Sho1 and Msb2 Play Complementary but Distinct Roles in Stress Responses, Sexual Differentiation, and Pathogenicity of Cryptococcus neoformans

The high-osmolarity glycerol response (HOG) pathway is pivotal in environmental stress response, differentiation, and virulence of Cryptococcus neoformans, which causes fatal meningoencephalitis. A putative membrane sensor protein, Sho1, has been postulated to regulate HOG pathway, but its regulatory mechanism remains elusive. In this study, we characterized the function of Sho1 with relation to the HOG pathway in C. neoformans. Sho1 played minor roles in osmoresistance, thermotolerance, and maintenance of membrane integrity mainly in a HOG-independent manner. However, it was dispensable for cryostress resistance, primarily mediated through the HOG pathway. A mucinlike transmembrane (TM) protein, Msb2, which interacts with Sho1 in Saccharomyces cerevisiae, was identified in C. neoformans, but found not to interact with Sho1. MSB2 codeletion with SHO1 further decreased osmoresistance and membrane integrity, but not thermotolerance, of sho1Δ mutant, indicating that both factors play to some level redundant but also discrete roles in C. neoformans. Sho1 and Msb2 played redundant roles in promoting the filamentous growth in sexual differentiation in a Cpk1-independent manner, in contrast to the inhibitory effect of the HOG pathway in the process. Both factors also played redundant roles in maintaining cell wall integrity in the absence of Mpk1. Finally, Sho1 and Msb2 play distinct but complementary roles in the pulmonary virulence of C. neoformans. Overall, Sho1 and Msb2 play complementary but distinct roles in stress response, differentiation, and pathogenicity of C. neoformans

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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The calcineurin pathway is required for glucosamine-induced self-filamentation.

<p>(<b>A</b>) A diagram depicting the calcineurin pathway. (<b>B</b>) The wild-type H99, the <i>cna1</i>Δ mutant, the <i>cnb1</i>Δ mutant, the <i>cbp1</i>Δ mutant, and two independent <i>crz1</i>Δ mutants were cultured on glucosamine medium for 7 days. (<b>C</b>) The wild-type H99 was cultured on glucosamine medium with or without the calcineurin inhibitor FK506. (<b>D</b>) The wild-type H99, the <i>crz1</i>Δ mutant, and the <i>CRZ1</i>^<i>oe</i> strain (P_<i>GPD1</i>-<i>CRZ1</i>) were cultured on glucosamine 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

Glucosamine stimulates pheromone-independent dimorphic transition in <i>Cryptococcus neoformans</i> by promoting Crz1 nuclear translocation

<div><p>Morphotype switch is a cellular response to external and internal cues. The <i>Cryptococcus neoformans</i> species complex can undergo morphological transitions between the yeast and the hypha form, and such morphological changes profoundly affect cryptococcal interaction with various hosts. Filamentation in <i>Cryptococcus</i> was historically considered a mating response towards pheromone. Recent studies indicate the existence of pheromone-independent signaling pathways but their identity or the effectors remain unknown. Here, we demonstrated that glucosamine stimulated the <i>C</i>. <i>neoformans</i> species complex to undergo self-filamentation. Glucosamine-stimulated filamentation was independent of the key components of the pheromone pathway, which is distinct from pheromone-elicited filamentation. Glucosamine stimulated self-filamentation in H99, a highly virulent serotype A clinical isolate and a widely used reference strain. Through a genetic screen of the deletion sets made in the H99 background, we found that Crz1, a transcription factor downstream of calcineurin, was essential for glucosamine-stimulated filamentation despite its dispensability for pheromone-mediated filamentation. Glucosamine promoted Crz1 translocation from the cytoplasm to the nucleus. Interestingly, multiple components of the high osmolality glycerol response (HOG) pathway, consisting of the phosphorelay system and some of the Hog1 MAPK module, acted as repressors of glucosamine-elicited filamentation through their calcineurin-opposing effect on Crz1’s nuclear translocation. Surprisingly, glucosamine-stimulated filamentation did not require Hog1 itself and was distinct from the conventional general stress response. The results demonstrate that <i>Cryptococcus</i> can resort to multiple genetic pathways for morphological transition in response to different stimuli. Given that the filamentous form attenuates cryptococcal virulence and is immune-stimulatory in mammalian models, the findings suggest that morphogenesis is a fertile ground for future investigation into novel means to compromise cryptococcal pathogenesis.</p></div

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

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