The Crz1/Sp1 Transcription Factor of Cryptococcus neoformans Is Activated by Calcineurin and Regulates Cell Wall Integrity

ABSTRACTCryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely,PKC1,BCK1,MKK2, andMPK1results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions ofBCK1,MKK2, andMPK1compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis.IMPORTANCECryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/protein kinase A pathway.

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Mechanism of Mpk1 Mitogen-Activated Protein Kinase Binding to the Swi4 Transcription Factor and Its Regulation by a Novel Caffeine-Induced Phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.00794-09 ◽

2009 ◽

Vol 29 (24) ◽

pp. 6449-6461 ◽

Cited By ~ 32

Author(s):

Andrew W. Truman ◽

Ki-Young Kim ◽

David E. Levin

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Protein Kinase ◽

Dna Binding ◽

Binding Site ◽

Mutational Analysis ◽

Cell Wall Integrity ◽

Mitogen Activated Protein Kinase ◽

Activation Mechanism ◽

Mitogen Activated Protein

ABSTRACT The Mpk1 mitogen-activated protein kinase (MAPK) of the cell wall integrity signaling pathway uses a noncatalytic mechanism to activate the SBF (Swi4/Swi6) transcription factor. Active Mpk1 forms a complex with Swi4, the DNA-binding subunit of SBF, conferring the ability to bind DNA. Because SBF activation is independent of Mpk1 catalytic activity but requires Mpk1 to be in an active conformation, we sought to understand how Mpk1 interacts with Swi4. Mutational analysis revealed that binding and activation of Swi4 by Mpk1 requires an intact D-motif-binding site, a docking surface common to MAPKs that resides distal to the phosphorylation loop but does not require the substrate-binding site, revealing a novel mechanism for MAPK target regulation. Additionally, we found that Mpk1 binds near the autoinhibitory C terminus of Swi4, suggesting an activation mechanism in which Mpk1 substitutes for Swi6 in promoting Swi4 DNA binding. Finally, we show that caffeine is an atypical activator of cell wall integrity signaling, because it induces phosphorylation of the Mpk1 C-terminal extension at Ser423 and Ser428. These phosphorylations were dependent on the DNA damage checkpoint kinases, Mec1/Tel1 and Rad53. Phosphorylation of Ser423 specifically blocked SBF activation by preventing Mpk1 association with Swi4, revealing a novel mechanism for regulating MAPK target specificity.

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The monothiol glutaredoxin Grx4 influences thermotolerance, cell wall integrity, and Mpk1 signaling in Cryptococcus neoformans

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab322 ◽

2021 ◽

Author(s):

Guanggan Hu ◽

Linda Horianopoulos ◽

Eddy Sánchez-León ◽

Mélissa Caza ◽

Wonhee Jung ◽

...

Keyword(s):

Cell Wall ◽

Cryptococcus Neoformans ◽

Iron Homeostasis ◽

Elevated Temperatures ◽

Stress Conditions ◽

Cell Wall Integrity ◽

Mitogen Activated Protein Kinase ◽

Calcofluor White ◽

Mitogen Activated Protein ◽

Increased Sensitivity

Abstract Monothiol glutaredoxins are important regulators of iron homeostasis that play conserved roles in the sensing and trafficking of iron-sulfur clusters. We previously characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis, the interaction with the iron regulator Cir1, and virulence in Cryptococcus neoformans. This important fungal pathogen causes cryptococcal meningoencephalitis in immunocompromised individuals worldwide. Here, we demonstrate that Grx4 is required for proliferation at elevated temperatures (both 37°C and 39°C) and under stress conditions. In particular, the grx4Δ mutant was hypersensitive to SDS, calcofluor white (CFW), and caffeine, suggesting that Grx4 is required for membrane and cell wall integrity (CWI). In this context, we found that Grx4 regulated the phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) of the CWI pathway in cells grown at elevated temperature or upon treatment with CFW, caffeine, or SDS. The grx4Δ mutant also displayed increased sensitivity to FK506 and cyclosporin A, two inhibitors of the calcineurin pathway, indicating that Grx4 may influence growth at higher temperatures in parallel with calcineurin signaling. Upon thermal stress or calcium treatment, loss of Grx4 also caused partial mis-localization of Crz1, the transcription factor that is a calcineurin substrate. The phenotypes of the grx4Δ, crz1Δ, and cna1Δ (calcineurin) mutants suggest shared contributions to the regulation of temperature, cell wall, and other stresses. In summary, we show that Grx4 is also a key regulator of the responses to a variety of stress conditions in addition to its roles in iron homeostasis in C. neoformans.

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MaAreB, a GATA Transcription Factor, Is Involved in Nitrogen Source Utilization, Stress Tolerances and Virulence in Metarhizium acridum

Journal of Fungi ◽

10.3390/jof7070512 ◽

2021 ◽

Vol 7 (7) ◽

pp. 512

Author(s):

Chaochuang Li ◽

Qipei Zhang ◽

Yuxian Xia ◽

Kai Jin

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Functional Characterization ◽

Turgor Pressure ◽

Nitrogen Utilization ◽

Cell Wall Integrity ◽

Homologous Proteins ◽

Calcofluor White ◽

Metarhizium Acridum ◽

Gata Transcription Factor

The nitrogen catabolite repression (NCR) pathway is involved in nitrogen utilization, in which the global GATA transcription factor AreA plays an indispensable role and has been reported in many fungi. However, relatively few studies are focused on AreB, another GATA transcription factor in the NCR pathway and the functions of AreB are largely unknown in entomopathogenic fungi. Here, we characterized MaAreB in the model entomopathogenic fungus Metarhizium acridum. Sequence arrangement found that MaAreB had a conserved GATA zinc finger DNA binding domain and a leucine zipper domain. Disruption of MaAreB affected the nitrogen utilization and led to decelerated conidial germination and hyphal growth, decreased conidial yield, and lower tolerances to UV-B irradiation and heat-shock. Furthermore, the MaAreB mutant (ΔMaAreB) exhibited increased sensitivity to CFW (Calcofluor white), decreased cell wall contents (chitin and β-1,3-glucan) and reduced expression levels of some genes related to cell wall integrity, indicating that disruption of MaAreB affected the cell wall integrity. Bioassays showed that the virulence of the ΔMaAreB strain was decreased in topical inoculation but not in intra-hemocoel injection. Consistently, deletion of MaAreB severely impaired the appressorium formation and reduced the turgor pressure of appressorium. These results revealed that MaAreB regulated fungal nitrogen utilization, cell wall integrity and biological control potential, which would contribute to the functional characterization of AreB homologous proteins in other insect fungal pathogens, and even filamentous fungi.

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Cell Wall Integrity Pathway Involved in Morphogenesis, Virulence and Antifungal Susceptibility in Cryptococcus neoformans

Journal of Fungi ◽

10.3390/jof7100831 ◽

2021 ◽

Vol 7 (10) ◽

pp. 831

Author(s):

Haroldo Cesar de Oliveira ◽

Suelen Andreia Rossi ◽

Irene García-Barbazán ◽

Óscar Zaragoza ◽

Nuria Trevijano-Contador

Keyword(s):

Cell Wall ◽

Cryptococcus Neoformans ◽

Morphological Changes ◽

Antifungal Susceptibility ◽

Pathogenic Fungi ◽

Cell Wall Integrity ◽

Cell Permeability ◽

Fungal Cell ◽

Cell Wall Integrity Pathway ◽

Fungal Kingdom

Due to its location, the fungal cell wall is the compartment that allows the interaction with the environment and/or the host, playing an important role during infection as well as in different biological functions such as cell morphology, cell permeability and protection against stress. All these processes involve the activation of signaling pathways within the cell. The cell wall integrity (CWI) pathway is the main route responsible for maintaining the functionality and proper structure of the cell wall. This pathway is highly conserved in the fungal kingdom and has been extensively characterized in Saccharomyces cerevisiae. However, there are still many unknown aspects of this pathway in the pathogenic fungi, such as Cryptococcus neoformans. This yeast is of particular interest because it is found in the environment, but can also behave as pathogen in multiple organisms, including vertebrates and invertebrates, so it has to adapt to multiple factors to survive in multiple niches. In this review, we summarize the components of the CWI pathway in C. neoformans as well as its involvement in different aspects such as virulence factors, morphological changes, and its role as target for antifungal therapies among others.

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Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase

Molecular Microbiology ◽

10.1046/j.1365-2958.2002.03198.x ◽

2002 ◽

Vol 46 (3) ◽

pp. 781-789 ◽

Cited By ~ 163

Author(s):

Un Sung Jung ◽

Andrew K. Sobering ◽

Martin J. Romeo ◽

David E. Levin

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Map Kinase ◽

Cell Wall Integrity

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Role of Protein O-Mannosyltransferase Pmt4 in the Morphogenesis and Virulence of Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.00182-06 ◽

2006 ◽

Vol 6 (2) ◽

pp. 222-234 ◽

Cited By ~ 51

Author(s):

Gillian M. Olson ◽

Deborah S. Fox ◽

Ping Wang ◽

J. Andrew Alspaugh ◽

Kent L. Buchanan

Keyword(s):

Cell Wall ◽

Cryptococcus Neoformans ◽

Cell Separation ◽

Sodium Dodecyl ◽

Protein Composition ◽

Cell Wall Integrity ◽

Abnormal Growth ◽

Transmission Electron ◽

A Cell ◽

And Function

ABSTRACT Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen Cryptococcus neoformans. Disruption of the C. neoformans PMT4 gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the pmt4 mutant compared to wild-type cells. The pmt4 mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the pmt4 mutant, as well as a global decrease in protein mannosylation. Heterologous expression of C. neoformans PMT4 in a Saccharomyces cerevisiae pmt1pmt4 mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in C. neoformans was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of C. neoformans.

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Susceptibility of Cryptococcus neoformans to Photodynamic Inactivation Is Associated with Cell Wall Integrity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00121-07 ◽

2007 ◽

Vol 51 (8) ◽

pp. 2929-2936 ◽

Cited By ~ 49

Author(s):

Beth Burgwyn Fuchs ◽

George P. Tegos ◽

Michael R. Hamblin ◽

Eleftherios Mylonakis

Keyword(s):

Cell Wall ◽

Photodynamic Therapy ◽

Cryptococcus Neoformans ◽

Cell Wall Integrity ◽

Photodynamic Inactivation ◽

Nucleotide Exchange ◽

Wild Type ◽

Kinase C ◽

Nucleotide Exchange Factor ◽

Cell Wall Integrity Pathway

ABSTRACT Photodynamic therapy is a rapidly developing antimicrobial technology which combines a nontoxic photoactivatable dye or photosensitizer with harmless visible light of the correct wavelength to excite the dye to its reactive triplet state to generate reactive oxygen species toxic to cells. In this report we present evidence that the fungal pathogen Cryptococcus neoformans is susceptible to photodynamic inactivation by use of a polycationic conjugate of polyethyleneimine and the photosensitizer chlorin(e6). A C. neoformans rom2 mutant, with a mutation involving a putative Rho1 guanyl nucleotide exchange factor that is part of the protein kinase C-cell wall integrity pathway, demonstrated a compromised cell wall and less (1,3)β-d glucan than the wild-type strain and increased accumulation of PEI-ce6 as assessed by fluorescence uptake and confocal microscopy. Interestingly, C. neoformans rom2 was hypersusceptible to photodynamic inactivation and coincubation of wild-type C. neoformans strain KN99α with caspofungin-enhanced photoinactivation. These studies demonstrated that C. neoformans is sensitive to photodynamic therapy and illustrated the significance of cell wall integrity in microbial susceptibility to antimicrobial photodynamic inactivation.

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Cryptococcus neoformans Rim101 Is Associated with Cell Wall Remodeling and Evasion of the Host Immune Responses

mBio ◽

10.1128/mbio.00522-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 55

Author(s):

Teresa R. O'Meara ◽

Stephanie M. Holmer ◽

Kyla Selvig ◽

Fred Dietrich ◽

J. Andrew Alspaugh

Keyword(s):

Immune Response ◽

Cell Wall ◽

Transcription Factor ◽

Immune System ◽

Immune Responses ◽

Cryptococcus Neoformans ◽

Host Immune Response ◽

Host Immune System ◽

Content Type ◽

Host Pathogen

ABSTRACTInfectious microorganisms often play a role in modulating the immune responses of their infected hosts. We demonstrate thatCryptococcus neoformanssignals through the Rim101 transcription factor to regulate cell wall composition and the host-pathogen interface. In the absence of Rim101,C. neoformansexhibits an altered cell surface in response to host signals, generating an excessive and ineffective immune response that results in accelerated host death. This host immune response to therim101Δ mutant strain is characterized by increased neutrophil influx into the infected lungs and an altered pattern of host cytokine expression compared to the response to wild-type cryptococcal infection. To identify genes associated with the observed phenotypes, we performed whole-genome RNA sequencing experiments under capsule-inducing conditions. We defined the downstream regulon of the Rim101 transcription factor and determined potential cell wall processes involved in the capsule attachment defects and altered mechanisms of virulence in therim101Δ mutant. The cell wall generates structural stability for the cell and allows the attachment of surface molecules such as capsule polysaccharides. In turn, the capsule provides an effective mask for the immunogenic cell wall, shielding it from recognition by the host immune system.IMPORTANCECryptococcus neoformansis an opportunistic human pathogen that is a significant cause of death in immunocompromised individuals. There are two major causes of death due to this pathogen: meningitis due to uncontrolled fungal proliferation in the brain in the face of a weakened immune system and immune reconstitution inflammatory syndrome characterized by an overactive immune response to subclinical levels of the pathogen. In this study, we examined howC. neoformansuses the conserved Rim101 transcription factor to specifically remodel the host-pathogen interface, thus regulating the host immune response. These studies explored the complex ways in which successful microbial pathogens induce phenotypes that ensure their own survival while simultaneously controlling the nature and degree of the associated host response.

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