Molecular Dissection of Crz1 and Its Dynamic Subcellular Localization in Cryptococcus neoformans

Chadwick, Benjamin J; Ross, Brittain Elizabeth; Lin, Xiaorong

doi:10.3390/jof9020252

Cited by 3 publications

(3 citation statements)

References 37 publications

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“…Intriguingly, the residue S103 of Crz1, which becomes phosphorylated upon CKA1 deletion, has been previously reported to be critical for Crz1 function ( 43 ). To further explore this, we deleted CKA1 in a strain expressing Crz1-mCherry ( 44 ) and assessed Crz1’s nuclear translocation efficiency (see Fig. S7 ).…”

Section: Resultsmentioning

confidence: 99%

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Casein kinase 2 complex: a central regulator of multiple pathobiological signaling pathways in Cryptococcus neoformans

Choi,

Yu,

Lee

et al. 2024

mBio

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The casein kinase 2 (CK2) complex has garnered extensive attention over the past decades as a potential therapeutic target for diverse human diseases, including cancer, diabetes, and obesity, due to its pivotal roles in eukaryotic growth, differentiation, and metabolic homeostasis. While CK2 is also considered a promising antifungal target, its role in fungal pathogens remains unexplored. In this study, we investigated the functions and regulatory mechanisms of the CK2 complex in Cryptococcus neoformans , a major cause of fungal meningitis. The cryptococcal CK2 complex consists of a single catalytic subunit, Cka1, and two regulatory subunits, Ckb1 and Ckb2. Our findings show that Cka1 plays a primary role as a protein kinase, while Ckb1 and Ckb2 have major and minor regulatory functions, respectively, in growth, cell cycle control, morphogenesis, stress response, antifungal drug resistance, and virulence factor production. Interestingly, triple mutants lacking all three subunits ( cka1 Δ ckb1 Δ ckb2 Δ) exhibited more severe phenotypic defects than the cka1 Δ mutant alone, suggesting that Ckb1/2 may have Cka1-independent functions. In a murine model of systemic cryptococcosis, cka1 Δ and cka1 Δ ckb1 Δ ckb2 Δ mutants showed severely reduced virulence. Transcriptomic, proteomic, and phosphoproteomic analyses further revealed that the CK2 complex controls a wide array of effector proteins involved in transcriptional regulation, cell cycle control, nutrient metabolisms, and stress responses. Most notably, CK2 disruption led to dysregulation of key signaling cascades central to C. neoformans pathogenicity, including the Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin signaling pathways. In summary, our study provides novel insights into the multifaceted roles of the fungal CK2 complex and presents a compelling case for targeting it in the development of new antifungal drugs. IMPORTANCE The casein kinase 2 (CK2) complex, crucial for eukaryotic growth, differentiation, and metabolic regulation, presents a promising therapeutic target for various human diseases, including cancer, diabetes, and obesity. Its potential as an antifungal target is further highlighted in this study, which explores CK2’s functions in C. neoformans , a key fungal meningitis pathogen. The CK2 complex in C. neoformans , comprising the Cka1 catalytic subunit and Ckb1/2 regulatory subunits, is integral to processes like growth, cell cycle, morphogenesis, stress response, drug resistance, and virulence. Our findings of CK2’s role in regulating critical signaling pathways, including Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin, underscore its importance in C. neoformans pathogenicity. This study provides valuable insights into the fungal CK2 complex, reinforcing its potential as a target for novel antifungal drug development and pointing out a promising direction for creating new antifungal agents.

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Section: Resultsmentioning

confidence: 99%

“…For visualizing the localization of Crz1-mCherry, P CRZ1 -Crz1-mCherry (NEO) (JL334) ( 44 ), and P CRZ1 -Crz1-mCherry (NEO) cka1 Δ (YSB11149) strains were first cultured in YPD at 30°C. They were then diluted with fresh YPD media to an OD 600 of 0.2 and incubated at 23°C until an OD 600 of 0.8 was achieved.…”

Section: Methodsmentioning

confidence: 99%

Casein kinase 2 complex: a central regulator of multiple pathobiological signaling pathways in Cryptococcus neoformans

Choi,

Yu,

Lee

et al. 2024

mBio

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“…Interestingly, combined mutation of Crz1 and P-body targets does not recapitulate the phenotypes of calcineurin deletion, suggesting that additional substrates may be important for calcineurin functions [ 24 ]. A recent study revealed that Crz1 also localizes to stress granules and septa upon heat stress, similar to calcineurin [ 26 ]. Interestingly, the study found that Crz1 localizes to stress granules prior to its translocation to the nucleus hinting at the possibility that calcineurin might be dephosphorylating Crz1 at stress granules.…”

Section: What Are Calcineurin’s Substrates?mentioning

confidence: 99%

Calcineurin: The Achilles’ heel of fungal pathogens

Yadav

Heitman

2023

PLoS Pathog

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Transcriptional and Post-Translational Roles of Calcineurin in Cationic Stress and Glycerol Biosynthesis in Cryptococcus neoformans

Santos,

Martins-Silva,

Padilla

et al. 2024

JoF

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Stress management is an adaptive advantage for survival in adverse environments. Pathogens face this challenge during host colonization, requiring an appropriate stress response to establish infection. The fungal pathogen Cryptococcus neoformans undergoes thermal, oxidative, and osmotic stresses in the environment and animal host. Signaling systems controlled by Ras1, Hog1, and calcineurin respond to high temperatures and osmotic stress. Cationic stress caused by Na+, K+, and Li+ can be overcome with glycerol, the preferred osmolyte. Deleting the glycerol phosphate phosphatase gene (GPP2) prevents cells from accumulating glycerol due to a block in the last step of its biosynthetic pathway. Gpp2 accumulates in a phosphorylated form in a cna1Δ strain, and a physical interaction between Gpp2 and Cna1 was found; moreover, the gpp2Δ strain undergoes slow growth and has attenuated virulence in animal models of infection. We provide biochemical evidence that growth in 1 M NaCl increases glycerol content in the wild type, whereas gpp2Δ, cna1Δ, and cnb1Δ mutants fail to accumulate it. The deletion of cnb1Δ or cna1Δ renders yeast cells sensitive to cationic stress, and the Gfp-Gpp2 protein assumes an abnormal localization. We suggest a mechanism in which calcineurin controls Gpp2 at the post-translational level, affecting its localization and activity, leading to glycerol biosynthesis. Also, we showed the transcriptional profile of glycerol-deficient mutants and established the cationic stress response mediated by calcineurin; among the biological processes differentially expressed are carbon utilization, translation, transmembrane transport, glutathione metabolism, oxidative stress response, and transcription regulation. To our knowledge, this is the first time that this transcriptional profile has been described. These results have implications for pathogen stress adaptability.

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Molecular Dissection of Crz1 and Its Dynamic Subcellular Localization in Cryptococcus neoformans

Cited by 3 publications

References 37 publications

Casein kinase 2 complex: a central regulator of multiple pathobiological signaling pathways in Cryptococcus neoformans

Casein kinase 2 complex: a central regulator of multiple pathobiological signaling pathways in Cryptococcus neoformans

Calcineurin: The Achilles’ heel of fungal pathogens

Transcriptional and Post-Translational Roles of Calcineurin in Cationic Stress and Glycerol Biosynthesis in Cryptococcus neoformans

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