Candida albicans cell walls contain the fluorescent cross-linking amino acid dityrosine

Abstract

Several clinical and laboratory isolates of Candida albicans have a natural blue surface fluorescence when cultured and observed with sensitive optics. The localization and color of the fluorescence are similar to those of the natural fluorescence of sporulated Saccharomyces cerevisiae which is caused by the generation and surface deposition of the cross-linking amino acid dityrosine. In S. cerevisiae, dityrosine production results from the direct action of at least two genes and is responsible for resistance of the ascospores to lytic enzymes and physicochemical trauma. Among the criteria for the identification of dityrosine is pH sensitivity of the fluorescence intensity and a highly characteristic shift of the fluorescence excitation maximum with a change in pH. Video microscopy of whole Candida organisms revealed the characteristic dityrosine intensity maximum at pH ϳ10 and the intensity minimum at pH ϳ2. Separation of an acid hydrolysate of Candida cell walls by reverse-phase high-performance liquid chromatography revealed a fluorescence peak that coelutes with the reagent dityrosine. At pH ϳ10, this peak has a fluorescence excitation maximum of 320 to 325 nm, while at pH ϳ2, the excitation maximum is 285 to 290 nm. This excitation maximum shift and the observed emission maximum of ϳ410 nm are characteristic of dityrosine. Two separate strains of C. albicans were injected intraperitoneally into mice and harvested at 24 h. Blue surface fluorescence was observed, suggesting that dityrosine generation occurs in vivo as well as in vitro. This is the first report of the presence of dityrosine in a human fungal pathogen. Candida albicans and several other Candida species are major pathogens in hospitalized, immunocompromised patients (4). The mechanisms allowing C. albicans to colonize mucosal surfaces and then invade the bloodstream and visceral organs have long been under intense investigation (32), with a large number of putative virulence factors proposed as contributing to pathogenicity (15). Studies of the Candida cell surface (36) have been of particular interest because of its importance in antigenicity (11, 39), in adherence to epithelial and endothelial cells (11), and in interactions with the host immune system (24, 38, 40, 41) and because the mechanisms of cell wall synthesis are critical for our understanding of morphogenesis and for development of new antifungal agents (25). The Candida cell wall is composed principally of mannan (linked with protein to form mannoprotein), ␣-glucan, ␤-glucan, and chitin, which are complex polymers of mannose, glucose, and N-acetylglucosamine, respectively. The content and distribution of these components vary with cell age, culture conditions, and morphology (25, 36). The linkages between the glucan, chitin, and mannoprotein are poorly understood but presumably play a critical role in maintaining the integrity of the organism. Dityrosine, a relatively rare amino acid, is well known as a crosslinking agent that is essential for the resistance of several species, inclu...

pH-Dependent Self-Assembly of Polyalanine Peptides

“…Whereas there was hardly any fluorescence at pH 7, an intense fluorescence band at ;410 nm was observed at pH 11. The wavelengths of the excitation and emission peaks of the high pH solution matched those of the cross-linked amino acid dityrosine, presumably formed by cross-linking of the tyrosyl groups at the C-termini of pairs of 11-ala peptide chains (26). The pH dependence of the fluorescence spectra of the other two peptides (7-ala and 17-ala) were similar to those shown in Figs.…”

“…By measuring the optical density (OD) of the peptide solutions at pH 11 and using the extinction coefficients (e) of tyrosine and dityrosine at 284 nm (10,26), the final concentrations where (C Tyr ) ini is the initial concentration of tyrosine and was obtained by measuring the OD at pH 7, where very little of the tyrosine is expected to be dimerized. It was thus found that for both the peptides 11-ala and 17-ala, ;45% of the initial popu-lation of tyrosines at pH 7 formed dityrosine at pH 11, whereas the rest (55%) existed in the monomeric state.…”

Evaluation of fluorescence in situ hybridisation (FISH) for the identification of Candida albicans in comparison with three phenotypic methods

“…It may be speculated that the lack of nutrients or pHshifts in the centre of a colony growing on an agar plate result in the synthesis of fluorophores like Dityrosin as described before, or in changed cell wall structures. [31][32][33] We were not able to determine a definitive reason for the autofluorescence, but were happy to find that there was hardly any autofluorescence, when yeasts were grown in broth. Under these conditions, all implemented probes were 100% sensitive ( Table 1).…”

A Novel Assay Reveals a Maturation Process during Ascospore Wall Formation

“…Moreover, the sporulation-induced mug72 gene of S. pombe encodes an enzyme related to OSW2 [ 35 ], raising the possibility that the OSW2 -generated component of the S. cerevisiae spore wall is present in the S. pombe wall as well. The outer surface of the S. pombe wall is constructed from a protein rather than dityrosine [ 36 ]; however, many other ascomycete genomes carry orthologs to the dityrosine synthesis genes DIT1 and DIT2 as well as the dityrosine exporter DTR1 and dityrosine has been detected in several other yeasts [ 37 , 38 ]. Thus, the mechanisms of assembly and properties of the spore wall in S. cerevisiae are likely to be a useful model for ascospore walls in other ascomycetes.…”