The structure of galactoxylomannan, a capsular polysaccharide from the opportunistic yeast Cryptococcus neoformans, was reexamined by NMR spectroscopy and GC–MS. The residue that is 3-linked to the side chain galactose and was previously assigned as β-D-xylose [Vaishnav, V. V.; Bacon, B. E.; O'Neill, M.; Cherniak, R., Carbohydr. Res., 1998, 306, 315–330] was determined to be β-D-glucuronic acid. A revised structure for this polymer is presented, along with a proposal that this compound be termed glucuronoxylomannogalactan (GXMGal).

Diagnosis of Epstein-Barr virus (EBV) infection is based on clinical symptoms and serological markers, including the following: immunoglobulin G (IgG)and IgM antibodies to the viral capsid antigen (VCA), heterophile antibodies, and IgG antibodies to the EBV early antigen-diffuse (EA-D) and nuclear antigen (EBNA-1). The use of all five markers results in 32 possible serological patterns. As a result, interpretation of EBV serologies remains a challenge. The purpose of this study was to use a large population of patients to develop evidence-based tools for interpreting EBV results. This study utilized 1,846 serum specimens sent to the laboratory for physician-ordered EBV testing. Chart review was performed for more than 800 patients, and diagnoses were assigned based on physician-ordered testing, clinical presentation, and patient history. Testing for all five EBV antibodies was performed separately on all serum samples using the Bio-Rad BioPlex 2200 system. Presumed EBV diagnosis (based on previous publications) was compared to EBV diagnosis based on a medical record review for each serological pattern. Interestingly, of the 32 possible serological patterns, only 12 occurred in >10 patients. The remaining 20 patterns were uninterpretable because they occurred with such infrequency. Two easy-to-use tables were created to interpret EBV serological patterns based on whether three (EBV VCA IgG, IgM, and heterophile) or five markers are utilized. The use of these two tables allows for interpretation of >95% of BioPlex serological results. This is the first evidence-based study of its kind for EBV serology.

The opportunistic yeast Cryptococcus neoformans causes serious disease in humans and expresses a prominent polysaccharide capsule that is required for its virulence. Little is known about how this capsule is synthesized. We previously identified a ␤1,2-xylosyltransferase (Cxt1p) with in vitro enzymatic activity appropriate for involvement in capsule synthesis. Here, we investigate C. neoformans strains in which the corresponding gene has been deleted (cxt1⌬). Loss of CXT1 does not affect in vitro growth of the mutant cells or the general morphology of their capsules. However, NMR structural analysis of the two main capsule polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), showed that both were missing ␤1,2-xylose residues. There was an ϳ30% reduction in the abundance of this residue in GXM in mutant compared with wild-type strains, and mutant GalXM was almost completely devoid of ␤1,2-linked xylose. The GalXM from the mutant strain was also missing a ␤1,3-linked xylose residue. Furthermore, deletion of CXT1 led to attenuation of cryptococcal growth in a mouse model of infection, suggesting that the affected xylose residues are important for normal host-pathogen interactions. Cxt1p is the first glycosyltransferase with a defined role in C. neoformans capsule biosynthesis, and cxt1⌬ is the only strain identified to date with structural alterations of the capsule polysaccharide GalXM.Cryptococcus neoformans is an opportunistic fungal pathogen of humans and other mammals that causes serious disease, including infections of both the lung and central nervous system (1). Neurological infections with C. neoformans typically occur in immunocompromised patients and are often debilitating or fatal. Anti-cryptococcal therapies that are currently available can resolve symptoms in many cases, but these drugs do not eradicate the organism from neural tissues (1). Recurrence is likely, and new therapies for this infection are needed.The main virulence factor of C. neoformans is the carbohydrate capsule surrounding the cell. Strains of C. neoformans lacking this structure do not cause disease in animal models (2), suggesting that the mechanisms of capsule synthesis are potential drug targets (3). The capsule is composed of two polysaccharides, glucuronoxylomannan (GXM) 2 and galactoxylomannan (GalXM), and a small amount of mannoproteins (3).The structures of both GXM and GalXM were resolved by Cherniak and colleagues (4, 5) a decade ago. GXM is a large polysaccharide (1-7 megadaltons) (6) composed of ␣1,3-linked mannan with glucuronic acid and xylose side chains (4) (Fig. 1A). The mannose residues of GXM are also variably 6-Oacetylated (7). GalXM is a smaller polymer (ϳ100 kDa), consisting of an ␣1,6-galactan backbone with galactomannan side chains that are further substituted with variable numbers of xylose residues (5) (Fig. 1B).GXM mediates diverse negative effects on the host immune response (8) and is well established as a critical virulence factor for C. neoformans. Our understanding of the importa...

Cryptococcus neoformans is a pathogenic fungus surrounded by an elaborate polysaccharide capsule that is strictly required for its virulence in humans and other mammals. Nearly half of the sugar residues in the capsule are derived from UDP-glucuronic acid or its metabolites. To examine the role of these nucleotide sugars in C. neoformans, the gene encoding UDP-glucose dehydrogenase was disrupted. Mass spectrometry analysis of nucleotide sugar pools showed that the resulting mutant lacked both UDP-glucuronic acid and its downstream product, UDP-xylose, thus confirming the effect of the knockout and indicating that an alternate pathway for UDP-glucuronic acid production was not used. The mutant was dramatically affected by the lack of specific sugar donors, demonstrating altered cell integrity, temperature sensitivity, lack of growth in an animal model of cryptococcosis, and morphological defects. Additionally, the polysaccharide capsule could not be detected on the mutant cells, although the possibility remains that abbreviated forms of capsule components are made, possibly without proper surface display. The capsule defect is largely independent of the other observed changes, as cells that are acapsular because of mutations in other genes show lack of virulence but do not exhibit alterations in cell integrity, temperature sensitivity, or cellular morphology. All of the observed alterations were reversed by correction of the gene disruption.