Scanning electron microscopy of encapsulated and non-encapsulated Cryptococcus neoformans and the effect of glucose on capsular polysaccharide release 1

Abstract

Cryptococcus neoformans has a polysaccharide capsule composed primarily of glucuronoxylomannan (GXM). This study focuses on the morphology of both encapsulated and non-encapsulated organisms in the presence and absence of monoclonal antibodies (mAbs) and serum proteins, and the effect of glucose on capsular polysaccharide release. Examination of the encapsulated C. neoformans strains 24067 and 34873 by scanning electron microscopy (SEM) revealed globular cells covered with a loose fibrillar network which was most prominent during the early stationary phase. In the presence of GXM-binding mAbs or serum the capsule border became distinct and bud scars were evident in the fibrillar network. In contrast, SEM of strain 52817, a non-encapsulated mutant of 34873 revealed ovoid cells devoid of a fibrillar network with bud scars and small surface protrusions. mAb 2H1 bound to cells from strains 24067 and 34873 but not 52817. No GXM was detected in supernatants of 52817 culture. For several strains, there was significantly more GXM in culture supernatants using high glucose media. In summary, our results indicate: i) SEM methods for studying capsular structure in C. neoformans; ii) no reactivity by GXM-binding mAb with a non-encapsulated strain; iii) the presence of distinctive bud scars in both encapsulated and non-encapsulated cells; and iv) dependence of GXM concentration on glucose concentration in culture media. The implications of these results are discussed.

Immunoglobulin M Efficacy AgainstCryptococcus neoformans: Mechanism, Dose Dependence, and Prozone-Like Effects in Passive Protection Experiments

“…At high concentrations, mAb 12A1 binding to the C. neoformans reduced susceptibility to oxidant killing. mAb 12A1 binding to the capsule is known to cause changes in the refractive index (16) and capsule structure (42). We interpret the reduced susceptibility to nitrogen-derived oxidant killing in the presence of high concentrations of mAb to reflect the existence of a proteinaceous coating to the capsule, which can have the paradoxical effect of protecting the yeast cell against oxidative killing.…”

Molecular Basis for Immunoglobulin M Specificity to Epitopes inCryptococcus neoformansPolysaccharide That Elicit Protective and Nonprotective Antibodies

“…However, scanning electron microscopy revealed differences in the fibrillar structure after binding of MAbs 12A1 and 13F1 and the 12A1V H 313F1V H mutants. Previous work has shown that fibrillar capsular structure is different after binding of MAbs 12A1 and 13F1 (12). Thus, each mutant with a single amino acid change displayed qualitative alterations in the annular IF pattern after binding to strain 24067.…”

“…At least 20 cells were imaged for each MAb tested, and representative photographs were taken. This methodology has previously been used to study the effects of MAbs to C. neoformans (12).…”

Antibody Interactions with the Capsule ofCryptococcus neoformans

“…Alternatively, tissue substances such as complement (14) or surfactant may bind to the capsule in the alveolar space and protect it during fixation and dehydration. Similarly, antibody or complement can protect the C. neoformans capsule from the dehydration steps that precede scanning EM (9). Another attraction of studying MAb binding to the capsule in tissue is that this technique approximates the state of C. neoformans cells at the time of experimental infection.…”