scholarly journals Lrg1 Regulates β (1,3)-Glucan Masking in Candida albicans through the Cek1 MAP Kinase Pathway

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Tian Chen ◽  
Andrew S. Wagner ◽  
Robert N. Tams ◽  
James E. Eyer ◽  
Sarah J. Kauffman ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Map Kinase ◽  
Fungal Virulence ◽  
Content Type ◽  
Map Kinase Cascade ◽  
Kinase Cascade ◽  
Map Kinase Pathway ◽  
Immune Detection ◽  
Kinase Pathway

ABSTRACT Candida albicans is among the most prevalent opportunistic human fungal pathogens. The ability to mask the immunogenic polysaccharide β (1,3)-glucan from immune detection via a layer of mannosylated proteins is a key virulence factor of C. albicans. We previously reported that hyperactivation of the Cek1 mitogen-activated protein (MAP) kinase pathway promotes β (1,3)-glucan exposure. In this communication, we report a novel upstream regulator of Cek1 activation and characterize the impact of Cek1 activity on fungal virulence. Lrg1 encodes a GTPase-activating protein (GAP) that has been suggested to inhibit the GTPase Rho1. We found that disruption of LRG1 causes Cek1 hyperactivation and β (1,3)-glucan unmasking. However, when GTPase activation was measured for a panel of GTPases, the lrg1ΔΔ mutant exhibited increased activation of Cdc42 and Ras1 but not Rho1 or Rac1. Unmasking and Cek1 activation in the lrg1ΔΔ mutant can be blocked by inhibition of the Ste11 MAP kinase kinase kinase (MAPKKK), indicating that the lrg1ΔΔ mutant acts through the canonical Cek1 MAP kinase cascade. In order to determine how Cek1 hyperactivation specifically impacts virulence, a doxycycline-repressible hyperactive STE11ΔN467 allele was expressed in C. albicans. In the absence of doxycycline, this allele overexpressed STE11ΔN467, which induced production of proinflammatory tumor necrosis factor alpha (TNF-α) from murine macrophages. This in vitro phenotype correlates with decreased colonization and virulence in a mouse model of systemic infection. The mechanism by which Ste11ΔN467 causes unmasking was explored with RNA sequencing (RNA-Seq) analysis. Overexpression of Ste11ΔN467 caused upregulation of the Cph1 transcription factor and of a group of cell wall-modifying proteins which are predicted to impact cell wall architecture. IMPORTANCE Candida albicans is an important source of systemic infections in humans. The ability to mask the immunogenic cell wall polymer β (1,3)-glucan from host immune surveillance contributes to fungal virulence. We previously reported that the hyperactivation of the Cek1 MAP kinase cascade promotes cell wall unmasking, thus increasing strain immunogenicity. In this study, we identified a novel regulator of the Cek1 pathway called Lrg1. Lrg1 is a predicted GTPase-activating protein (GAP) that represses Cek1 activity by downregulating the GTPase Cdc42 and its downstream MAPKKK, Ste11. Upregulation of Cek1 activity diminished fungal virulence in the mouse model of infection, and this correlates with increased cytokine responses from macrophages. We also analyzed the transcriptional profile determined during β (1,3)-glucan exposure driven by Cek1 hyperactivation. Our report provides a model where Cek1 hyperactivation causes β (1,3)-glucan exposure by upregulation of cell wall proteins and leads to more robust immune detection in vivo, promoting more effective clearance.

scholarly journals Yeast Protein Kinases and the RHO1 Exchange Factor TUS1 Are Novel Components of the Cell Integrity Pathway in Yeast

2002 ◽  
Vol 22 (5) ◽  
pp. 1329-1339 ◽  
Author(s):  
Tobias Schmelzle ◽  
Stephen B. Helliwell ◽  
Michael N. Hall
Keyword(s):  
Cell Wall ◽  
Actin Cytoskeleton ◽  
Map Kinase ◽  
Cell Integrity ◽  
Exchange Factor ◽  
Downstream Effectors ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Map Kinase Pathway ◽  
Kinase Pathway

ABSTRACT The PKC1-associated mitogen-activated protein (MAP) kinase pathway of Saccharomyces cerevisiae regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. Activation of PKC1 occurs via the GTPase RHO1 and the kinase pair PKH1 and PKH2. Here we report that YPK1 and YPK2, an essential pair of homologous kinases and proposed downstream effectors of PKH and sphingolipids, are also regulators of the PKC1-controlled MAP kinase cascade. ypk mutants display random distribution of the actin cytoskeleton and severely reduced activation of the MAP kinase MPK1. Upregulation of the RHO1 GTPase switch or the PKC1 effector MAP kinase pathway suppresses the growth and actin defects of ypk cells. ypk lethality is also suppressed by overexpression of an uncharacterized gene termed TUS1. TUS1 is a novel RHO1 exchange factor that contributes to cell wall integrity-mediated modulation of RHO1 activity. Thus, TUS1 and the YPKs add to the growing complexity of RHO1 and PKC1 regulation in the cell integrity signaling pathway. Furthermore, our findings suggest that the YPKs are a missing link between sphingolipid signaling and the cell integrity pathway.

scholarly journals Fus3p and Kss1p Control G1 Arrest in Saccharomyces cerevisiae Through a Balance of Distinct Arrest and Proliferative Functions That Operate in Parallel With Far1p

Genetics ◽  
1999 ◽  
Vol 151 (3) ◽  
pp. 989-1004 ◽  
Author(s):  
Vera Cherkasova ◽  
David M Lyons ◽  
Elaine A Elion
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Map Kinase ◽  
Map Kinases ◽  
G1 Arrest ◽  
Multiple Functions ◽  
Map Kinase Cascade ◽  
Kinase Cascade ◽  
Map Kinase Pathway ◽  
Kinase Pathway ◽  
Cyclin Genes

AbstractIn Saccharomyces cerevisiae, mating pheromones activate two MAP kinases (MAPKs), Fus3p and Kss1p, to induce G1 arrest prior to mating. Fus3p is known to promote G1 arrest by activating Far1p, which inhibits three Clnp/Cdc28p kinases. To analyze the contribution of Fus3p and Kss1p to G1 arrest that is independent of Far1p, we constructed far1 CLN strains that undergo G1 arrest from increased activation of the mating MAP kinase pathway. We find that Fus3p and Kss1p both control G1 arrest through multiple functions that operate in parallel with Far1p. Fus3p and Kss1p together promote G1 arrest by repressing transcription of G1/S cyclin genes (CLN1, CLN2, CLB5) by a mechanism that blocks their activation by Cln3p/Cdc28p kinase. In addition, Fus3p and Kss1p counteract G1 arrest through overlapping and distinct functions. Fus3p and Kss1p together increase the expression of CLN3 and PCL2 genes that promote budding, and Kss1p inhibits the MAP kinase cascade. Strikingly, Fus3p promotes proliferation by a novel function that is not linked to reduced Ste12p activity or increased levels of Cln2p/Cdc28p kinase. Genetic analysis suggests that Fus3p promotes proliferation through activation of Mcm1p transcription factor that upregulates numerous genes in G1 phase. Thus, Fus3p and Kss1p control G1 arrest through a balance of arrest functions that inhibit the Cdc28p machinery and proliferative functions that bypass this inhibition.

scholarly journals Metabolic Reprogramming in the Opportunistic Yeast Candida albicans in Response to Hypoxia

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Anaïs Burgain ◽  
Faiza Tebbji ◽  
Inès Khemiri ◽  
Adnane Sellam
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Metabolic Pathways ◽  
Membrane Lipids ◽  
Oxygen Depletion ◽  
Metabolic Reprogramming ◽  
Pathogenic Yeast ◽  
Fungal Virulence ◽  
Content Type ◽  
The Impact

ABSTRACT Hypoxia is the predominant condition that the human opportunistic fungus Candida albicans encounters in the majority of the colonized niches within the host. So far, the impact of such a condition on the overall metabolism of this important human-pathogenic yeast has not been investigated. Here, we have undertaken a time-resolved metabolomics analysis to uncover the metabolic landscape of fungal cells experiencing hypoxia. Our data showed a dynamic reprogramming of many fundamental metabolic pathways, such as glycolysis, the pentose phosphate pathway, and different metabolic routes related to fungal cell wall biogenesis. The C. albicans lipidome was highly affected by oxygen depletion, with an increased level of free fatty acids and biochemical intermediates of membrane lipids, including phospholipids, lysophospholipids, sphingolipids, and mevalonate. The depletion of oxygen-dependent lipids such as ergosterol or phosphatidylcholine with longer and polyunsaturated lateral fatty acid chains was observed only at the later hypoxic time point (180 min). Transcriptomics data supported the main metabolic response to hypoxia when matched to our metabolomic profiles. The hypoxic metabolome reflected different physiological alterations of the cell wall and plasma membrane of C. albicans under an oxygen-limiting environment that were confirmed by different approaches. This study provided a framework for future in vivo investigations to examine relevant hypoxic metabolic trajectories in fungal virulence and fitness within the host. IMPORTANCE A critical aspect of cell fitness is the ability to sense and adapt to variations in oxygen levels in their local environment. Candida albicans is an opportunistic yeast that is the most prevalent human fungal pathogen. While hypoxia is the predominant condition that C. albicans encounters in most of its niches, its impact on fungal metabolism remains unexplored so far. Here, we provided a detailed landscape of the C. albicans metabolome that emphasized the importance of many metabolic routes for the adaptation of this yeast to oxygen depletion. The fungal hypoxic metabolome identified in this work provides a framework for future investigations to assess the contribution of relevant metabolic pathways in the fitness of C. albicans and other human eukaryotic pathogens with similar colonized human niches. As hypoxia is present at most of the fungal infection foci in the host, hypoxic metabolic pathways are thus an attractive target for antifungal therapy.

Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1193-1204 ◽  
Author(s):  
Iwona Wojda ◽  
Rebeca Alonso-Monge ◽  
Jan-Paul Bebelman ◽  
Willem H. Mager ◽  
Marco Siderius
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Osmotic Stress ◽  
Map Kinase ◽  
Growth Temperature ◽  
Wild Type ◽  
Hog Pathway ◽  
Map Kinase Pathway ◽  
Kinase Pathway ◽  
Intracellular Glycerol

In the yeast Saccharomyces cerevisiae, response to an increase in external osmolarity is mediated by the HOG (high osmolarity glycerol) MAP kinase pathway. HOG pathway mutant strains display osmosensitive phenotypes. Recently evidence has been obtained that the osmosensitivity of HOG pathway mutants is reduced during growth at elevated temperature (37 °C). A notable exception is the ste11ssk2ssk22 mutant, which displays hypersensitivity to osmotic stress at 37 °C. This paper reports that overexpression of FPS1 or GPD1 (encoding the glycerol transport facilitator and glycerol-3-phosphate dehydrogenase, respectively, and both affecting intracellular glycerol levels) reduces the hypersensitivity to osmotic stress of ste11ssk2ssk22 at 37 °C. Although in this particular HOG pathway mutant a correlation between suppression of the phenotype and glycerol content could be demonstrated, the absolute level of intracellular glycerol per se does not determine whether a strain is osmosensitive or not. Rather, evidence was obtained that the glycerol level may have an indirect effect, viz. by influencing signalling through the PKC (protein kinase C) MAP kinase pathway, which plays an important role in maintenance of cellular integrity. In order to validate the data obtained with a HOG pathway mutant strain for wild-type yeast cells, MAP kinase signalling under different growth conditions was examined in wild-type strains. PKC pathway signalling, which is manifest at elevated growth temperature by phosphorylation of MAP kinase Mpk1p, is rapidly lost when cells are shifted to high external osmolarity conditions. Expression of bck1-20 or overexpression of WSC3 in wild-type cells resulted in restoration of PKC signalling. Both PKC and HOG signalling, cell wall phenotypes and high osmotic stress responses in wild-type cells were found to be influenced by the growth temperature. The data taken together indicate the intricate interdependence of growth temperature, intracellular glycerol, cell wall structure and MAP kinase signalling in the hyperosmotic stress response of yeast.

scholarly journals A Chemical Genetics Screen Reveals Influence of p38 Mitogen-Activated Protein Kinase and Autophagy on Phagosome Development and Intracellular Replication ofBrucella neotomaein Macrophages

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Yoon-Suk Kang ◽  
James E. Kirby
Keyword(s):  
Map Kinase ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
Chemical Genetics ◽  
P38 Map Kinase ◽  
Intracellular Replication ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Kinase Pathway

ABSTRACTBrucellais an intracellular bacterial pathogen that causes chronic systemic infection in domesticated livestock and poses a zoonotic infectious risk to humans. The virulence ofBrucellais critically dependent on its ability to replicate and survive within host macrophages.Brucellamodulates host physiological pathways and cell biology in order to establish a productive intracellular replicative niche. Conversely, the host cell presumably activates pathways that limit infection. To identify host pathways contributing to this yin and yang during host cell infection, we performed a high-throughput chemical genetics screen of known inhibitors and agonists of host cell targets to identify host factors that contribute to intracellular growth of the model pathogenBrucella neotomae. Using this approach, we identified the p38 mitogen-activated protein (MAP) kinase pathway and autophagy machinery as both a linchpin and an Achilles’ heel inB. neotomae’s ability to coopt host cell machinery and replicate within macrophages. Specifically,B. neotomaeinduced p38 MAP kinase phosphorylation and autophagy in a type IV secretion system-dependent fashion. Both p38 MAP kinase stimulation and an intact autophagy machinery in turn were required for phagosome maturation and intracellular replication. These findings contrasted with those forLegionella pneumophila, where chemical inhibition of the p38 MAP kinase pathway and autophagy factor depletion failed to block intracellular replication. Therefore, results from a chemical genetics screen suggest that intersections of the MAP kinase pathways and autophagy machinery are critical components ofBrucella’s intracellular life cycle.

scholarly journals Signaling Domains of Mucin Msb2 in Candida albicans

2015 ◽  
Vol 14 (4) ◽  
pp. 359-370 ◽  
Author(s):  
Marc Swidergall ◽  
Lasse van Wijlick ◽  
Joachim F. Ernst
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Map Kinase ◽  
Cytoplasmic Tail ◽  
Wall Stress ◽  
Mitogen Activated Protein Kinase ◽  
Basal Resistance ◽  
Interacting Protein ◽  
Content Type ◽  
Cell Wall Stress

ABSTRACTCandida albicansadapts to the human host by environmental sensing using the Msb2 signal mucin, which regulates fungal morphogenesis and resistance characteristics. Msb2 is anchored within the cytoplasmic membrane by a single transmembrane (TM) region dividing it into a large N-terminal exodomain, which is shed, and a small cytoplasmic domain. Analyses of strains carrying deleted Msb2 variants revealed an exodomain segment required for cleavage, shedding, and all functions of Msb2. Phosphorylation of the mitogen-activated protein kinase (MAP kinase) Cek1 was regulated by three distinct regions in Msb2: in unstressed cells, N-terminal sequences repressed phosphorylation, while its induction under cell wall stress required the cytoplasmic tail (C-tail) and sequences N-terminally flanking the TM region, downstream of the proposed cleavage site. Within the latter Msb2 region, overlapping but not identical sequences were also required for hyphal morphogenesis, basal resistance to antifungals, and, in unstressed cells, downregulation of thePMT1transcript, encoding proteinO-mannosyltransferase-1. Deletion of two-thirds of the exodomain generated a truncated Msb2 variant with a striking ability to induce hyperfilamentous growth, which depended on the presence of the Msb2-interacting protein Sho1, the MAP kinase Cek1, and the Efg1 transcription factor. Under cell wall stress, the cytoplasmic tail relocalized partially to the nucleus and contributed to regulation of 117 genes, as revealed by transcriptomic analyses. Genes regulated by the C-tail contained binding sites for the Ace2 and Azf1 transcription factors and included theALScell wall genes. We concluded that Msb2 fulfills its numerous functions by employing functional domains that are distributed over its entire length.

scholarly journals Methylglyoxal Activates the Target of Rapamycin Complex 2-Protein Kinase C Signaling Pathway in Saccharomyces cerevisiae

2015 ◽  
Vol 35 (7) ◽  
pp. 1269-1280 ◽  
Author(s):  
Wataru Nomura ◽  
Yoshiharu Inoue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Map Kinase ◽  
Target Of Rapamycin ◽  
Dependent Manner ◽  
Content Type ◽  
Protein Levels ◽  
Map Kinase Cascade ◽  
Phosphorylated Akt ◽  
Mitogen Activated Protein ◽  
Kinase Cascade

Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeastSaccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr1125and Ser1143. Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser1143, which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1T1125affected the phosphorylation of Pkc1 at Ser1143, in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser473. Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.

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