Elucidation of the mechanism underlying capsularpolysaccharide biosynthesis in Cryptococcus neoformans
Chihiro KADOOKA
Faculty of Biotechnology and Life Sciences, Sojo University
Research objective
Cryptococcus neoformans, a yeast belonging to the division Basidiomycota, is the primary
etiological agent of cryptococcosis. This pathogen synthesizes a thick capsular polysaccharide
on the surface layer of its cell wall, which is considered the main virulence factor contributing
to the pathogenicity of the organism as it aids in evasion of the immune system of the host.
The capsule is composed of two polysaccharides, namely glucuronoxylomannan andglucuronoxylomannogalactan; however, the mechanism underlying their biosynthesis remains
ambiguous. Therefore, the objective of this study was to identify the glycosyltransferases
responsible for capsule biosynthesis in C. neoformans.

Structures of GXM and GXMGal
Methods
In C. neoformans, disruption of the GDP-mannose (Man), UDP-xylose (Xyl), UDPglucuronic acid (GlcA), and UDP-galactose (Gal) transporter genes results in aberrations incapsule size1,2,3). This led to the hypothesis that glycosyltransferases involved in capsularproduction might exist among the proteins localized in the Golgi apparatus. The candidate
proteins were expressed as recombinant proteins using a bacterial expression system, and theirglycosyltransferase activities were evaluated. Furthermore, an analysis was conducted to
examine the impact of disrupting candidate genes on capsular synthesis in C. neoformans.
Results
The candidate proteins that were effectively expressed in a soluble form by the bacterial
expression system were assayed for activity with varying combinations of 4methylumbelliferylated sugar (4MU) and sugar-nucleotides. Consequently, among these
proteins, two were discovered to generate reaction products when exposed to the combinationof 4MU-β-Gal and GDP-Man. These proteins were designated Cryptococcal β-Galactoside
Mannosyltransferase 1 (Cgm1) and Cgm2, owing to their shared homology of approximately
40%. The reaction product of Cgm1 was prepared and purified via high-performance liquid
chromatography, and its structure was analyzed using 1H-NMR and methylation gas
chromatography-mass spectrometry (GC-MS). The Cgm1 products structurally resembled
4MU-β-Gal with the C1-position of α-Man attached to the C4-position, indicating that Cgm1
is a β-Gal α-(1→4)-Man transferase. Subsequently, cgm1 and cgm2 disruptants of C.
neoformans H99 were constructed using the CRISPR/Cas9 system and their phenotypes wereassessed. The cgm1 disruptant strain exhibited a minor discrepancy in growth from the wildtype strain at 30°C; nonetheless, growth was substantially delayed at 37°C. Conversely, thecgm2 disruptant strain displayed no discernible phenotype. As some acapsular mutants areknown to be temperature-sensitive at 37°C3, 4), we hypothesized that Cgm1 might be involvedin capsule biosynthesis. Based on the results of Cgm1 enzyme activity analysis, Cgm1 waspredicted to be involved in the biosynthesis of the galactomannan side chain of GXMGal.
GXMGal exhibits a weak interaction with GXM, and in the GXM-deficient strain, it diffuses
into the culture supernatant, facilitating purification. Therefore, a double disruptant of cgm1
and cap59 was engineered, which was an α-(1→3)-Man transferase gene presumed to beinvolved in the biosynthesis of GXM. A comparative analysis of the structure of GXMGalusing 13C-NMR and methylation GC-MS revealed that the cgm1 disruptant strain-generated
GXMGal appeared to have lost or eliminated the galactomannan side chain. These results
indicate that Cgm1 is a β-Gal α-(1→4)-Man transferase involved in the biosynthesis of thegalactomannan side chain of GXMGal.
Conclusion
The study findings established Cgm1 and Cgm2 as novel β-Gal α-(1→4)-Man transferases,
among which Cgm1 was found to be involved in the biosynthesis of GXMGal in C.
neoformans. Considering the absence of Cgm1 homologs in mammals, including humans, andthe exhibition of a temperature-sensitive phenotype in the cgm1-disrupted strain at 37°C,
Cgm1 may be considered an effective potential target for the development of novel antifungal
agents to combat cryptococcosis.
References
1) Cottrell et al. (2007) The pathogenic fungus Cryptococcus neoformans expresses twofunctional GDP-mannose transporters with distinct expression patterns and roles incapsule synthesis. Eukaryot Cell. 6: 776-785.
2) Li et al. (2017) Cryptococcus neoformans UGT1 encodes a UDP-Galactose/UDP-GalNActransporter. Glycobiology. 27: 87–98.
3) Li et al. (2018) UDP-Glucuronic Acid Transport Is Required for Virulence of
Cryptococcus neoformans. mBio. 9: e02319-17
4) Wang et al. (2014) Cryptococcus neoformans Dual GDP-Mannose Transporters and Their
Role in Biology and Virulence. Eukaryot Cell. 13: 832–842.