Christina M. Hull

Elucidating the Pathogenesis of Spores from the Human Fungal Pathogen Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans was first described as a human fungal pathogen more than a century ago. One aspect of the C. neoformans infectious life cycle that has been the subject of earnest debate is whether the spores are pathogenic. Despite much speculation, no direct evidence has been presented to resolve this outstanding question. We present evidence that C. neoformans spores are pathogenic in a mouse intranasal inhalation model of infection. In addition, we provide mechanistic insights into spore-host interactions. We found that C. neoformans spores were phagocytosed by alveolar macrophages via interactions between fungal β-(1,3)-glucan and the host receptors Dectin-1 and CD11b. Moreover, we discovered an important link between spore survival and macrophage activation state: intracellular spores were susceptible to reactive oxygen-nitrogen species. We anticipate these results will serve as the basis for a model to further investigate the pathogenic implications of infections caused by fungal spores.

Sex-Specific Homeodomain Proteins Sxi1α and Sxi2a Coordinately Regulate Sexual Development in Cryptococcus neoformans

ABSTRACT Homeodomain proteins are central regulators of development in eukaryotes. In fungi, homeodomain proteins have been shown to control cell identity and sexual development. Cryptococcus neoformans is a human fungal pathogen with a defined sexual cycle that produces spores, the suspected infectious particles. Previously, only a single homeodomain regulatory protein involved in sexual development, Sxi1α, had been identified. Here we present the discovery of Sxi2a, a predicted but heretofore elusive cell-type-specific homeodomain protein essential for the regulation of sexual development. Our studies reveal that Sxi2a is necessary for proper sexual development and sufficient to drive this development in otherwise haploid α cells. We further show that Sxi1α and Sxi2a interact with one another and impart similar expression patterns for two key mating genes. The discovery of Sxi2a and its relationship with Sxi1α leads to a new model for how the sexual cycle is controlled in C. neoformans, with implications for virulence.

Disruption of Ergosterol Biosynthesis Confers Resistance to Amphotericin B in Candida lusitaniae

ABSTRACT Candida lusitaniae is an emerging human pathogen that, unlike other fungal pathogens, frequently develops resistance to the commonly used antifungal agent amphotericin B. Amphotericin B is a member of the polyene class of antifungal drugs, which impair fungal cell membrane integrity. Here we analyzed mechanisms contributing to amphotericin B resistance in C. lusitaniae. Sensitivity to polyenes in the related fungi Saccharomyces cerevisiae and Candida albicans requires the ergosterol biosynthetic gene ERG6. In an effort to understand the mechanisms contributing to amphotericin B resistance in C. lusitaniae, we isolated the ERG6 gene and created a C. lusitaniae erg6Δ strain. This mutant strain exhibited a growth defect, was resistant to amphotericin B, and was hypersensitive to other sterol inhibitors. Based on the similarities between the phenotypes of the erg6Δ mutant and clinical isolates of C. lusitaniae resistant to amphotericin B, we analyzed ERG6 expression levels and ergosterol content in multiple clinical isolates. C. lusitaniae amphotericin B-resistant isolates were found to have increased levels of ERG6 transcript as well as reduced ergosterol content. These changes suggest that another gene in the ergosterol biosynthetic pathway could be mutated or misregulated. Further transcript analysis showed that expression of the ERG3 gene, which encodes C-5 sterol desaturase, was reduced in two amphotericin B-resistant isolates. Our findings reveal that mutation or altered expression of ergosterol biosynthetic genes can result in resistance to amphotericin B in C. lusitaniae.

Isolation and Characterization of Cryptococcus neoformans Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis

ABSTRACT Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus Cryptococcus neoformans. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for C. neoformans virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that C. neoformans spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts.

Aspergillus fumigatus LaeA-Mediated Phagocytosis Is Associated with a Decreased Hydrophobin Layer

ABSTRACT Aspergillus fumigatus is the causal agent of the life-threatening disease invasive aspergillosis. A. fumigatus laeA deletants, aberrant in toxin biosynthesis and spore development, are decreased in virulence. Among other characteristics, the decreased virulence is associated with increased spore susceptibility to macrophage phagocytosis. Three characteristics, cell wall microbe-associated molecular patterns (MAMPs), secreted metabolites, and rodlet content, thought to be important in macrophage-Aspergillus spore interactions were examined. Flow cytometry analysis of wild-type and ΔlaeA spores did not reveal any differences in surface-accessible MAMPs, including β-(1,3)-glucan, α-mannose, chitin, and other carbohydrate ligands. Blocking experiments with laminarin and mannan supported the conclusion that differences in cell wall carbohydrates were not responsible for enhanced ΔlaeA spore phagocytosis. Aspergillus spores have been reported to secrete metabolites affecting phagocytosis. Neither spent culture exchange, transwell, nor coincubation internalization experiments supported a role for secreted metabolites in the differential uptake of wild-type and ΔlaeA spores. However, sonication assays implicated a role for surface rodlet protein/hydrophobin (RodAp) in differential spore phagocytosis. A possible role of RodAp in enhanced ΔlaeA spore uptake was further assessed by RodAp extraction and quantification, where wild-type spores were found to contain 60% more RodAp than ΔlaeA spores. After removal of the surface rodlet layer, wild-type spores were phagocytosed at similar rates as ΔlaeA spores. We conclude that increased uptake of ΔlaeA resting spores is not associated with changes in secreted metabolite production of this mutant or surface carbohydrate availability but, rather, due to a decrease in the surface RodAp content of ΔlaeA spores. We theorize that RodAp acts as an antiphagocytic molecule, possibly via physicochemical means and/or by impeding MAMP recognition by macrophage receptors.

Dueling in the lung: how Cryptococcus spores race the host for survival.

Many human fungal pathogens infect people when they are inhaled as spores. Despite the serious impact of fungal spores on human health, little is known about their basic properties or how they interact with the host. This is particularly true for Cryptococcus neoformans, a human fungal pathogen that causes more than 600,000 deaths annually. Spores of C. neoformans have not been well characterized previously because of technical challenges in isolating them; however, recent advances in spore isolation have lead to the first direct analyses of spores. Novel insights into the spore-host interaction, specifically how spores interact with alveolar macrophages, have provided a new model of cryptococcosis that could have broad implications for human fungal pathogenesis.

The mating type-specific homeodomain genes SXI1α and SXI2a coordinately control uniparental mitochondrial inheritance in Cryptococcus neoformans

In the great majority of sexual eukaryotes, mitochondrial genomes are inherited almost exclusively from a single parent. While many hypotheses have been proposed to explain this phenomenon, very little is known about the genetic elements controlling uniparental mitochondria inheritance. In the bipolar, isogamous basidiomycete yeast Cryptococcus neoformans, progeny from crosses between strains of mating type a (MATa) and mating type α (MATα) typically inherit mitochondrial DNA (mtDNA) from the MATa parent. We recently demonstrated that a mating type α (MATα)-specific gene SXI1α, controls mitochondrial inheritance in C. neoformans. Here, we show that another homeodomain gene SXI2a in the alternative mating type MATa is also required for uniparental mtDNA inheritance in this fungus. Disruption of SXI2a resulted in biparental mtDNA inheritance in the zygote population with significant numbers of progeny inheriting mtDNA from the MATa parent, the MATα parent, and both the MATa and the MATα parents. In addition, progeny from same-sex mating between MATα strains showed a biparental mitochondrial inheritance pattern. Our results suggest that SXI1α and SXI2a coordinately control uniparental mitochondrial inheritance in C. neoformans.

Defects in Conidiophore Development and Conidium-Macrophage Interactions in a Dioxygenase Mutant of Aspergillus fumigatus

ABSTRACT Oxygenated fatty acids, or oxylipins, play an essential role in physiological signaling and developmental processes in animals, plants, and fungi. Previous characterization of three Aspergillus fumigatus dioxygenases (PpoA, PpoB, and PpoC), similar in sequence to mammalian cyclooxygenases, showed that PpoA is responsible for the production of the oxylipins 8R-hydroperoxyoctadecadienoic acid and 5S,8R-dihydroxy-9Z,12Z-octadecadienoic acid and that PpoC is responsible for 10R-hydroxy-8E,12Z-hydroperoxyoctadecadienoic acid. Here, Δppo mutants were characterized to elucidate the role of fungal dioxygenases in A. fumigatus development and host interactions. The ΔppoC strain displayed distinct phenotypes compared to those of other Δppo mutants and the wild type, including altered conidium size, germination, and tolerance to oxidative stress as well as increased uptake and killing by primary alveolar macrophages. These experiments implicate oxylipins in pathogen development and suggest that ΔppoC represents a useful model for studying the A. fumigatus-host interaction.

The α-Specific Cell Identity Factor Sxi1α Is Not Required for Virulence of Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans is a human fungal pathogen that has two mating types (a and α). Experiments have shown that in some backgrounds α strains are more virulent than a strains. Our studies reveal that the only known α-specific factor, SXI1α, is not necessary for virulence.

Allelic Exchange of Pheromones and Their Receptors Reprograms Sexual Identity in Cryptococcus neoformans

Cell type specification is a fundamental process that all cells must carry out to ensure appropriate behaviors in response to environmental stimuli. In fungi, cell identity is critical for defining “sexes” known as mating types and is controlled by components of mating type (MAT) loci. MAT–encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The human fungal pathogen Cryptococcus neoformans has two mating types (a and α) that are specified by an extremely unusual MAT locus. The complex architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created strains in which the pheromone and pheromone receptor from one mating type (a) replaced the pheromone and pheromone receptor of the other (α). We discovered that these “αa” cells effectively adopt a new mating type (that of a cells); they sense and respond to α factor, they elicit a mating response from α cells, and they fuse with α cells. In addition, αa cells lose the α cell type-specific response to pheromone and do not form germ tubes, instead remaining spherical like a cells. Finally, we discovered that exogenous expression of the diploid/dikaryon-specific transcription factor Sxi2a could then promote complete sexual development in crosses between α and αa strains. These data reveal that cell identity in C. neoformans is controlled fully by three kinds of MAT–encoded proteins: pheromones, pheromone receptors, and homeodomain proteins. Our findings establish the mechanisms for maintenance of distinct cell types and subsequent developmental behaviors in this unusual human fungal pathogen.