.1017\/S0950268809991014","article-title":"The epidemiology of primary amoebic meningoencephalitis in the USA 1962\u20132008","volume":"138","author":"Yoder","year":"2010","journal-title":"Epidemiol. Infect."}],"container-title":["Frontiers in Cellular and Infection Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2025.1585448\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,6]],"date-time":"2025-05-06T05:26:03Z","timestamp":1746509163000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2025.1585448\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,6]]},"references-count":42,"alternative-id":["10.3389\/fcimb.2025.1585448"],"URL":"https:\/\/doi.org\/10.3389\/fcimb.2025.1585448","relation":{},"ISSN":["2235-2988"],"issn-type":[{"value":"2235-2988","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,6]]},"article-number":"1585448"}}