Deletion of Cryptococcus neoformans AIF Ortholog Promotes Chromosome Aneuploidy and Fluconazole-Resistance in a Metacaspase-Independent Manner

Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLCR) in vitro and in vivo. FLCR isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLCR subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug resistance

Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is the leading cause of death by fungal meningoencephalitis; however, treatment options remain limited. Here we report the construction of 264 signature-tagged gene-deletion strains for 129 putative kinases, and examine their phenotypic traits under 30 distinct in vitro growth conditions and in two different hosts (insect larvae and mice). Clustering analysis of in vitro phenotypic traits indicates that several of these kinases have roles in known signalling pathways, and identifies hitherto uncharacterized signalling cascades. Virulence assays in the insect and mouse models provide evidence of pathogenicity-related roles for 63 kinases involved in the following biological categories: growth and cell cycle, nutrient metabolism, stress response and adaptation, cell signalling, cell polarity and morphology, vacuole trafficking, transfer RNA (tRNA) modification and other functions. Our study provides insights into the pathobiological signalling circuitry of C. neoformans and identifies potential anticryptococcal or antifungal drug targets.OAIID:RECH_ACHV_DSTSH_NO:T201615370RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:A003535CITE_RATE:11.329FILENAME:4. ncomms12766.pdfDEPT_NM:농생명공학부EMAIL:[email protected]_YN:YFILEURL:https://srnd.snu.ac.kr/eXrepEIR/fws/file/fce63c4a-7de7-4741-996f-d8d24af38905/linkCONFIRM:

<i>Candida albicans</i> strains used in this study.

<p><i>Candida albicans</i> strains used in this study.</p

Depletion of zinc induces filamentous growth of <i>C</i>. <i>albicans</i>.

<p><b>A)</b> DTPA and the zinc chelator TPEN induce filamentation. Wild-type cells (SN95) were grown in synthetic defined medium with 100 μM DTPA or 10 μM TPEN at 30°C for 24 hours. <b>B)</b> Zinc depletion induces filamentation. Wild-type cells were grown in untreated synthetic defined medium (Complete) or that treated with Chelex 100 resin to deplete metals (Metal-depleted). All metals, all metals except for zinc, or only zinc were restored, as indicated. DTPA-treated cells were grown in 50 μM DTPA, in the presence or absence of 8.7 mg/L ZnSO₄. Cells were grown for 24 hours at 30°C. <b>C)</b> Transcriptional repression of the zinc transporters <i>ZRT1</i> or <i>ZRT2</i> renders cells hyperfilamentous in DTPA. Strains in which the only allele of <i>ZRT1</i> or <i>ZRT2</i> is under the control of a tetracycline-repressible promoter, and their wild-type counterpart (CaSS1), were grown for 24 hours in YPD at 30°C in the presence of 20 μg/mL doxycycline (DOX) and the presence or absence of 10 μM DTPA, and subcultured into YPD with 20 μg/mL DOX, in the presence or absence of 50 μM DTPA for 4.5 hours at 30°C. In both the <i>ZRT1</i> and <i>ZRT2</i> depletion strains, <i>HWP1</i> expression is increased in the presence of DOX and DTPA, relative to the wild type (P<0.0001, two-way ANOVA, Bonferroni correction). All scale bars are 20 μM.</p