A Candida albicans cyclic nucleotide phosphodiesterase: cloning and expression in Saccharomyces cerevisiae and biochemical characterization of the recombinant enzyme

Hoyer, Lois L.; Cieslinski, Lenora B.; McLaughlin, Megan M.; Torphy, Theodore J.; Shatzman, Allan R.; Livi, George P.

doi:10.1099/13500872-140-7-1533

Cited by 46 publications

(46 citation statements)

References 49 publications

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“…CaPDE2 is functional in S. cerevisiae, but is toxic upon overexpression Previous attempts to clone the C. albicans homologue of PDE2 by functional complementation have been unsuccessful (Hoyer et al, 1994;Taylor, 1999). However, a putative CaPDE2 ORF, with the conserved signature motif of a class I cAMP PDE (HDVGHPGTTNDF) (Charbonneau et al, 1986), 1716 bp long and with 28 % aa identity to ScPDE2, was revealed by the Candida genome sequence project (Tait et al, 1997).…”

Section: Resultsmentioning

confidence: 99%

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The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans

Jung

Stateva

2003

Microbiology

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The cAMP-dependent pathway, which regulates yeast-to-hypha morphogenesis in Candida albicans, is controlled by changes in cAMP levels determined by the processes of synthesis and hydrolysis. Both low-and high-affinity cAMP phosphodiesterases are encoded in the C. albicans genome. CaPDE2, encoding the high-affinity cAMP phosphodiesterase, has been cloned and shown to be toxic in Saccharomyces cerevisiae upon overexpression under pGAL1, but functional under the moderate pMET3. Deletion of CaPDE2 causes elevated cAMP levels and responsiveness to exogenous cAMP, higher sensitivity to heat shock, severe growth defects at 42 6C and highly reduced levels of EFG1 transcription. In vitro in hypha-inducing liquid medium CaPDE2, deletion prohibits normal hyphal, but not pseudohyphal growth. On solid medium capde2 mutants form aberrant hyphae, with fewer branches and almost no lateral buds, which are deficient in hypha-to-yeast reversion. The phenotypic defects of capde2 mutants show that the cAMP-dependent pathway plays specific roles in hyphal and pseudohyphal development, its regulatory role however, being greater in liquid than on solid medium in vitro. The increased expression of CaPDE2 after serum addition correlates well with a drop in cAMP levels following the initial rise in response to the hyphal inducer. These results suggest that Capde2p mediates a desensitization mechanism by lowering basal cAMP levels in response to environmental stimuli in C. albicans.

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Section: Resultsmentioning

confidence: 99%

“…7). CaPDE1 has been cloned, but no capde1 mutants have yet been constructed (Hoyer et al, 1994). Analysing such mutants might help to elucidate the more specific roles of the low-affinity cAMP PDE in C. albicans.…”

Section: Discussionmentioning

confidence: 99%

The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans

Jung

Stateva

2003

Microbiology

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“…However, there is one additional phosphodiesterase in Candida, the low-affinity cAMP phosphodiesterase, encoded by PDE1 (Hoyer et al, 1994). At present there are no mutants available and the phenotypic consequences of its deletion are unknown.…”

Section: Discussionmentioning

confidence: 99%

“…Two phosphodiesterase-encoding genes PDE1 and PDE2 have been found in the fungal pathogen C. albicans (Bahn et al, 2003;Hoyer et al, 1994;Jung and Stateva, 2003). Mutants lacking PDE1 are not yet available; however, hetero-and homozygous pde2 null mutants have been generated and characterized (Bahn et al, 2003;Jung and Stateva, 2003).…”

Section: Introductionmentioning

confidence: 99%

Deletion of PDE2, the gene encoding the high‐affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans

Jung

Warn

Ragni

et al. 2005

Yeast

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A role for the cAMP-dependent pathway in regulation of the cell wall in the model yeast Saccharomyces cerevisiae has recently been demonstrated. In this study we report the results of a phenotypic analysis of a Candida albicans mutant, characterized by a constitutive activation of the cAMP pathway due to deletion of PDE2, the gene encoding the high cAMP-affinity phosphodiesterase. Unlike wild-type strains, this mutant has an increased sensitivity to cell wall and membrane perturbing agents such as SDS and CFW, and antifungals such as amphotericin B and flucytosine. Moreover, the mutant is characterized by an altered sensitivity and a significantly reduced tolerance to fluconazole. The mutant's membrane has around 30% higher ergosterol content and the cell wall glucan was 22% lower than in the wild-type. These cell wall and membrane changes are manifested by a considerable reduction in the thickness of the cell wall, which in the mutant is on average 60-65 nm, compared to 80-85 nm in the wild-type strains as revealed by electron microscopy. These results suggest that constitutive activation of the cAMP pathway affects cell wall and membrane structure, and biosynthesis, not only in the model yeast S. cerevisiae but also in the human fungal pathogen C. albicans.

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“…As a group they have been designated as class I PDEs. Additional forms of PDEs have been described in Saccharomyces cerevisiae (8), Dictyostelium discoideum (9), Vibrio fisheri (10), and Candida albicans (11), which exhibit very little amino acid sequence identity to the class I enzymes. These enzymes, currently designated as class II PDEs, likely have a different evolutionary origin because, in contrast to all other eukaryotic PDEs, they have catalytic domains unlike those in mammalian class I enzymes (8).…”

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confidence: 99%

Cloning and characterization of a cAMP-specific phosphodiesterase (TbPDE2B) from Trypanosoma brucei

Rascón

Soderling

Schaefer

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Here we report the cloning, expression, and characterization of a cAMP-specific phosphodiesterase (PDE) from Trypanosoma brucei (TbPDE2B). Using a bioinformatic approach, two different expressed sequence tag clones were identified and used to isolate the complete sequence of two identical PDE genes arranged in tandem. Each gene consists of 2,793 bases that predict a protein of 930 aa with a molecular mass of 103.2 kDa. Two GAF (for cGMP binding and stimulated PDEs, Anabaena adenylyl cyclases, and Escherichia coli FhlA) domains, similar to those contained in many signaling molecules including mammalian PDE2, PDE5, PDE6, PDE10, and PDE11, were located N-terminal to a consensus PDE catalytic domain. The catalytic domain is homologous to the catalytic domain of all 11 mammalian PDEs, the Dictyostelium discoideum RegA, and a probable PDE from Caenorhabditis elegans. It is most similar to the T. brucei PDE2A (89% identity). TbPDE2B has substrate specificity for cAMP with a K m of 2.4 M. cGMP is not hydrolyzed by TbPDE2B nor does this cyclic nucleotide modulate cAMP PDE activity. The nonselective PDE inhibitors 3-isobutyl-1-methylxanthine, papaverine and pentoxifyline are poor inhibitors of TbPDE2B. Similarly, PDE inhibitors selective for the mammalian PDE families 2, 3, 5, and 6 (erythro-9-[3-(2-hydroxynonyl)]-adenine, enoximone, zaprinast, and sildenafil) were also unable to inhibit this enzyme. However, dipyridamole was a reasonably good inhibitor of this enzyme with an IC50 of 27 M. cAMP plays key roles in cell growth and differentiation in this parasite, and PDEs are responsible for the hydrolysis of this important second messenger. Therefore, parasite PDEs, including this one, have the potential to be attractive targets for selective drug design.inhibitors ͉ GAF domain

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A Candida albicans cyclic nucleotide phosphodiesterase: cloning and expression in Saccharomyces cerevisiae and biochemical characterization of the recombinant enzyme

Cited by 46 publications

References 49 publications

The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans

The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans

Deletion of PDE2, the gene encoding the high‐affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans

Cloning and characterization of a cAMP-specific phosphodiesterase (TbPDE2B) from Trypanosoma brucei

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