Jennifer L. Reedy

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/α2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections

The number of immunocompromised patients with invasive fungal infections continues to increase and new antifungal therapies are not keeping pace with the growing incidence of these infections and their associated mortality. Calcineurin inhibition is currently used to exert effective immunosuppression following organ transplantation and in treating various other conditions. However, the calcineurin pathway is also intricately involved in the growth and pathogenesis of the three major fungal pathogens of humans, Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, and the exploitation of fungal calcineurin pathways holds great promise for the future development of novel antifungal agents. This Review summarizes our current understanding of calcineurin biology in these fungal species, and its exciting potential role in treating invasive fungal infections.

Cryptococcus neoformans Virulence Gene Discovery through Insertional Mutagenesis

ABSTRACT Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37°C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37°C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu+-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

Mechanistic Plasticity of Sexual Reproduction and Meiosis in the Candida Pathogenic Species Complex

BACKGROUND Candida species are microbial pathogens originally thought to be asexual, but several are now recognized as sexual or parasexual. Candida albicans, the most common fungus infecting humans, is an obligate diploid with a parasexual cycle involving mating, recombination, and genome reduction but no recognized meiosis. Others (C. lusitaniae, C. guilliermondii) are haploid, and their mating produces spores, suggestive of complete meiotic sexual cycles. However, comparative genomic analysis reveals that these species lack key meiotic components, including the recombinase Dmc1 and cofactors (Mei5/Sae3), synaptonemal-complex proteins (Zip1-Zip4/Hop1), and the crossover interference pathway (Msh4/5). RESULTS Here we elucidate the structure and functions of the mating-type (MAT) locus and establish that C. lusitaniae undergoes meiosis during its sexual cycle. The MAT-encoded a2 (high-mobility group) and alpha1 (alpha domain) factors specify a and alpha cell identity, whereas the a1 homeodomain protein drives meiosis and sporulation and functions without its canonical heterodimeric partner, alpha2. Despite the apparent loss of meiotic genes, C. lusitaniae undergoes meiosis during sexual reproduction involving diploid intermediates, frequent SPO11-dependent recombination, and whole-genome reduction generating haploid progeny. The majority of meiotic progeny are euploid, but approximately one-third are diploid/aneuploid. CONCLUSIONS The cell identity and meiotic pathways have been substantially rewired, and meiotic generation of both recombinant and aneuploid progeny may expand genetic diversity. These findings inform our understanding of sexual reproduction in pathogenic microbes and the evolutionary plasticity of the meiotic machinery, with implications for the sexual nature of C. albicans and the generation and consequences of aneuploidy in biology and medicine.

Identification of ENA1 as a Virulence Gene of the Human Pathogenic Fungus Cryptococcus neoformans through Signature-Tagged Insertional Mutagenesis

ABSTRACT A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals.

Elucidating the Candida albicans calcineurin signaling cascade controlling stress response and virulence.

The protein phosphatase calcineurin is a key mediator of virulence and antifungal susceptibility of multiple fungal pathogens including Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, and has clinical potential as a therapeutic target to increase the efficacy of the current antifungal armamentarium. Despite the importance of this signaling pathway, few components of the calcineurin-signaling pathway are known in C. albicans. Here we identified and analyzed additional components of the C. albicans calcineurin cascade, including the RCN1 (regulator of calcineurin1), MID1, and CCH1 genes, which mediate calcineurin functions in other species. When heterologously expressed in Saccharomyces cerevisiae, C. albicans Rcn1 inhibited calcineurin function. Although rcn1/rcn1, mid1/mid1, and cch1/cch1 mutant strains share some phenotypes with calcineurin mutants, they do not completely recapitulate the phenotypes of a calcineurin mutant strain. These studies extend our understanding of the C. albicans calcineurin signaling cascade and its host-niche specific role in virulence.

Dectin-1 Activation Controls Maturation of β-1,3-Glucan-containing Phagosomes

Background: Dectin-1 is able to recognize and phagocytose the fungal carbohydrate, β-1,3-glucan, but its contribution to phagosomal maturation has not been explored. Results: Dectin-1-dependent Syk activation promotes phagolysosomal fusion and acidification. Conclusion: Dectin-1-dependent Syk-activation permits egress of early phagosomes to mature phagolysosomes. Significance: The surface recognition receptor, Dectin-1 shapes anti-fungal responses by controlling fungal phagosome maturation. Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.

Immunotherapy with Tacrolimus (FK506) Does Not Select for Resistance to Calcineurin Inhibitors in Candida albicans Isolates from Liver Transplant Patients

ABSTRACT In Candida albicans, calcineurin mediates tolerance to azole antifungal drugs, survival in serum, and virulence. In this study, we examined 24 Candida isolates from liver transplant recipients receiving a calcineurin inhibitor as a component of their immunosuppressive therapy. We were unable to detect a difference in susceptibility to calcineurin inhibitors in combination with fluconazole, serum, or calcium in these isolates.

Dectin-1 Controls TLR9 Trafficking to Phagosomes Containing β-1,3 Glucan

Dectin-1 and TLR9 play distinct roles in the recognition and induction of innate immune responses to Aspergillus fumigatus and Candida albicans. Dectin-1 is a receptor for the major fungal cell wall carbohydrate β-1,3 glucan that induces inflammatory cytokines and controls phagosomal maturation through spleen tyrosine kinase activation. TLR9 is an endosomal TLR that also modulates the inflammatory cytokine response to fungal pathogens. In this study, we demonstrate that β-1,3 glucan beads are sufficient to induce dynamic redistribution and accumulation of cleaved TLR9 to phagosomes. Trafficking of TLR9 to A. fumigatus and C. albicans phagosomes requires Dectin-1 recognition. Inhibition of phagosomal acidification blocks TLR9 accumulation on phagosomes containing β-1,3 glucan beads. Dectin-1–mediated spleen tyrosine kinase activation is required for TLR9 trafficking to β-1,3 glucan–, A. fumigatus–, and C. albicans–containing phagosomes. In addition, Dectin-1 regulates TLR9-dependent gene expression. Collectively, our study demonstrates that recognition of β-1,3 glucan by Dectin-1 triggers TLR9 trafficking to β-1,3 glucan–containing phagosomes, which may be critical in coordinating innate antifungal defense.

The Carbohydrate Lectin Receptor Dectin-1 Mediates the Immune Response to Exserohilum rostratum

ABSTRACT Dematiaceous molds are found ubiquitously in the environment and cause a wide spectrum of human disease, including infections associated with high rates of mortality. Despite this, the mechanism of the innate immune response has been less well studied, although it is key in the clearance of fungal pathogens. Here, we focus on Exserohilum rostratum, a dematiaceous mold that caused 753 infections during a multistate outbreak due to injection of contaminated methylprednisolone. We show that macrophages are incapable of phagocytosing Exserohilum. Despite a lack of phagocytosis, macrophage production of tumor necrosis factor alpha is triggered by hyphae but not spores and depends upon Dectin-1, a C-type lectin receptor. Dectin-1 is specifically recruited to the macrophage-hyphal interface but not the macrophage-spore interface due to differences in carbohydrate antigen expression between these two fungal forms. Corticosteroid and antifungal therapy perturb this response, resulting in decreased cytokine production. In vivo soft tissue infection in wild-type mice demonstrated that Exserohilum provokes robust neutrophilic and granulomatous inflammation capable of thwarting fungal growth. However, coadministration of methylprednisolone acetate results in robust hyphal tissue invasion and a significant reduction in immune cell recruitment. Our results suggest that Dectin-1 is crucial for macrophage recognition and the macrophage response to Exserohilum and that corticosteroids potently attenuate the immune response to this pathogen.