TY - JOUR
T1 - Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe
AU - Crawford, Conor J.
AU - Cordero, Radamés J.B.
AU - Guazzelli, Lorenzo
AU - Wear, Maggie P.
AU - Bowen, Anthony
AU - Oscarson, Stefan
AU - Casadevall, Arturo
N1 - Funding Information:
This work was supported by Irish Research Council Postgraduate Award GOIPG/2016/998 (to C. J. C.), by JHU CFAR National Institutes of Health/ NIAID Grants P30AI094189 (to R. J. B. C.) and AI007417 (to M. P. W.), by National Institutes of Health Grant GM00728847 (to A. B.), by Science Foun-dation Ireland Award 13/IA/1959 (to S. O.), and by National Institutes of Health Grants AI052733 16 and HL059842 19 (to A. C.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2020 Crawford et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2020/3/27
Y1 - 2020/3/27
N2 - Chemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights. The opportunistic fungal pathogen Cryptococcus neoformans possesses a polysaccharide capsule that is a major virulence factor, but is challenging to study. We report here the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the C. neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall-membrane interface, implying the presence of reducing-end glycans at this location where the capsule is attached to the cell body. In contrast, no fluorescence signal was detected in the capsule body. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear stress resulted in capsule alterations that affected the binding of the probe, as reducing ends were exposed and cell membrane integrity was compromised. Unlike the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly whereby reducing ends localize to the cell wall surface, supporting previous findings suggesting that this is an initiation point for capsular assembly. We propose that chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides to unravel their roles in fungal virulence.
AB - Chemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights. The opportunistic fungal pathogen Cryptococcus neoformans possesses a polysaccharide capsule that is a major virulence factor, but is challenging to study. We report here the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the C. neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall-membrane interface, implying the presence of reducing-end glycans at this location where the capsule is attached to the cell body. In contrast, no fluorescence signal was detected in the capsule body. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear stress resulted in capsule alterations that affected the binding of the probe, as reducing ends were exposed and cell membrane integrity was compromised. Unlike the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly whereby reducing ends localize to the cell wall surface, supporting previous findings suggesting that this is an initiation point for capsular assembly. We propose that chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides to unravel their roles in fungal virulence.
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U2 - 10.1074/jbc.RA119.012251
DO - 10.1074/jbc.RA119.012251
M3 - Article
C2 - 32005661
AN - SCOPUS:85082539125
VL - 295
SP - 4327
EP - 4340
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 13
ER -