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:

9-O-butyl-13-(4-isopropylbenzyl)berberine, KR-72, is a potent antifungal agent that inhibits the growth of Cryptococcus neoformans by regulating gene expression.

In this study we explored the mode of action of KR-72, a 9-O-butyl-13-(4-isopropylbenzyl)berberine derivative previously shown to exhibit potent antifungal activity against a variety of human fungal pathogens. The DNA microarray data revealed that KR-72 treatment significantly changed the transcription profiles of C. neoformans, affecting the expression of more than 2,000 genes. Genes involved in translation and transcription were mostly upregulated, whereas those involved in the cytoskeleton, intracellular trafficking, and lipid metabolism were downregulated. KR-72 also exhibited a strong synergistic effect with the antifungal agent FK506. KR-72 treatment regulated the expression of several essential genes, including ECM16, NOP14, HSP10 and MGE1, which are required for C. neoformans growth. The KR-72-mediated induction of MGE1 also likely reduced the viability of C. neoformans by impairing cell cycle or the DNA repair system. In conclusion, KR-72 showed antifungal activity by modulating diverse biological processes through a mode of action distinct from those of clinically available antifungal drugs such as polyene and azole drugs

Visualization of basilar artery atherosclerotic plaques by conventional T2-weighted magnetic resonance imaging: A case-control study.

ObjectiveIn vivo visualization of intracranial atherosclerotic plaque has been performed only with high-resolution magnetic resonance imaging (HRMR). We investigated whether atherosclerotic plaque of the basilar artery (BA) can be identified in conventional magnetic resonance imaging (MRI).MethodsPatients with acute ischemic stroke who had BA stenosis ("symptomatic BAA") were retrospectively recruited using the prospective stroke registry. In the HRMR databank, subjects without BA stenosis were recruited and classified as those with silent plaque ("silent BAA") and without any plaque ("normal controls"). Outer diameter of the BA and T2 plaque sign (an eccentric or complete obscuration of normal flow-void) within the BA were assessed by two blinded raters using conventional T2 MRI.ResultsSeventy-five patients with symptomatic BAA, 40 with asymptomatic BAA, and 36 normal controls were included in the study. Maximal BA diameter was significantly larger in symptomatic BAA patients with 70% stenosis (all pConclusionsOur data suggest that BA atherosclerosis can be detected by conventional MRI. When the use of HRMR is limited, conventional MR imaging may give additive information to clinicians

<i>In vitro</i> antifungal activities of KR-72 combined with commercially available antifungal agents against <i>C. neoformans.</i>

a<p>KR, KR-72; FDX, Fludioxonil; 5Fc, 5-flucytosine; FCZ, fluconazole. MIC₅₀ was determined by CLSI method. For calculation purposes, >32, >4, >2, and >0.5 were assumed to be 32, 4, 2, and 0.5. For all checkerboard assays, <i>C. neoformans</i> H99 strain was used.</p>b<p>FICI≤0.5 = synergy, FICI>4.0 = antagonism, 0.5[38].</p><p><i>In vitro</i> antifungal activities of KR-72 combined with commercially available antifungal agents against <i>C. neoformans.</i></p

KR-72 treatment reduced <i>NCR1</i> expression and affected sterol metabolism.

<p>(a) Expression level changes of <i>NCR1</i> during KR-72 treatment (0, 30, 60 min) were measured by northern blot analysis. (b and c) To examine whether KR-72 affects the sterol transport to the cell membrane, the WT strain (H99) was exposed to 1 µg/mL KR-72 or 2.5 mM H₂O₂ for 60 min (b) or indicated incubation time (c), stained with 12.5 mM Filipin working solution and monitored by a fluorescence microscope (b) or fluorescence microplate reader (c) for quantitatively measuring fluorescence intensities. Bar, 10 µm. Fluorescence was calculated at OD_{485–515 nm}/OD_{595 nm}. Three independent triplicate experiments were performed. Standard deviations are presented as error bars. Statistical analysis was performed by Bonferroni’s multiple comparison test. Each symbol in (c) indicates the following: *, <i>P</i><0.05; NS, not significant (<i>P</i>>0.05). (d) Expression level changes of <i>ERG11</i> by KR-72 treatment (90 min) in the WT strain (H99) and <i>hog1Δ</i> mutant were measured by northern blot analysis.</p

<i>C. neoformans</i> mutants defective in cell wall and membrane integrity exhibited increased susceptibility to KR-72.

<p>(a) Each <i>C. neoformans</i> strain [WT (H99), <i>hog1</i>Δ (YSB64), <i>cac1</i>Δ (YSB42), <i>cpk1</i>Δ (YSB127), <i>mpk1</i>Δ (KK3), <i>ras1</i>Δ (YSB53), <i>cna1</i>Δ (KK1), and <i>cnb1</i>Δ (KK2)] was grown overnight at 30°C in liquid YPD medium, 10-fold serially diluted (1 to 10⁴ dilutions), and spotted (3 µL) onto YPD agar containing the indicated concentrations of KR-72. Cells were incubated at 30°C for 3 days and then photographed. (b) The WT H99 strain and <i>cna1</i>Δ (KK1) and <i>cnb1</i>Δ (KK2) mutants were cultured in YPD medium at 30°C for 16–20 h, 10-fold serially diluted (1 to 10⁴ dilutions), and spotted (3 µL) onto YPD agar containing the indicated concentrations of KR-72 (5 [KR-72₅], 10 [KR-72₁₀], or 30 µg/mL [KR-72₃₀]), FK506 (1 µg/mL, FK506₁), or a combination of both (KR-72₅+FK506₁, KR-72₁₀+FK506₁, or KR-72₃₀+FK506₁). Cells were then incubated at 30°C or 37°C for 3 days and then photographed.</p

Functional categories of KR-72-responsive genes in <i>C. neoformans.</i>

<p>(a) Venn diagrams showing number of upregulated (left diagram) and downregulated <i>C. neoformans</i> genes (right diagram) with 30 min and 60 min treatment of KR-72. The number was counted only for genes whose expression levels were significantly changed (ANOVA, <i>P</i><0.05). (b) Functional categories of KR-72 responsive genes in <i>C. neoformans</i>. Among the KR-72 responsive genes, genes whose expression was changed more than 1.5-fold were functionally categorized based on the COG (eukaryotic Cluster of Orthologous Groups of proteins, <a href="http://www.ncbi.nlm/nih.gov/COG/" target="_blank">http://www.ncbi.nlm/nih.gov/COG/</a>) functional description. The red and blue bars indicate the number of up-regulated and down-regulated genes by KR-72, respectively.</p

Overexpression of <i>MGE1</i> promoted cell lethality and susceptibility to genotoxic stresses in the presence of KR-72.

<p>(a) WT H99, P<i>_CTR4:ECM16</i> (YSB2596 and YSB2597), P<i>_CTR4:HSP10</i> (YSB2688 and YSB2689), P<i>_CTR4:NOP14</i> (YSB2604 and YSB2605), and P<i>_CTR4:MGE1</i> (YSB3172 and YSB3173) strains were grown in liquid YNB medium at 30°C overnight, 10-fold serially diluted (1 to 10⁴ dilutions) in sterile distilled water. Then cell suspensions (3 µL) of each strain were spotted onto solid YNB agar medium containing the indicated concentration of KR-72. Cells were incubated at 30°C for 3 days and then photographed. (b) The genotoxic response of the <i>MGE1</i> overexpression strain in <i>C. neoformans</i>. WT H99 and P<i>_CTR4</i>:<i>MGE1</i> (YSB3172 and YSB3173) strains were grown in liquid YNB medium at 30°C overnight and 10-fold serially diluted (1 to 10⁴ dilutions) in dH₂O. Cell suspensions (3 µL) of each strain were then either spotted onto solid YNB agar medium containing the indicated concentration of hydroxyurea (HU) and methyl methanesulfonate (MMS) or exposed to UV. Cells were incubated at 30°C for 3 days and then photographed.</p