Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

Conor J Crawford,Radamés J.B Cordero,Lorenzo Guazzelli,Maggie P Wear,Anthony Bowen,Stefan Oscarson,Arturo Casadevall

doi:10.1074/jbc.ra119.012251

Conor J Crawford, Radamés J.B Cordero + Show 5 more

Open Access

https://doi.org/10.1074/jbc.ra119.012251

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Abstract

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.

Highlights

Chemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights
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
We describe the synthesis of a hydroxylamine-armed fluorescent probe that selectively labels reducing sugars, and we describe the use of this probe to study the capsule architecture and biosynthesis in C. neoformans

Summary

The abbreviations used are

GalXM, galactoxylomannan; GXM, glucuronoxylomannan; R.E probe; reducing-end probe, hydroxylamine-armed fluorescent probe; CPS, capsular polysaccharide; EPS, exopolysaccharide; RFU, relative fluorescence unit; TRITC, tetramethylrhodamine isothiocyanate; ESI, electrospray ionization. The importance of vesicles in C. neoformans is further demonstrated by mutations in the C. neoformans secretory system known as cap mutants [23, 25] These mutants lack capsular GXM but secrete other polysaccharides suggesting the presence of other transport mechanisms independent of that for the capsule, possibly a process with similarities to that of bacterial capsule biosynthesis [21, 26, 27]. Under chemical or biological settings, hydroxylamine nucleophiles are known to react with a high selectively with the reducing end of glycans (which contain a transient aldehyde), to a give the corresponding oxime conjugate (Fig. 1B) [33, 35,36,37,38] This stable adduct, when attached to a fluorophore, would allow imaging of cryptococcal cells and give insight into the distribution of reducing ends in the cell, cell wall, and capsule. We use the probe to examine the effect of different cellular processing techniques on capsular architecture and that the shed EPS contains reducing ends

Results

Discussion

Experimental procedures