Plasma membrane phosphatidylinositol-4-phosphate is not necessary for Candida albicans viability, yet is key for cell wall integrity and systemic infection

Phosphatidylinositol phosphates are key phospholipids with a range of regulatory roles, including membrane trafficking and cell polarity. Phosphatidylinositol-4-phosphate [PI(4)P] at the Golgi is required for the budding to filamentous growth transition in the human pathogenic fungus Candida albicans, however the role of plasma membrane PI(4)P is unclear. We have investigated the importance of this phospholipid in C. albicans growth, stress response, and virulence by generating mutant strains with decreased levels of plasma membrane PI(4)P, via deletion of components of the PI-4-kinase complex, i.e. Efr3, Ypp1 and Stt4. The amount of plasma membrane PI(4)P in the efr3∆/∆ and ypp1∆/∆ mutant was ~60% and ~40% of the wild-type strain, respectively, whereas it was nearly undetectable in the stt4∆/∆ mutant. All three mutants had reduced plasma membrane phosphatidylserine (PS). Although these mutants had normal yeast phase growth, they were defective in filamentous growth, exhibited defects in cell wall integrity and had an increased exposure of cell wall β(1,3)-glucan, yet they induced a range of hyphal specific genes. In a mouse model of hematogenously disseminated candidiasis, fungal plasma membrane PI(4)P levels directly correlated with virulence; the efr3∆/∆ had wild-type virulence, the ypp1∆/∆ mutant had attenuated virulence and the stt4∆/∆ mutant caused no lethality. In the mouse model of orpharyngeal candidiasis, only the ypp1∆/∆ mutant had reduced virulence, indicating that plasma membrane PI(4)P is less important for proliferation in the oropharynx. Collectively, these results demonstrate that plasma membrane PI(4)P levels play a central role in filamentation, cell wall integrity and virulence in C. albicans.

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Attenuation of the Activity of Caspofungin at High Concentrations against Candida albicans: Possible Role of Cell Wall Integrity and Calcineurin Pathways

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.12.5146-5148.2005 ◽

2005 ◽

Vol 49 (12) ◽

pp. 5146-5148 ◽

Cited By ~ 96

Author(s):

Nathan P. Wiederhold ◽

Dimitrios P. Kontoyiannis ◽

Randall A. Prince ◽

Russell E. Lewis

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Deletion Mutant ◽

Cell Wall Integrity ◽

Short Exposure ◽

Wild Type ◽

High Concentrations

ABSTRACT Caspofungin had diminished activity in vitro against Candida albicans at concentrations of 8 to 32 μg/ml. This phenomenon was markedly attenuated in a Δmkc1/Δmkc1 deletion mutant and by the addition of cyclosporine to the wild type. Short exposure to these caspofungin concentrations resulted in MKC1 up-regulation, suggesting roles of cell wall integrity and calcineurin pathways.

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Putative PmrA and PmcA are important for normal growth, morphogenesis and cell wall integrity, but not for viability in Aspergillus nidulans

Microbiology ◽

10.1099/mic.0.080119-0 ◽

2014 ◽

Vol 160 (11) ◽

pp. 2387-2395 ◽

Cited By ~ 7

Author(s):

Hechun Jiang ◽

Feifei Liu ◽

Shizhu Zhang ◽

Ling Lu

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Aspergillus Nidulans ◽

Normal Growth ◽

Cell Wall Integrity ◽

Plasma Membrane Atpase ◽

Growth Defects ◽

Double Deletion ◽

Intracellular Ca ◽

P Type

P-type Ca2+-transporting ATPases are Ca2+ pumps, extruding cytosolic Ca2+ to the extracellular environment or the intracellular Ca2+ store lumens. In budding yeast, Pmr1 (plasma membrane ATPase related), and Pmc1 (plasma membrane calcium-ATPase) cannot be deleted simultaneously for it to survive in standard medium. Here, we deleted two putative Ca2+ pumps, designated AnPmrA and AnPmcA, from Aspergillus nidulans, and obtained the mutants ΔanpmrA and ΔanpmcA, respectively. Then, using ΔanpmrA as the starting strain, the promoter of its anpmcA was replaced with the alcA promoter to secure the mutant ΔanpmrAalcApmcA or its anpmcA was deleted completely to produce the mutant ΔanpmrAΔpmcA. Different from the case in Saccharomyces cerevisiae, double deletion of anpmrA and anpmcA was not lethal in A. nidulans. In addition, deletion of anpmrA and/or anpmcA had produced growth defects, although overexpression of AnPmc1 in ΔanpmrAalcApmcA could not restore the growth defects that resulted from the loss of AnPmrA. Moreover, we found AnPmrA was indispensable for maintenance of normal morphogenesis, especially in low-Ca2+/Mn2+ environments. Thus, our findings suggest AnPmrA and AnPmcA might play important roles in growth, morphogenesis and cell wall integrity in A. nidulans in a different way from that in yeasts.

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UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

PLoS Pathogens ◽

10.1371/journal.ppat.1002384 ◽

2011 ◽

Vol 7 (11) ◽

pp. e1002384 ◽

Cited By ~ 10

Author(s):

Manimala Sen ◽

Bhavin Shah ◽

Srabanti Rakshit ◽

Vijender Singh ◽

Bhavna Padmanabhan ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Cell Wall Integrity ◽

Dehydratase Activity

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Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22158165 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8165

Author(s):

Amanda Chantziou ◽

Kostas Theodorakis ◽

Hara Polioudaki ◽

Eelco de Bree ◽

Marilena Kampa ◽

...

Keyword(s):

Breast Cancer ◽

Plasma Membrane ◽

Subcellular Localization ◽

Cancer Cells ◽

Membrane Trafficking ◽

Breast Cancer Cells ◽

Endocytic Pathway ◽

Cell Periphery ◽

Wild Type ◽

Mcf 7

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

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Aspergillus nidulans Protein O-Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

Eukaryotic Cell ◽

10.1128/ec.00040-09 ◽

2009 ◽

Vol 8 (10) ◽

pp. 1475-1485 ◽

Cited By ~ 33

Author(s):

Thanyanuch Kriangkripipat ◽

Michelle Momany

Keyword(s):

Cell Wall ◽

Aspergillus Nidulans ◽

Double Mutant ◽

Elevated Temperatures ◽

Cell Wall Integrity ◽

Wild Type ◽

Spatial Cues ◽

Developmental Patterning ◽

In The Wild ◽

Increased Sensitivity

ABSTRACT Protein O-mannosyltransferases (Pmts) initiate O-mannosyl glycan biosynthesis from Ser and Thr residues of target proteins. Fungal Pmts are divided into three subfamilies, Pmt1, -2, and -4. Aspergillus nidulans possesses a single representative of each Pmt subfamily, pmtA (subfamily 2), pmtB (subfamily 1), and pmtC (subfamily 4). In this work, we show that single Δpmt mutants are viable and have unique phenotypes and that the ΔpmtA ΔpmtB double mutant is the only viable double mutant. This makes A. nidulans the first fungus in which all members of individual Pmt subfamilies can be deleted without loss of viability. At elevated temperatures, all A. nidulans Δpmt mutants show cell wall-associated defects and increased sensitivity to cell wall-perturbing agents. The Δpmt mutants also show defects in developmental patterning. Germ tube emergence is early in ΔpmtA and more frequent in ΔpmtC mutants than in the wild type. In ΔpmtB mutants, intrahyphal hyphae develop. All Δpmt mutants show distinct conidiophore defects. The ΔpmtA strain has swollen vesicles and conidiogenous cells, the ΔpmtB strain has swollen conidiophore stalks, and the ΔpmtC strain has dramatically elongated conidiophore stalks. We also show that AN5660, an ortholog of Saccharomyces cerevisiae Wsc1p, is modified by PmtA and PmtC. The Δpmt phenotypes at elevated temperatures, increased sensitivity to cell wall-perturbing agents and restoration to wild-type growth with osmoticum suggest that A. nidulans Pmts modify proteins in the cell wall integrity pathway. The altered developmental patterns in Δpmt mutants suggest that A. nidulans Pmts modify proteins that serve as spatial cues.

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Yeast cell wall integrity sensors form specific plasma membrane microdomains important for signalling

Cellular Microbiology ◽

10.1111/cmi.12635 ◽

2016 ◽

Vol 18 (9) ◽

pp. 1251-1267 ◽

Cited By ~ 24

Author(s):

Christian Kock ◽

Henning Arlt ◽

Christian Ungermann ◽

Jürgen J. Heinisch

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Yeast Cell ◽

Cell Wall Integrity ◽

Yeast Cell Wall ◽

Membrane Microdomains ◽

Plasma Membrane Microdomains

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The Candida albicans pH-regulated KER1 gene encodes a lysine/glutamic-acid-rich plasma-membrane protein that is involved in cell aggregation

Microbiology ◽

10.1099/mic.0.26339-0 ◽

2004 ◽

Vol 150 (8) ◽

pp. 2641-2651 ◽

Cited By ~ 4

Author(s):

Amparo Galán ◽

Manuel Casanova ◽

Amelia Murgui ◽

Donna M. MacCallum ◽

Frank C. Odds ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Plasma Membrane ◽

Glutamic Acid ◽

Germ Tube ◽

Cell Surface Hydrophobicity ◽

Cell Aggregation ◽

Surface Hydrophobicity ◽

Amino Acid Sequences ◽

Calcofluor White

Immunoscreening of a Candida albicans cDNA library with a polyclonal germ-tube-specific antibody (pAb anti-gt) resulted in the isolation of a gene encoding a lysine/glutamic-acid-rich protein, which was consequently designated KER1. The nucleotide and deduced amino acid sequences of this gene displayed no significant homology with any other known sequence. KER1 encodes a 134 kDa lysine (14·5 %)/glutamic acid (16·7 %) protein (Ker1p) that contains two potential transmembrane segments. KER1 was expressed in a pH-conditional manner, with maximal expression at alkaline pH and lower expression at pH 4·0, and was regulated by RIM101. A Δker1/Δker1 null mutant grew normally but was hyperflocculant under germ-tube-inducing conditions, yet this behaviour was also observed in stationary-phase cells grown under other incubation conditions. Western blotting analysis of different subcellular fractions, using as a probe a monospecific polyclonal antibody raised against a highly antigenic domain of Ker1p (pAb anti-Ker1p), revealed the presence of a 134 kDa band in the purified plasma-membrane fraction from the wild-type strain that was absent in the homologous preparation from Δker1/Δker1 mutant. The pattern of cell-wall protein and mannoprotein species released by digestion with β-glucanases, reactive towards pAbs anti-gt and anti-Ker1p, as well as against concanavalin A, was also different in the Δker1/Δker1 mutant. Mutant strains also displayed an increased cell-surface hydrophobicity and sensitivity to Congo red and Calcofluor white. Overall, these findings indicate that the mutant strain was affected in cell-wall composition and/or structure. The fact that the ker1 mutant had attenuated virulence in systemic mouse infections suggests that this surface protein is also important in host–fungus interactions.

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A small G protein Rhb1 and a GTPase-activating protein Tsc2 involved in nitrogen starvation-induced morphogenesis and cell wall integrity of Candida albicans

Fungal Genetics and Biology ◽

10.1016/j.fgb.2008.11.008 ◽

2009 ◽

Vol 46 (2) ◽

pp. 126-136 ◽

Cited By ~ 37

Author(s):

Chang-Chih Tsao ◽

Yu-Ting Chen ◽

Chung-Yu Lan

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

G Protein ◽

Nitrogen Starvation ◽

Cell Wall Integrity ◽

Gtpase Activating Protein ◽

Small G Protein

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Cross Talk between the Cell Wall Integrity and Cyclic AMP/Protein Kinase A Pathways in Cryptococcus neoformans

mBio ◽

10.1128/mbio.01573-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 18

Author(s):

Maureen J. Donlin ◽

Rajendra Upadhya ◽

Kimberly J. Gerik ◽

Woei Lam ◽

Laura G. VanArendonk ◽

...

Keyword(s):

Cell Wall ◽

Protein Kinase ◽

Cyclic Amp ◽

Protein Kinase A ◽

Cryptococcus Neoformans ◽

Signaling Pathway ◽

Cell Wall Integrity ◽

Wild Type ◽

Content Type ◽

Kinase A

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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