Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.

SummaryCell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae , the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae b -1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans . Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.

The fungal pathogen Cryptococcus neoformans survives phagocytosis by macrophages and proliferates within, ultimately establishing latent infection as a facultative intracellular pathogen that can escape macrophage control to cause disseminated disease. This process is hypothesized to be important for C. neoformans pathogenesis; however, it is poorly understood how C. neoformans adapts to and overcomes the hostile intracellular environment of the macrophage. Using DNA microarray technology, we have investigated the transcriptional response of C. neoformans to phagocytosis by murine macrophages. The expression profiles of several genes were verified using quantitative reverse transcription-PCR and a green fluorescent protein reporter strain. Multiple membrane transporters for hexoses, amino acids, and iron were up-regulated, as well as genes involved in responses to oxidative stress. Genes involved in autophagy, peroxisome function, and lipid metabolism were also induced. Interestingly, almost the entire mating type locus displayed increased expression 24 h after internalization, suggesting an intrinsic connection between infection and the MAT locus. Genes in the Gpa1-cyclic AMP-protein kinase A pathway were also up-regulated. Both gpa1 and pka1 mutants were found to be compromised in macrophage infection, confirming the important role of this virulence pathway. A large proportion of the repressed genes are involved in ribosome-related functions, rRNA processing, and translation initiation/elongation, implicating a reduction in translation as a central response to phagocytosis. In summary, this gene expression profile allows us to interpret the adaptation of C. neoformans to the intracellular infection process and informs the search for genes encoding novel virulence attributes.

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.

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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.

The function of calcium as a signaling molecule is conserved in eukaryotes from fungi to humans. Previous studies have identified the calcium-activated phosphatase calcineurin as a critical factor in governing growth of the human pathogenic fungus Cryptococcus neoformans at mammalian body temperature. Here, we employed insertional mutagenesis to identify new genes required for growth at 37°C. One insertion mutant, cam1-ts, that displayed a growth defect at 37°C and hypersensitivity to the calcineurin inhibitor FK506 at 25°C was isolated. Both phenotypes were linked to the dominant marker in genetic crosses, and molecular analysis revealed that the insertion occurred in the 3 untranslated region of the gene encoding the calcineurin activator calmodulin (CAM1) and impairs growth at 37°C by significantly reducing calmodulin mRNA abundance. The CAM1 gene was demonstrated to be essential using genetic analysis of a CAM1/cam1⌬ diploid strain. In the absence of calcineurin function, the cam1-ts mutant displayed a severe morphological defect with impaired bud formation. Expression of a calmodulin-independent calcineurin mutant did not suppress the growth defect of the cam1-ts mutant at 37°C, indicating that calmodulin promotes growth at high temperature via calcineurin-dependent and -independent pathways. In addition, a Ca 2؉ -binding-defective allele of CAM1 complemented the 37°C growth defect, FK506 hypersensitivity, and morphogenesis defect of the cam1-ts mutant. Our findings reveal that calmodulin performs Ca 2؉ -and calcineurin-independent and -dependent roles in controlling C. neoformans morphogenesis and high-temperature growth.

The 14-3-3 family of proteins function as small adaptors that facilitate a diverse array of cellular processes by mediating specific protein interactions. One such process is the DNA damage checkpoint, where these proteins prevent inappropriate activation of cyclin-dependent kinases. The filamentous fungus Aspergillus nidulans possesses a highly conserved 14-3-3 homologue (artA) that may function in an analogous manner to prevent septum formation. However, instead of blocking septation, over-expression of artA causes a severe delay in the polarization of conidiospores. This observation suggests that these proteins play an important role in hyphal morphogenesis. ß