Cryptococcosis is primarily caused by Cryptococcus neoformans and Cryptococcus gattii. These two pathogenic species each divide into four distinct molecular genotypes. In this study, we examined whether genotype influenced susceptibility to antifungal drugs used to treat cryptococcosis using the broth microdilution method described by the Clinical and Laboratory Standards Institute. C. gattii isolates belonging to molecular genotype VGII had significantly higher MIC values for flucytosine and all azole antifungal agents tested, particularly fluconazole, than isolates of other C. gattii genotypes. In an extended analysis of fluconazole susceptibility, VGII isolates from the north and west of Australia required higher drug levels for inhibition than those from Vancouver Island, Canada. Within C. neoformans, genotype VNII had significantly lower geometric mean MICs for fluconazole than genotype VNI. These results indicate that cryptococcal species, molecular genotype, and region of origin may be important when deciding treatment options for cryptococcosis.

The cell cycle and the circadian clock communicate with each other, resulting in circadian-gated cell division cycles. Alterations in this network may lead to diseases such as cancer. Therefore, it is critical to identify molecular components that connect these two oscillators. However, molecular mechanisms between the clock and the cell cycle remain largely unknown. A model filamentous fungus, Neurospora crassa, is a multinucleate system used to elucidate molecular mechanisms of circadian rhythms, but not used to investigate the molecular coupling between these two oscillators.In this report, we show that a conserved coupling between the circadian clock and the cell cycle exists via serine/threonine protein kinase-29 (STK-29), the Neurospora homolog of mammalian WEE1 kinase. Based on this finding, we established a mathematical model that predicts circadian oscillations of cell cycle components and circadian clock-dependent synchronized nuclear divisions. We experimentally demonstrate that G1 and G2 cyclins, CLN-1 and CLB-1, respectively, oscillate in a circadian manner with bioluminescence reporters. The oscillations of clb-1 and stk-29 gene expression are abolished in a circadian arrhythmic frq ko mutant. Additionally, we show the light-induced phase shifts of a core circadian component, frq, as well as the gene expression of the cell cycle components clb-1 and stk-29, which may alter the timing of divisions. We then used a histone hH1-GFP reporter to observe nuclear divisions over time, and show that a large number of nuclear divisions occur in the evening. Our findings demonstrate the circadian clock-dependent molecular dynamics of cell cycle components that result in synchronized nuclear divisions in Neurospora. M olecular mechanisms of circadian rhythms provide temporal information to other cellular processes, such as metabolism, to optimize their outcomes (1-3). For instance, circadian oscillations of rate-limiting genes in glucose metabolism suggest time-of-day specific regulatory mechanisms that maintain glucose homeostasis in mammals (3). Circadian clockgated cell division cycles have been observed in various organisms, including mammals, indicating that cell divisions preferentially occur at specific times of the day (4-7). In the mouse liver, expression of the cell cycle kinase-encoding gene, wee1, is directly activated by a heterodimeric circadian transcription factor, CLOCK-BMAL1, providing a molecular link between the cell cycle and circadian rhythms (5). This suggests that circadian clock-regulated WEE1 promotes periodic inhibition of mitotic cycles between G2 and M phase by phosphorylating and inactivating the mitotic cyclin-dependent kinase (CDK) (8). On the other hand, circadian-independent cell divisions have been reported in rat-1 fibroblasts despite the fact that these cells maintain robust circadian rhythms (9). These data suggest that not all cells with circadian rhythms may display circadian-gated cell division cycles.The multinucleate fungus Neurospora crassa has played a pivotal role i...

The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1bd. These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output.

Cryptococcus gattii is an encapsulated fungus capable of causing fatal disease in immunocompetent humans and animals. As current antifungal therapies are few and limited in efficacy, and resistance is an emerging issue, the development of new treatment strategies is urgently required. The current study undertook a time-course analysis of the proteome of C. gattii during treatment with fluconazole (FLC), which is used widely in prophylactic and maintenance therapies. The aims were to analyze the overall cellular response to FLC, and to find fungal proteins involved in this response that might be useful targets in therapies that augment the antifungal activity of FLC. During FLC treatment, an increase in stress response, ATP synthesis and mitochondrial respiratory chain proteins, and a decrease in most ribosomal proteins was observed, suggesting that ATP-dependent efflux pumps had been initiated for survival and that the maintenance of ribosome synthesis was differentially expressed. Two proteins involved in fungal specific pathways were responsive to FLC. An integrative network analysis revealed co-ordinated processes involved in drug response, and highlighted hubs in the network representing essential proteins that are required for cell viability. This work demonstrates the dynamic cellular response of a typical susceptible isolate of C. gattii to FLC, and identified a number of proteins and pathways that could be targeted to augment the activity of FLC.

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