Production of Extracellular Polysaccharides by CAP Mutants of Cryptococcus neoformans

The human pathogen Cryptococcus neoformans causes meningoencephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). How capsular polysaccharides are synthesized, transported, and assembled is largely unknown. Previously, it was shown that mutations in the CAP10, CAP59, CAP60, and CAP64 genes result in an acapsular phenotype. Here, it is shown that these acapsular mutants do secrete GalXM and GXM-like polymers. GXM and GalXM antibodies specifically reacted with whole cells and the growth medium of the wild type and CAP mutants, indicating that the capsule polysaccharides adhere to the cell wall and are shed into the environment. These polysaccharides were purified from the medium, either with or without anion-exchange chromatography. Monosaccharide analysis of polysaccharide fractions by gas-liquid chromatography/mass spectrometry showed that wild-type cells secrete both GalXM and GXM. The CAP mutants, on the other hand, were shown to secrete GalXM and GXM-like polymers. Notably, the GalXM polymers were shown to contain glucuronic acid. One-dimensional ¹H nuclear magnetic resonance confirmed that the CAP mutants secrete GalXM and also showed the presence of O-acetylated polymers. This is the first time it is shown that CAP mutants secrete GXM-like polymers in addition to GalXM. The small amount of this GXM-like polymer, 1 to 5% of the total amount of secreted polysaccharides, may explain the acapsular phenotype.Cryptococcus neoformans of the A (var. grubii [24]) and D (var. neoformans [36]) serotypes are the causative agents of cryptococcosis, of which the most common clinical form is meningoencephalitis. This disease is related to immunocompromised patients but can also occur in immunocompetent individuals (4, 19, 38). One of the main virulence factors is the polysaccharide capsule (2, 5, 17, 21, 27, 35). This capsule enables the yeast-like fungus to survive the harsh environment of the human body by using its immunomodulatory properties that enable immune evasion and by preventing killing through phagocytosis by macrophages (44, 45).The capsule consists of a low percentage of mannoproteins (46) and the polysaccharides glucuronoxylomannan (GXM) and galactoxylomannan (GalXM) in a mass ratio of about 10:1 (14, 16, 17). Little is known about the synthesis of GXM and GalXM and their transport toward the cell surface. A mutation in the Sec4/Rab8 GTPase homologue was recently shown to affect protein secretion as well as polysaccharide secretion and resulted in intracellular accumulation of vesicles containing GXM (51). From this and the fact that GXM has been detected in extracellular vesicles, it was proposed that polysaccharides are packaged in such vesicles to cross the cell wall to reach the extracellular environment (47).Mutation analysis has revealed four genes, called CAP10, CAP59, CAP60, and CAP64, which give an acapsular phenotype when inactivated (7, 9-13). The precise role of the encoded Cap proteins is unknown. Cap59 has been suggested to play a role in extracellular trafficking of multimeric forms of GXM molecules (26). Moreover, it may play a role in the assembly of GXM, since it shares homology with a mannosyltransferase (48). Like Cap59, Cap60 is a putative mannosyltransferase. Cap10 shares homology with a xylosyltransferase and therefore may also be involved in capsule assembly (34), like the recently identified xylosyltransferase encoded by CXT1 (33). This transferase has been shown to play a direct role in the synthesis of both of the capsular polysaccharides but is especially active in the addition of xyloses to the GalXM polysaccharide. CAP64 shares homology with so-called CAS genes, encoding proteins involved in O acetylation of GXM (40).Structural analysis has revealed a relatively clear picture of the buildup of the GXM and GalXM polysaccharides (14, 50) (Fig. ​(Fig.1).1). Some variability in the chemical structures of the capsular polysaccharides has been described, even within the capsule of a particular strain (40, 50). In addition, GalXM has been shown to also contain, besides galactopyranose, galactofuranose in trace amounts (1, 29). The two C. neoformans serotypes A and D are distinguished based on variation in the position of the different xylose residues in the GXM repeating unit (30). The structure of the GalXM repeating unit was analyzed by using a fraction of purified polysaccharides secreted in the medium by a mutant of the D serotype called the CAP67 mutant. This strain is mutated in the same gene as a serotype A CAP59 mutant. The number of xylose residues can vary from zero up to six within the GalXM repeating unit (Fig. ​(Fig.1)1) (50).Open in a separate windowFIG. 1.Chemical structure of GXM and GalXM monomers. Large strands of these monomers form polymers of up to 1 × 10⁶ to 7 × 10⁶ daltons for GXM and 1 × 10⁵ daltons for GalXM. Ratios vary between serotypes. Shown are serotype A GXM, Man 3/Xyl 2/GlcA 1, and GalXM, Gal 6/Man 4/Xyl 1.6 (shown are three xyloses). The degree of O acetylation is not shown. The picture is based on data from reference 3.So far, secreted polysaccharides in the medium of the serotype D CAP67 mutant and the corresponding serotype A CAP59 mutant have been analyzed (41, 50). It was shown that these mutants secrete GalXM but not GXM in the medium. However, it is shown here that these mutants, as well as the serotype A CAP10, CAP60, and CAP64 mutants, also secrete GXM-like polymers in addition to GalXM. Moreover, part of GalXM seems to contain glucuronic acid, supporting earlier findings (16, 49).