The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.

To produce chronic infection, microbial pathogens must escape host immune defenses. Infection with the human pathogenic fungus Cryptococcus neoformans is typically chronic. To understand the mechanism by which C. neoformans survives in tissue after the infection of immunocompetent hosts, we systematically studied the course of pulmonary infection in mice by electron microscopy. The macrophage was the primary phagocytic cell at all times of infection, but neutrophils also ingested yeast.

Inhalation of fungal spores (conidia) occurs commonly and, in specific circumstances, can result in invasive disease. We investigated the murine inflammatory response to conidia of Aspergillus fumigatus, the most common invasive mold in immunocompromised hosts. In contrast to dormant spores, germinating conidia induce neutrophil recruitment to the airways and TNF-α/MIP-2 secretion by alveolar macrophages. Fungal β-glucans act as a trigger for the induction of these inflammatory responses through their time-dependent exposure on the surface of germinating conidia. Dectin-1, an innate immune receptor that recognizes fungal β-glucans, is recruited in vivo to alveolar macrophage phagosomes that have internalized conidia with exposed β-glucans. Antibody-mediated blockade of Dectin-1 partially inhibits TNF-α/MIP-2 induction by metabolically active conidia. TLR-2- and MyD88-mediated signals provide an additive contribution to macrophage activation by germinating conidia. Selective responsiveness to germinating conidia provides the innate immune system with a mechanism to restrict inflammatory responses to metabolically active, potentially invasive fungal spores.

Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ΔhacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ΔhacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ΔhacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.

The pathogenesis of Cryptococcus neoformans infection has been studied extensively with respect to inflammatory and pathological changes, but very little information is available regarding the morphology of yeast cells during the course of infection. Electron microscopy of Cryptococcus neoformans in murine pulmonary infection revealed increased cell wall thickness with time, but this difference was only partially accounted for by increases in cell diameter. Cell walls of melanized cells were thicker than those of nonmelanized cells 2 h after infection, and the cell wall of yeast became blacker with time, suggesting that melanization contributes to the increased cell wall thickness. Heterogeneous cell populations emerged, with the appearance of giant forms. While for C. neoformans ATCC strain 24067 (serotype D) the full spectrum of cell sizes were observed, for strains H99 (serotype A) and 3501 (serotype D) cells were divisible into two populations, giant and micro forms. In contrast to cellular heterogeneity, the epitope recognized by a protective mAb on the capsular glucuronoxylomannan (GXM) was found at all times of infection. Immunoelectron microscopy using mAbs to GXM demonstrated reactivity with intracellular structures, suggesting that synthesis of capsular polysaccharide occurs, at least in part, in the cytoplasm. In summary, the results indicate that : (i) the infection is dynamic with respect to yeast cell morphology ; (ii) giant cell forms arise in tissue during the course of infection ; (iii) cell walls blacken and thicken during the course of infection, consistent with melanin synthesis during infection ; and (iv) GXM epitopes are found in the capsule, cell wall and cytoplasm, consistent with intracellular polysaccharide synthesis. The results indicate that the population of C. neoformans cells in tissue is in a highly dynamic state, implying that the immune system must confront cells with varying characteristics during the course of infection.