Genealogy can illuminate the evolutionary path of important human pathogens. In some microbes, strict clonal reproduction predominates, as with the worldwide dissemination of Mycobacterium leprae, the cause of leprosy. In other pathogens, sexual reproduction yields clones with novel attributes, for example, enabling the efficient, oral transmission of the parasite Toxoplasma gondii. However, the roles of clonal or sexual propagation in the origins of many other microbial pathogen outbreaks remain unknown, like the recent fungal meningoencephalitis outbreak on Vancouver Island, Canada, caused by Cryptococcus gattii. Here we show that the C. gattii outbreak isolates comprise two distinct genotypes. The majority of isolates are hypervirulent and have an identical genotype that is unique to the Pacific Northwest. A minority of the isolates are significantly less virulent and share an identical genotype with fertile isolates from an Australian recombining population. Genotypic analysis reveals evidence of sexual reproduction, in which the majority genotype is the predicted offspring. However, instead of the classic a-alpha sexual cycle, the majority outbreak clone appears to have descended from two alpha mating-type parents. Analysis of nuclear content revealed a diploid environmental isolate homozygous for the major genotype, an intermediate produced during same-sex mating. These studies demonstrate how cryptic same-sex reproduction can enable expansion of a human pathogen to a new geographical niche and contribute to the ongoing production of infectious spores. This has implications for the emergence of other microbial pathogens and inbreeding in host range expansion in the fungal and other kingdoms.
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 -(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.
Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved G␣ protein/cyclic AMP (cAMP) pathway controls melanin and capsule production. To identify targets of this pathway, we used an expression profiling approach to define genes that are transcriptionally regulated by the G␣ protein Gpa1. This approach revealed that Gpa1 transcriptionally regulates multiple genes involved in capsule assembly and identified two additional genes with a marked dependence on Gpa1 for transcription. The first is the LAC1 gene, encoding the laccase enzyme that catalyzes a rate-limiting step in diphenol oxidation and melanin production. The second gene identified (LAC2) is adjacent to the LAC1 gene and encodes a second laccase that shares 75% nucleotide identity with LAC1. Similar to the LAC1 gene, LAC2 is induced in response to glucose deprivation. However, LAC2 basal transcript levels are much lower than those for LAC1. Accordingly, a lac2 mutation results in only a modest delay in melanin formation. LAC2 overexpression suppresses the melanin defects of gpa1 and lac1 mutants and partially restores virulence of these strains. These studies provide mechanistic insights into the regulation of capsule and melanin production by the C. neoformans cAMP pathway and demonstrate that multiple laccases contribute to C. neoformans melanin production and pathogenesis.
Invasive aspergillosis (IA) is a common and life-threatening infection in immunocompromised individuals. A number of environmental and epidemiologic risk factors for developing IA have been identified. However, genetic factors that affect risk for developing IA have not been clearly identified. We report that host genetic differences influence outcome following establishment of pulmonary aspergillosis in an exogenously immune suppressed mouse model. Computational haplotype-based genetic analysis indicated that genetic variation within the biologically plausible positional candidate gene plasminogen (Plg; Gene ID 18855) correlated with murine outcome. There was a single nonsynonymous coding change (Gly110Ser) where the minor allele was found in all of the susceptible strains, but not in the resistant strains. A nonsynonymous single nucleotide polymorphism (Asp472Asn) was also identified in the human homolog (PLG; Gene ID 5340). An association study within a cohort of 236 allogeneic hematopoietic stem cell transplant (HSCT) recipients revealed that alleles at this SNP significantly affected the risk of developing IA after HSCT. Furthermore, we demonstrated that plasminogen directly binds to Aspergillus fumigatus. We propose that genetic variation within the plasminogen pathway influences the pathogenesis of this invasive fungal infection.
Cryptococcus neoformans is a fungal pathogen that has evolved over the past 40 million years into three distinct varieties or sibling species (gattii, grubii, and neoformans). Each variety manifests differences in epidemiology and disease, and var. grubii strains are responsible for the vast majority of human disease. In previous studies, ␣ strains were more virulent than congenic a strains in var. neoformans, whereas var. grubii congenic a and ␣ strains exhibited equivalent levels of virulence. Here the role of mating type in the virulence of var. grubii was further characterized in a panel of model systems. Congenic var. grubii a and ␣ strains had equivalent survival rates when cultured with amoebae, nematodes, and macrophages. No difference in virulence was observed between a and ␣ congenic strains in multiple inbred-mouse genetic backgrounds, and there was no difference in accumulations in the central nervous system (CNS) late in infection. In contrast, during coinfections, a and ␣ strains are equivalent in peripheral tissues but ␣ cells have an enhanced predilection to penetrate the CNS. These studies reveal the first virulence difference between congenic a and ␣ strains in the most common pathogenic variety and suggest an explanation for the prevalence of ␣ strains in clinical isolates.
The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the G␣ subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.Cryptococcus neoformans is a heterothallic, basidiomycetous, pathogenic fungus that causes serious infections of the central nervous system in individuals immunocompromised by AIDS or undergoing organ transplantation, cytotoxic chemotherapy, or corticosteroid therapy (6, 31). The virulence of C. neoformans is influenced by several factors, including the production of an antiphagocytic polysaccharide capsule (7,20,27), the use of melanin as an antioxidant (26, 51), growth at host physiological temperature (37 to 39°C) (27, 29), and prototrophy (41). Although not directly involved in the virulence of C. neoformans, mating and filamentous growth may play a survival role in the environment and also promote dissemination of the pathogen into the host. Signaling cascades regulating virulence and differentiation of C. neoformans have been extensively studied, including a mitogen-activated protein kinase (MAPK) pathway, a G-protein-regulated cyclic AMP (cAMP) pathway, a Ras-specific pathway, and the calcineurin pathway (for reviews, see references 29 and 48).The MAPK cascade regulates mating processes involving morphological differentiation, such as the dikaryotic mycelia, basidia, and basidiospores, which are produced in response to peptide pheromones secreted by opposite mating-type cells (24,32,43). The MAPK pathway is composed of mating-typespecific (Ste3␣/a, Ste20␣/a, Ste11␣/a, and Ste12␣/a) and nonspecific (Gpb1, Ste7, and Cpk1) elements (12, 49). Gene disruption experiments revealed that the MAPK pathway is required for mating and cell type-specific differentiation but not for virulence (12). However, mating type has been associated with the virulence of serotype D (variety neoformans) strains by Kwon-Chung et al., who showed that the ␣-mating type is more virulent than the a-mating type (25). Furthermore, Del Poeta et al. demonstrated that the MF␣1 pheromone gene is induced during the late stages of central nervous system infection...
Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2⌬ mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2⌬ mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformans cells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 M) at 30°C, but not at 37°C. Furthermore, sod2⌬ mutant cells that were incubated for 24 h at 37°C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25°C in the presence of air. These data suggest that the C. neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.Eukaryotic cells possess several specialized enzymes that provide protection against reactive oxygen species produced as a by-product of physiologic metabolic processes (46). The mitochondria are an especially important source of endogenous reactive oxygen species in the cell. Under physiological conditions, a fraction of the oxygen consumed by the mitochondria is incompletely reduced, resulting in the formation of superoxide radical (9). Although superoxide radical can undergo spontaneous degradation, mitochondria depend on a manganese-containing superoxide dismutase (Sod2) to catalyze the rapid conversion of superoxide radical to hydrogen peroxide and oxygen (15). Loss of this enzyme activity has profound consequences for the viability of many organisms. For example, mice with a homozygous SOD2 disruption exhibited heart and liver defects, metabolic acidosis, and early neonatal death (23,24). Drosophila melanogaster SOD2 mutants exhibited increased sensitivity to oxidative stress and early onset death in young adults (21), and Saccharomyces cerevisiae SOD2 mutants exhibited a rapid loss of viability in the early stationary phase (25). These studies demonstrate a conserved and essential function for Sod2 as a component of the mitochondrial antioxidant defense system of many phylogenetically diverse organisms.Cryptococcus neoformans is an opportunistic human pathogen. Most cases of C. neoformans disease involve immunocompromised patients, such as those with human immunodeficiency virus infection or receiving corticosteroid therapy. In these patients, C. neoformans ca...
The ability to survive and proliferate at 37°C is an essential virulence attribute of pathogenic microorganisms. A partial-genome microarray was used to profile gene expression in the human-pathogenic fungus Cryptococcus neoformans during growth at 37°C. Genes with orthologs involved in stress responses were induced during growth at 37°C, suggesting that a conserved transcriptional program is used by C. neoformans to alter gene expression during stressful conditions. A gene encoding the transcription factor homolog Mga2 was induced at 37°C and found to be important for high-temperature growth. Genes encoding fatty acid biosynthetic enzymes were identified as potential targets of Mga2, suggesting that membrane remodeling is an important component of adaptation to high growth temperatures. mga2⌬ mutants were extremely sensitive to the ergosterol synthesis inhibitor fluconazole, indicating a coordination of the synthesis of membrane component precursors. Unexpectedly, genes involved in amino acid and pyrimidine biosynthesis were repressed at 37°C, but components of these pathways were found to be required for high-temperature growth. Our findings demonstrate the utility of even partial-genome microarrays for delineating regulatory cascades that contribute to microbial pathogenesis.
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