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TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus.

ABSTRACT: Small peptides formed from non-ribosomal peptide synthetases (NRPS) are bioactive molecules produced by many fungi including the genus Aspergillus. A subset of NRPS utilizes tryptophan and its precursor, the non-proteinogenic amino acid anthranilate, in synthesis of various metabolites such as Aspergillus fumigatus fumiquinazolines (Fqs) produced by the fmq gene cluster. The A. fumigatus genome contains two putative anthranilate synthases - a key enzyme in conversion of anthranilic acid to tryptophan - one beside the fmq cluster and one in a region of co-linearity with other Aspergillus spp. Only the gene found in the co-linear region, trpE, was involved in tryptophan biosynthesis. We found that site-specific mutations of the TrpE feedback domain resulted in significantly increased production of anthranilate, tryptophan, p-aminobenzoate and fumiquinazolines FqF and FqC. Supplementation with tryptophan restored metabolism to near wild type levels in the feedback mutants and suggested that synthesis of the tryptophan degradation product kynurenine could negatively impact Fq synthesis. The second putative anthranilate synthase gene next to the fmq cluster was termed icsA for its considerable identity to isochorismate synthases in bacteria. Although icsA had no impact on A. fumigatus Fq production, deletion and over-expression of icsA increased and decreased respectively aromatic amino acid levels suggesting that IcsA can draw from the cellular chorismate pool.

SUBMITTER: Wang PM

PROVIDER: S-EPMC4789178 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Project description:Inhibition of fungal growth by Congo Red (CR) has been known for a long time and claimed to be associated to a specific binding to fibrillar chains of β-1,3-glucans which blocks cell wall polysaccharide synthesis. It has also been suggested that the effect of CR could be pleiotropic, especially because of its oxido-reductive properties. One way to elucidate the mechanism of action of CR is to identify transcription factors (TF) which regulate the response to CR and interrogate their regulon. The analysis of TF mutants with differential sensitivity to CR would be also a way to identify new regulatory pathways involved in cell wall biosynthesis. During the investigation of the sensitivity to CR of a TF mutant library, several mutants either sensitive or resistant to CR were analysed . The morphology of the conidial germination was affected differently by CR in the parental strain and TF mutants. Abnormal distorted swollen conidia called Quasimodo cells were seen in presence of the CR. The size of the Quasimodo cells in the resistant mutants are bigger and adsorb more CR than the germinating conidia of the sensitive or parental strain. This is associated to a difference in permeability of the mutants. The clearing of the drug from the medium by the resistant strain was mainly responsible for the survival and later growth of some of resting conidia of these mutants which did not adsorb intracellularly the drug whereas in the sensitive mutants all conidia adsorbed the drug and were consequently killed. Although there are some modifications of the composition of the cell wall, a transcriptomic analysis of the mutant showed that CR affected many different pathways, including iron and calcium metabolism. Accordingly, the susceptibility to other cell wall drugs of the sensitive and the resistant TF mutants varied with the mutants and not always in connexion with their sensitivity to CR. Interestingly, a new paradoxical effect of the CR was discovered in the resistant mutant since the growth of one of them could be stimulated after recovering from the CR stress.

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TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus.

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