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PLoS One
2015 Jan 01;107:e0132369. doi: 10.1371/journal.pone.0132369.
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The Role of Amino Acid Permeases and Tryptophan Biosynthesis in Cryptococcus neoformans Survival.
Fernandes JD
,
Martho K
,
Tofik V
,
Vallim MA
,
Pascon RC
.
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Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (Nitrogen Catabolism Repression, NCR) and carbon sources (Carbon Catabolism Repression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (Global Amino Acid Control, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro.
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Fig 1. Tryptophan biosynthetic pathway in C. neoformans and S. cerevisiae.Genes are depicted on the right side. The dashed line indicates feedback regulation by the end product.
Fig 2. Effect of anti-metabolites in C. neoformans.Serial dilutions of the H99 strain were spotted in rich (YEPD) and synthetic medium (YNB supplemented with ammonium sulfate and glucose) containing 5μg/mL of anti-metabolite. 5-FAA = 5-fluoroanthranilic acid; 3-HAA = 3-hydroxianthranilic acid; 5-MAA = 5-methylanthranilic acid and NI = no inhibitor.
Fig 3. Position of the fragments used to induce RNA interference in TRP3 and TRP5.The numbers and black lines represent the coding region of TRP3 and TRP5. The position and length of RNAi fragments in the coding region are represented as open boxes. trp3.1i and trp5.1i fragments are smaller (280 pb and 233 pb, respectively) than trp3.2i and trp5.2i fragments (402 pb and 353 pb, respectively). Gray boxes indicate the position of the fragments amplified in qPCR.
Fig 4. (A) TRP3 and (B) TRP5 RNA interference.Ten-fold serial dilutions (104 to 1 cell) were spotted in YEP medium with dextrose (RNAi repressing condition) or galactose (RNAi inducing condition), with or without 20 mg/L tryptophan supplementation. CNU026 and CNU007 are control strains (empty pIBB103 plasmids) and CNU031 and CNU004 are test strains (trp3.1i and trp5.1i in pIBB103 plasmids). The graphs in Fig 4A and 4B show the relative quantification (RQ) of TRP3 and TRP5 transcripts in RNAi inducing (YEPG, galactose) and repressing (YEPD, dextrose) conditions for control strains (CNU026 and CNU007) and mutant strains (CNU031 and CNU004). The RNAi mutants CNU031 and CNU004 are representative of 39 mutants obtained in a screen comprising 100 transformants.
Fig 5.
trp3i and trp5i phenotype in synthetic medium.Tryptophan uptake by trp3.1i (CNU031) and trp5.1i (CNU004) mutants was compared to the wild-type (CNU026) in synthetic medium (YNB) under preferred (NH4)2SO4 and non preferred nitrogen sources (proline) at 25º and 30ºC. Five serial dilutions (104 to 1 cell) were spotted on plates containing YNB plus (NH4)2SO4 or proline, dextrose (repressing condition) or galactose (inducing condition), with and without 20 mg/L tryptophan.
Alspaugh,
Virulence mechanisms and Cryptococcus neoformans pathogenesis.
2015, Pubmed
Alspaugh,
Virulence mechanisms and Cryptococcus neoformans pathogenesis.
2015,
Pubmed
Antinori,
New Insights into HIV/AIDS-Associated Cryptococcosis.
2013,
Pubmed
Braus,
Aromatic amino acid biosynthesis in the yeast Saccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway.
1991,
Pubmed
Brown,
Hidden killers: human fungal infections.
2012,
Pubmed
Brown,
Cryptococcus neoformans, a fungus under stress.
2007,
Pubmed
Chittur,
Mechanism for acivicin inactivation of triad glutamine amidotransferases.
2001,
Pubmed
Cox,
Superoxide dismutase influences the virulence of Cryptococcus neoformans by affecting growth within macrophages.
2003,
Pubmed
Datta,
Spread of Cryptococcus gattii into Pacific Northwest region of the United States.
2009,
Pubmed
Davidson,
A PCR-based strategy to generate integrative targeting alleles with large regions of homology.
2002,
Pubmed
Fan,
Cryptococcus neoformans gene expression during murine macrophage infection.
2005,
Pubmed
Fernandez,
Mechanisms of nutrient acquisition and utilization during fungal infections of leaves.
2014,
Pubmed
Fraser,
Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak.
2005,
Pubmed
Gahan,
Listeria monocytogenes: survival and adaptation in the gastrointestinal tract.
2014,
Pubmed
Godard,
Effect of 21 different nitrogen sources on global gene expression in the yeast Saccharomyces cerevisiae.
2007,
Pubmed
Hinnebusch,
Translational regulation of GCN4 and the general amino acid control of yeast.
2005,
Pubmed
Hu,
Metabolic adaptation in Cryptococcus neoformans during early murine pulmonary infection.
2008,
Pubmed
Jones,
The spectrum of Trp- mutants isolated as 5-fluoroanthranilate-resistant clones in Saccharomyces bayanus, S. mikatae and S. paradoxus.
2008,
Pubmed
Jung,
Iron and fungal pathogenesis: a case study with Cryptococcus neoformans.
2008,
Pubmed
Jung,
Iron source preference and regulation of iron uptake in Cryptococcus neoformans.
2008,
Pubmed
Jung,
The iron-responsive, GATA-type transcription factor Cir1 influences mating in Cryptococcus neoformans.
2011,
Pubmed
Jung,
Role of ferroxidases in iron uptake and virulence of Cryptococcus neoformans.
2009,
Pubmed
Kim,
An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers.
2009,
Pubmed
Kingsbury,
Cryptococcus neoformans Ilv2p confers resistance to sulfometuron methyl and is required for survival at 37 degrees C and in vivo.
2004,
Pubmed
Kingsbury,
Threonine biosynthetic genes are essential in Cryptococcus neoformans.
2008,
Pubmed
Kingsbury,
Novel chimeric spermidine synthase-saccharopine dehydrogenase gene (SPE3-LYS9) in the human pathogen Cryptococcus neoformans.
2004,
Pubmed
Kingsbury,
Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.
2010,
Pubmed
Kingsbury,
Fungal homoserine kinase (thr1Delta) mutants are attenuated in virulence and die rapidly upon threonine starvation and serum incubation.
2010,
Pubmed
Kmetzsch,
The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans.
2011,
Pubmed
Kretschmer,
Defects in phosphate acquisition and storage influence virulence of Cryptococcus neoformans.
2014,
Pubmed
Kronstad,
Adaptation of Cryptococcus neoformans to mammalian hosts: integrated regulation of metabolism and virulence.
2012,
Pubmed
Lee,
Nitrogen regulation of virulence in clinically prevalent fungal pathogens.
2013,
Pubmed
Lee,
Nitrogen metabolite repression of metabolism and virulence in the human fungal pathogen Cryptococcus neoformans.
2011,
Pubmed
Lee,
Characterization of the complete uric acid degradation pathway in the fungal pathogen Cryptococcus neoformans.
2013,
Pubmed
Li,
Cryptococcus.
2010,
Pubmed
Lin,
The biology of the Cryptococcus neoformans species complex.
2006,
Pubmed
Livak,
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
2001,
Pubmed
Ljungdahl,
Regulation of amino acid, nucleotide, and phosphate metabolism in Saccharomyces cerevisiae.
2012,
Pubmed
Ljungdahl,
Amino-acid-induced signalling via the SPS-sensing pathway in yeast.
2009,
Pubmed
Maurer,
New regulatory mutation affecting some of the tryptophan genes in Pseudomonas putida.
1971,
Pubmed
Miozzari,
Action of tryptophan analogues in Saccharomyces cerevisiae.
1977,
Pubmed
Moran,
Comparative genomics and the evolution of pathogenicity in human pathogenic fungi.
2011,
Pubmed
Nazi,
Role of homoserine transacetylase as a new target for antifungal agents.
2007,
Pubmed
Nelissen,
Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae.
1997,
Pubmed
Nelissen,
Phylogenetic classification of the major superfamily of membrane transport facilitators, as deduced from yeast genome sequencing.
1995,
Pubmed
Nosanchuk,
Glyphosate inhibits melanization of Cryptococcus neoformans and prolongs survival of mice after systemic infection.
2001,
Pubmed
Park,
Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS.
2009,
Pubmed
Pascon,
Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence.
2004,
Pubmed
Paulsen,
Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae.
1998,
Pubmed
Payne,
Synthesis and evaluation of 2,5-dihydrochorismate analogues as inhibitors of the chorismate-utilising enzymes.
2009,
Pubmed
Payne,
Design and synthesis of aromatic inhibitors of anthranilate synthase.
2005,
Pubmed
Payne,
Inhibition of chorismate-utilising enzymes by 2-amino-4-carboxypyridine and 4-carboxypyridone and 5-carboxypyridone analogues.
2010,
Pubmed
Perfect,
Cloning the Cryptococcus neoformans TRP1 gene by complementation in Saccharomyces cerevisiae.
1992,
Pubmed
Peter,
Carbon catabolite repression regulates amino acid permeases in Saccharomyces cerevisiae via the TOR signaling pathway.
2006,
Pubmed
Raboni,
Tryptophan synthase: a mine for enzymologists.
2009,
Pubmed
Regenberg,
Substrate specificity and gene expression of the amino-acid permeases in Saccharomyces cerevisiae.
1999,
Pubmed
Reilly,
A novel xylosylphosphotransferase activity discovered in Cryptococcus neoformans.
2009,
Pubmed
Sasaki,
Growth inhibitory effects of anthranilic acid and its derivatives against Legionella pneumophila.
2012,
Pubmed
Sassetti,
Genetic requirements for mycobacterial survival during infection.
2003,
Pubmed
Shen,
A novel inhibitor of indole-3-glycerol phosphate synthase with activity against multidrug-resistant Mycobacterium tuberculosis.
2009,
Pubmed
Shen,
Altered protein expression patterns of Mycobacterium tuberculosis induced by ATB107.
2010,
Pubmed
Skowyra,
RNA interference in Cryptococcus neoformans.
2012,
Pubmed
Smith,
Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates.
2001,
Pubmed
Tilby,
Tryptophan biosynthesis in Coprinus lagopus: a genetic analysis of mutants.
1976,
Pubmed
Toffaletti,
Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA.
1993,
Pubmed
Toyn,
A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance.
2000,
Pubmed
Webby,
Synergistic allostery, a sophisticated regulatory network for the control of aromatic amino acid biosynthesis in Mycobacterium tuberculosis.
2010,
Pubmed
Yang,
Molecular and genetic analysis of the Cryptococcus neoformans MET3 gene and a met3 mutant.
2002,
Pubmed
Ziebart,
Targeting multiple chorismate-utilizing enzymes with a single inhibitor: validation of a three-stage design.
2010,
Pubmed
de Gontijo,
The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence.
2014,
Pubmed
,
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