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:

Major Sensing Proteins in Pathogenic Fungi: The Hybrid Histidine Kinase Family

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Crz1 acts upstream of Znf2 in regulating filamentation induced by glucosamine.

<p>(<b>A</b>) Transcript levels of <i>CRZ1</i> and <i>ZNF2</i> in the wild-type H99, the <i>crz1</i>Δ mutant, and the <i>znf2</i>Δ mutant cultured on glucosamine medium for 2 days, 4 days, and 6 days compared to the control of 0 days. (<b>B</b>) The wild-type H99, the <i>znf2</i>Δ mutant, the <i>ZNF2</i>^<i>oe</i> <i>znf2</i>Δ strain, the <i>CRZ1</i>^<i>oe</i> <i>znf2</i>Δ strain, the <i>crz1</i>Δ mutant, the <i>ZNF2</i>^<i>oe</i> <i>crz1</i>Δ strain, and the <i>CRZ1</i>^<i>oe</i> <i>crz1</i>Δ strain were cultured on glucosamine medium for 7 days. The overexpression of both <i>CRZ1</i> and <i>ZNF2</i> was driven by the constitutively active <i>GPD1</i> promoter and the inducible <i>CTR4</i> promoter respectively. (<b>C</b>) The subcellular localization of mCherry-Znf2 in the <i>crz1</i>Δ mutant and in the wild-type strain H99 on glucosamine medium. DAPI was used to indicate nuclear localization.</p

Glucosamine stimulates Crz1 translocation to the nucleus.

<p>(<b>A</b>) Localization of mCherry-Crz1 under different the indicated conditions. To test temperature’s effect on the subcellular localization of mCrz1, the strain P_<i>GPD1</i>-mCherry-<i>CRZ1</i> was cultured in YPD liquid at 22°C (first row, or 37°C (2^nd row) with shaking overnight. To test the effect of calcium or NaCl on the subcellular localization of mCrz1, cells of the strain P_<i>GPD1</i>-mCherry-<i>CRZ1</i> were collected from an overnight culture in liquid YPD at 22°C and then suspended in 100 mM CaCl₂ or 1.5 M NaCl solution for 10–20 min (3^rd and 5^th rows). To test the effect of glucosamine on mCrz1’s localization, the strain P_<i>GPD1</i>-mCherry-<i>CRZ1</i> was cultured in YPGlcN liquid medium for 12 hours at 22°C (4^th row). (<b>B</b>) Quantification of the percent of cells with mCherry-Crz1 located to the nucleus under the conditions used in panel A. (n≥60) (*** p<0.001). (<b>C</b>) Cells of the strain mCherry-<i>CRZ1</i>/<i>znf2</i>Δ were tested for the effect of glucosamine and CaCl₂ on the localization of mCherry-Crz1 as described in panel A. (<b>D</b>) Quantification of the percentage of cells with mCherry-Crz1 located to the nucleus under the same conditions used in panel C.</p

Crz1 depends on calcineurin for its nuclear localization and its regulation of filamentation on glucosamine medium.

<p>(<b>A</b>) Strains <i>cna1</i>Δ, <i>cna1</i>ΔP_<i>GPD1</i>-mCherry-Crz1, <i>cnb1</i>Δ, <i>cnb1</i>ΔP_<i>GPD1</i>-mCherry-Crz1, <i>cbp1</i>Δ, and <i>cbp1</i>ΔP_<i>GPD1</i>-mCherry-Crz1 were cultured on YP+GlcN medium for 7 days. (<b>B</b>) a diagram depicting the different localization patterns of Crz1: diffused in the cytosol with nuclear exclusion (left); localized to the nucleus (middle); localized to both cytoplasm and the nucleus but more concentrated in the nucleus (right), (<b>C</b>) Strains <i>cna1</i>ΔP_<i>GPD1</i>-mCherry-Crz1, <i>cnb1</i>ΔP_<i>GPD1</i>-mCherry-Crz1, and <i>cbp1</i>ΔP_<i>GPD1</i>-mCherry-Crz1 were cultured either in YPD medium or in YP+GlcN medium overnight. The mCherry-Crz1 signal showed diffused cytoplasmic localization in the <i>cna1</i>Δ and <i>cnb1</i>Δ mutants under both conditions. The mCherry-Crz1 signal showed diffused cytoplasmic localization but with more concentrated signals in the nucleus in the <i>cbp1</i>Δ mutant.</p

Glucosamine stimulates self-filamentation in H99 and other cryptococcal isolates.

<p>(<b>A</b>) The effect of the addition of different six-carbon sugars and hexamines at 2% to YP base medium. H99 was cultured on YP, YP+Glc (glucose), YP+Gal (galactose), YP+Inositol, YP+GlcNMe (N-Methyl-glucosamine), YP+GlcNAc (N-Acetyl-glucosamine), YP+GlcN (glucosamine), and YP+2-Dexoyl-Glc (2-Deoxyl-glucose) for 7 days. (<b>B</b>) The dose-dependent effect of glucosamine on self-filamentation in H99. H99 was cultured on YP+GlcN at final concentration of 0, 0.2%, 0.5%, 1%, and 2% for 7 days. (<b>C</b>) The inhibitory effect of other carbon sources on GlcN-induced filamentation. H99 was cultured on the indicated media for 7 days. H99 cultured on YP+GlcN (2%) was used as control. Other carbon sources, such as glucose, galactose, or N-Acetyl-glucosamine (2%) were added to the YP+GlcN medium. (<b>D</b>) The effect of glucosamine on filamentation is not specific to H99. 92BC2-45 (serotype A), 92BC2-11 (serotype A), 93BC1-53 (serotype D), 35–20 VNI (serotype A), 98BC1-50 (serotype D), Bt3 strain (serotype A), and XL280α (serotype D) were cultured on YP+GlcN medium for 7 days.</p

Crz1 acts upstream of Znf2 in regulating filamentation induced by glucosamine.

<p>(<b>A</b>) Transcript levels of <i>CRZ1</i> and <i>ZNF2</i> in the wild-type H99, the <i>crz1</i>Δ mutant, and the <i>znf2</i>Δ mutant cultured on glucosamine medium for 2 days, 4 days, and 6 days compared to the control of 0 days. (<b>B</b>) The wild-type H99, the <i>znf2</i>Δ mutant, the <i>ZNF2</i>^<i>oe</i> <i>znf2</i>Δ strain, the <i>CRZ1</i>^<i>oe</i> <i>znf2</i>Δ strain, the <i>crz1</i>Δ mutant, the <i>ZNF2</i>^<i>oe</i> <i>crz1</i>Δ strain, and the <i>CRZ1</i>^<i>oe</i> <i>crz1</i>Δ strain were cultured on glucosamine medium for 7 days. The overexpression of both <i>CRZ1</i> and <i>ZNF2</i> was driven by the constitutively active <i>GPD1</i> promoter and the inducible <i>CTR4</i> promoter respectively. (<b>C</b>) The subcellular localization of mCherry-Znf2 in the <i>crz1</i>Δ mutant and in the wild-type strain H99 on glucosamine medium. DAPI was used to indicate nuclear localization.</p

The HOG components function upstream of Crz1 and suppress Crz1’s nuclear localization.

<p>(<b>A</b>) Cells of the wild-type KN99<b>a</b>, <i>crz1</i>Δ (<b>a</b>), <i>ssk2</i>Δ (<b>a</b>), and <i>pbs2</i>Δ (<b>a</b>) were cultured on YP+GlcN (0.5%) medium for 7 days. (<b>B</b>) Cells of the wild-type H99, the <i>ssk2</i>Δ<i>crz1</i>Δ double mutant, and the <i>pbs2</i>Δ<i>crz1</i>Δ double mutant were cultured on YP+GlcN medium for 7 days. (<b>C</b>) The control strain XW252 (GFP-Nop1, P_<i>CRZ1</i>-Crz1-mCherry) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006982#pgen.1006982.ref079" target="_blank">79</a>] and the corresponding <i>ssk1</i>Δ, <i>ssk2</i>Δ, and <i>pbs2</i>Δ mutants in the XW252 background were cultured in YPD or YP+GlcN medium overnight at 22°C. GFP-Nop1 labels the nucleolus within the nucleus [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006982#pgen.1006982.ref079" target="_blank">79</a>]. (<b>D</b>) The strain JL408 (<i>MAT</i>α, <i>hog</i>1Δ; P_<i>GPD1</i>-mCherry-<i>CRZ1</i>) was generated from a cross between the <i>hog1</i>Δ mutant in the mating type <b>a</b> background and the strain JL410 (<i>MAT</i>α, P_<i>GPD1</i>-mCherry-<i>CRZ1</i>). JL408 was cultured in YP or YP+GlcN medium overnight at 22°C. (<b>E</b>) The wild-type strain H99 was grown to mid-logarithmic phase and then exposed to 1 M NaCl or 2% glucosamine (YPGlcN) for the indicated time points. Hog1 phosphorylation levels were monitored using anti-P-p38 antibody. The blot was stripped and then used for detection of Hog1 protein level with a polyclonal anti-Hog1 antibody as a loading control.</p