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Determination of chitin content in fungal cell wall: an alternative flow cytometric method

dc.contributor.authorCosta-de-Oliveira, Sofia
dc.contributor.authorSilva, Ana P.
dc.contributor.authorMiranda, Isabel M.
dc.contributor.authorSalvador, Alexandre
dc.contributor.authorAzevedo, Maria M.
dc.contributor.authorMunro, Carol A.
dc.contributor.authorRodrigues, Acácio G.
dc.contributor.authorPina-Vaz, Cidália
dc.date.accessioned2013-04-23T13:55:51Z
dc.date.available2013-04-23T13:55:51Z
dc.date.issued2013-03
dc.description.abstractThe conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted Glycosyl Phosphatidyl Inositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi.por
dc.identifier.citationCosta-de-Oliveira S, Silva AP, Miranda IM, Salvador A, Azevedo MM, Munro CA, et al. Determination of chitin content in fungal cell wall: an alternative flow cytometric method. Cytometry A. 2013;83(3):324-8.por
dc.identifier.issn1552-4930
dc.identifier.urihttp://hdl.handle.net/10400.21/2456
dc.language.isoengpor
dc.peerreviewedyespor
dc.publisherWileypor
dc.relation.publisherversionhttp://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22250/abstract;jsessionid=2D028C321898E58E662B73712A906CAA.d03t01por
dc.subjectChitinpor
dc.subjectFlow cytometrypor
dc.subjectParadoxical effectpor
dc.subjectEchinocandinspor
dc.titleDetermination of chitin content in fungal cell wall: an alternative flow cytometric methodpor
dc.typejournal article
dspace.entity.typePublication
oaire.citation.endPage328por
oaire.citation.startPage324por
oaire.citation.titleCytometry Part Apor
oaire.citation.volume83por
rcaap.rightsrestrictedAccesspor
rcaap.typearticlepor

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