The Golgi GDPase of the Fungal Pathogen Candida albicans Affects Morphogenesis, Glycosylation, and Cell Wall Properties

Self Cite

Lumenal ecto-nucleoside tri-and di-phosphohydrolases (ENTPDases) of the secretory pathway of eukaryotes hydrolyze nucleoside diphosphates resulting from glycosyltransferase-mediated reactions, yielding nucleoside monophosphates. The latter are weaker inhibitors of glycosyltransferases than the former and are also antiporters for the transport of nucleotide sugars from the cytosol to the endoplasmic reticulum (ER) and Golgi apparatus (GA) lumen. Here we describe the presence of two cation-dependent nucleotide phosphohydrolase activities in membranes of Caenorhabditis elegans: one, UDA-1, is a UDP/GDPase encoded by the gene uda-1, whereas the other is an apyrase encoded by the gene ntp-1. UDA-1 shares significant amino acid sequence similarity to yeast GA Gda1p and mammalian UDP/GDPases and has a lumenal active site in vesicles displaying an intermediate density between those of the ER and GA when expressed in S. cerevisiae. NTP-1 expressed in COS-7 cells appeared to localize to the GA. The transcript of uda-1 but not those of two other C. elegans ENTPDase mRNAs (ntp-1 and mig-23) was induced up to 3.5-fold by high temperature, tunicamycin, and ethanol. The same effectors triggered the unfolded protein response as shown by the induction of expression of green fluorescent protein under the control of the BiP chaperone promoter and the UDP-glucose:glycoprotein glucosyltransferase. Up-regulation of uda-1 did not occur in ire-1-deficient mutants, demonstrating the role of this ER stress sensor in this event. We hypothesize that up-regulation of uda-1 favors hydrolysis of the glucosyltransferase inhibitory product UDP to UMP, and that the latter product then exits the lumen of the ER or pre-GA compartment in a coupled exchange with the entry of UDP-glucose, thereby further relieving ER stress by favoring protein re-glycosylation.The occurrence of the eukaryotic endomembrane system has brought forth a need for a mechanism to coordinate the metabolic flux of nucleotides, nucleotide sugars, and nucleotide sulfate between the cytosol and the lumen of secretory organelles. E-type ATPases play an important role in this function. They have been conserved through evolution with their catalytic site in the ecto-position, facing the outer surface of the plasma membrane or its topological equivalent, the lumen of intracellular organelles such as the endoplasmic reticulum (ER), 1 Golgi apparatus (GA), and lysosomes/vacuoles. These enzymes, members of the ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) family, hydrolyze nucleoside tri-and/or diphosphates in the presence of cations and share, at the amino acid sequence level, five conserved motifs called apyrase-conserved regions (ACRs) (1).Extracellular nucleotides such as ATP and ADP are intercellular signaling molecules in virtually every tissue where, modulated by ecto-apyrases, they participate in a broad range of biological processes such as the regulation of immune responses (2) and modulation of signaling by neuronal cells (3). Specifically, mammalian ENTPDase1/CD...

Section: Nucleotide Hydrolyzing Activities Of C Elegans Membranes-mentioning

confidence: 59%

ire-1-dependent Transcriptional Up-regulation of a Lumenal Uridine Diphosphatase from Caenorhabditis elegans

Uccelletti

O'Callaghan

Berninsone

et al. 2004

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“…Null Mutant-Previously, the electrophoretic mobility of secreted acid phosphatase on native gels has been used as a maker of N-glycosylation status (13,25,27). However, no acid phosphatase activity was detected in zymograms of the Caoch1⌬ null mutant.…”

Section: Glycosylation Defects In the Caoch1⌬mentioning

confidence: 99%

“…The O-linked oligosaccharides, attached to serine or threonine, consist of a linear chain of one to five ␣1,2-linked mannose residues (12)(13)(14) and are known to be required for full virulence (14). The process of N-glycosylation has been studied extensively in Saccharomyces cerevisiae.…”

mentioning

confidence: 99%

Outer Chain N-Glycans Are Required for Cell Wall Integrity and Virulence of Candida albicans

Bates

Hughes

Munro

et al. 2006

215

272

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated mannoproteins that are the immediate point of contact with the host and strongly influence the host-fungal interaction. N-Glycans are the major form of mannoprotein modification and consist of a core structure, common to all eukaryotes, that is further elaborated in the Golgi to form the highly branched outer chain that is characteristic of fungi. In yeasts, outer chain branching is initiated by the action of the ␣1,6-mannosyltransferase Och1p; therefore, we disrupted the C. albicans OCH1 homolog to determine the importance of outer chain N-glycans on the host-fungal interaction. Loss of CaOCH1 resulted in a temperature-sensitive growth defect and cellular aggregation. Outer chain elongation of N-glycans was absent in the null mutant, demonstrated by the lack of the ␣1,6-linked polymannose backbone and the underglycosylation of N-acetylglucosaminidase. A null mutant lacking OCH1 was hypersensitive to a range of cell wall perturbing agents and had a constitutively activated cell wall integrity pathway. These mutants had near normal growth rates in vitro but were attenuated in virulence in a murine model of systemic infection. However, tissue burdens for the Caoch1⌬ null mutant were similar to control strains with normal N-glycosylation, suggesting the host-fungal interaction was altered such that high burdens were tolerated. This demonstrates the importance of N-glycan outer chain epitopes to the host-fungal interaction and virulence.Candida albicans is a commensal organism carried by a significant proportion of healthy individuals. It is the most common opportunistic fungal pathogen of humans causing superficial infections of the mucosa and in the immunocompromised host life-threatening systemic infections (1-4). The cell wall is the immediate point of contact between fungus and host and plays an important role in adherence, antigenicity, and the modulation of the host immune response (5-9). The outer layer of the cell wall is enriched in highly glycosylated mannoproteins (10), and both the protein and carbohydrate components have been implicated in the host-fungal interaction (5, 6, 11). The study of glycosylation in C. albicans therefore has its own relevance in identifying the carbohydrate epitopes involved in pathogenesis.Cell surface mannoproteins contain both O-and N-linked oligosaccharides. The O-linked oligosaccharides, attached to serine or threonine, consist of a linear chain of one to five ␣1,2-linked mannose residues (12)(13)(14) and are known to be required for full virulence (14). The process of N-glycosylation has been studied extensively in Saccharomyces cerevisiae. N-Linked glycosylation is initiated in the endoplasmic reticulum with the transfer of the Glc 3 Man 9 GlcNAc 2 oligosaccharide precursor to the protein target (15, 16). The oligosaccharide precursor is then processed by endoplasmic reticulum-resident glucosidases and a mannosidase to yield the mature triantennary Man 8 GlcNAc 2 core (17). Outer chain ...

“…2 and CaSRB1, which encode proteins required for supplying the Golgi with GDP-mannose, the mannose donor, are essential in C. albicans, indicating the overall importance of glycosylation to cell viability (27)(28)(29)(30). However, to date, no single glycosylation event in the Golgi has been shown to be essential.…”

Section: Cavrg4mentioning

confidence: 99%

“…The essential nature of both CaSRB1 and CaVRG4, while emphasizing the importance of glycosylation, means that the role of the glycans in host-fungus interactions and virulence cannot be assessed. The Golgi GDPase CaGDA1 has also been studied (30); this enzyme converts GDP to GMP, which is then exported from the Golgi by Vrg4p in an antiport process with GDP-mannose. However, CaGDA1 is nonessential, presumably due to functional redundancy, and does not exhibit a gross glycosylation defect.…”

Section: Fig 8 Attenuation Of Virulence In the Capmr1⌬ Null Mutantmentioning

confidence: 99%

Candida albicans Pmr1p, a Secretory Pathway P-type Ca2+/Mn2+-ATPase, Is Required for Glycosylation and Virulence

Bates

MacCallum

Bertram

et al. 2005

172

213

The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn 2؉ as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca 2؉ and Mn 2؉ ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn 2؉ -dependent mannosyltransferases. The Capmr1⌬ null mutant was viable in vitro and had no growth defect even on media containing low Ca 2؉ /Mn 2؉ ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans.