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.
Table 1.
Summary of genome organization of the TRP genes in C. neoformans (strains H99 and JEC21) and C. gattii (strains WM276 and R265).
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.
Fig 6.
Expression profile of C. neoformans permeases by qPCR.
(A) graph showing the effect of tryptophan supplementation (SD–AA vs. SD+tryptophan) on permease AAP1 and AAP2 expression; (B) shows the strong inducing effect of amino acids as the sole nitrogen source (SD-N+AA) on the expression of permeases AAP2, 4, 5 and 8, and the NCR effect of ammonium sulfate on permeases AAP2, 5 and 8 (SD-N+AA vs. SD-AA and SD+AA); (C) shows the effect of carbon catabolite repression on amino acid permeases. SD = synthetic dextrose with ammonium sulfate; SD+AA = synthetic dextrose added with ammonium sulfate and amino acids (histidine, tryptophan and methionine); SD-AA = synthetic dextrose added with ammonium sulfate without amino acids; SD+W = synthetic dextrose added with ammonium sulfate and tryptophan; SD-N+AA = synthetic dextrose without ammonium sulfate added with amino acids; SD-N-AA = synthetic dextrose without ammonium sulfate and without amino acids; YEPD = yeast extract peptone and dextrose; YEPG = yeast extract, peptone and galactose. Bars represent standard errors of 3 biological replicates. Differences are significant if p < 0.05 (***).
Fig 7.
Expression profile of the tryptophan biosynthetic genes.
TRP2, TRP3, TRP4 and TRP5 transcripts were evaluated by qPCR in response to nutritional deprivation of the nitrogen source (SD-N-AA). Bars represent standard errors of 3 biological replicates. Differences are significant if p < 0.05 (***).
Table 2.
Minimum inhibitory concentration (in μM) of 6-diazo-5-oxo-L-norleucine (DON) determined by broth microdilution method.