Abstract:Terms of Useengineering transcriptional circuits in filamentous fungi, we used our strategy for improving cassette stability by promoter replacement in the A. niger Tet-on system, which resulted in a modified Tet-on cassette with higher stability in recipient genomes.-3 -
“…Genes TRP3 and TRP5 of the C. neoformans tryptophan biosynthetic pathway, have also been reported to be essential but their roles in pathogenicity remain untested [14], as do those of all C. neoformans amino acid biosynthetic gene products thus far found to be essential in vitro. It is important to note that (as for studies of aromatic amino acid biosynthesis in A. fumigatus where tetracycline-responsive promoter tools have been developed [15][16][17]) the ability to harness regulatable expression of the essential gene products during mammalian infection is a critical step in discerning utility as antifungal drug targets because such an approach allows investigators to formally rule out complementation of auxotrophies via uptake of exogenous amino acids in the host niche. For example, in the case of C. neoformans TRP3 and TRP5 genes, in vitro supplementation of null mutants with exogenous tryptophan under nitrogen catabolite depressing conditions (proline as nitrogen source) led to partial rescue of growth, presumably via upregulated tryptophan uptake.…”
Section: Essential Genes In Fungal Amino Acid Biosynthesismentioning
confidence: 99%
“…To better address questions surrounding in vivo acquisition of amino acids essential for fungal viability novel genetic tools which allow temporal manipulation of gene expression during infection are required. A good example is the Tet-OFF system, recently established in A. fumigatus [17].…”
Section: Perspectives For Drug Developmentmentioning
Amongst 1.5 million fatal mycoses of humans occurring annually [1], the vast majority involve the human lung as the primary site of pathogenesis, and are derived from organisms which occupy environmental niches. On entry into the respiratory system pathogenic fungi must draw upon metabolic versatility for survival and proliferation as the mammalian lung is a nutritionally limiting environment. The nutritional stresses encountered have exposed vulnerabilities which have long been viewed as potential antifungal targets, since humans lack several of the metabolic pathways which fungi rely upon for pathogenic growth. However the ability of saprophytic fungi to proteolytically liberate amino acids from exogenous protein sources, and the differential availabilities of amino acids in diverse host niches have undermined confidence in amino acid metabolism as a target for selectively toxic antifungal therapies. Recent studies have reopened this debate by revealing a number of anabolic amino acid pathways in pathogenic fungi as being essential for viability per se. This review examines new knowledge on fungal amino acid metabolism in fungal pathogens of the human lung with a view to highlighting important new advances and gaps in understanding.
TITLE
Role of amino acid metabolism in fungal pathogenicityThroughout the kingdoms of life, amino acids are important building blocks of proteins and critical sources of macroelements such as nitrogen and sulphur which, if not acquired via intrinsic anabolic metabolism, must be sourced from extrinsic sources. Although fungi can biosynthesise all 20 of the proteinogenic amino acids the uptake of exogenous amino acids is significantly energetically preferable [2]. Accordingly, saprophytic fungal genomes harbour a multitude of protease and peptidase, and amino acid transporter encoding genes to ensure the liberation and uptake of amino acids from exogenous proteinaceous substrates.Amongst the hierarchy of preferred nitrogen sources the nitrogen-rich amino acids glutamine and arginine are dominant, but other amino acids can also be utilised when starvation threatens. Several wide domain regulatory mechanisms operate to ensure that starvation for nitrogen, carbon or amino acids is met with an appropriate metabolic response. In response to intracellular glutamine levels AreA-type GATA factors interact with other transcription factors specific for the biosynthesis of alternative nitrogen sources eg proline [3,4], while amino acid starvation increases translation of the transcriptional activator CpcA to generate a broad-acting cellular response governing amino acid biosynthesis and uptake [5]. Cellular levels of cysteine and methionine reflect intracellular sulphur content and modulate sulphur uptake via sulphur metabolite repression involving the positive-acting
“…Genes TRP3 and TRP5 of the C. neoformans tryptophan biosynthetic pathway, have also been reported to be essential but their roles in pathogenicity remain untested [14], as do those of all C. neoformans amino acid biosynthetic gene products thus far found to be essential in vitro. It is important to note that (as for studies of aromatic amino acid biosynthesis in A. fumigatus where tetracycline-responsive promoter tools have been developed [15][16][17]) the ability to harness regulatable expression of the essential gene products during mammalian infection is a critical step in discerning utility as antifungal drug targets because such an approach allows investigators to formally rule out complementation of auxotrophies via uptake of exogenous amino acids in the host niche. For example, in the case of C. neoformans TRP3 and TRP5 genes, in vitro supplementation of null mutants with exogenous tryptophan under nitrogen catabolite depressing conditions (proline as nitrogen source) led to partial rescue of growth, presumably via upregulated tryptophan uptake.…”
Section: Essential Genes In Fungal Amino Acid Biosynthesismentioning
confidence: 99%
“…To better address questions surrounding in vivo acquisition of amino acids essential for fungal viability novel genetic tools which allow temporal manipulation of gene expression during infection are required. A good example is the Tet-OFF system, recently established in A. fumigatus [17].…”
Section: Perspectives For Drug Developmentmentioning
Amongst 1.5 million fatal mycoses of humans occurring annually [1], the vast majority involve the human lung as the primary site of pathogenesis, and are derived from organisms which occupy environmental niches. On entry into the respiratory system pathogenic fungi must draw upon metabolic versatility for survival and proliferation as the mammalian lung is a nutritionally limiting environment. The nutritional stresses encountered have exposed vulnerabilities which have long been viewed as potential antifungal targets, since humans lack several of the metabolic pathways which fungi rely upon for pathogenic growth. However the ability of saprophytic fungi to proteolytically liberate amino acids from exogenous protein sources, and the differential availabilities of amino acids in diverse host niches have undermined confidence in amino acid metabolism as a target for selectively toxic antifungal therapies. Recent studies have reopened this debate by revealing a number of anabolic amino acid pathways in pathogenic fungi as being essential for viability per se. This review examines new knowledge on fungal amino acid metabolism in fungal pathogens of the human lung with a view to highlighting important new advances and gaps in understanding.
TITLE
Role of amino acid metabolism in fungal pathogenicityThroughout the kingdoms of life, amino acids are important building blocks of proteins and critical sources of macroelements such as nitrogen and sulphur which, if not acquired via intrinsic anabolic metabolism, must be sourced from extrinsic sources. Although fungi can biosynthesise all 20 of the proteinogenic amino acids the uptake of exogenous amino acids is significantly energetically preferable [2]. Accordingly, saprophytic fungal genomes harbour a multitude of protease and peptidase, and amino acid transporter encoding genes to ensure the liberation and uptake of amino acids from exogenous proteinaceous substrates.Amongst the hierarchy of preferred nitrogen sources the nitrogen-rich amino acids glutamine and arginine are dominant, but other amino acids can also be utilised when starvation threatens. Several wide domain regulatory mechanisms operate to ensure that starvation for nitrogen, carbon or amino acids is met with an appropriate metabolic response. In response to intracellular glutamine levels AreA-type GATA factors interact with other transcription factors specific for the biosynthesis of alternative nitrogen sources eg proline [3,4], while amino acid starvation increases translation of the transcriptional activator CpcA to generate a broad-acting cellular response governing amino acid biosynthesis and uptake [5]. Cellular levels of cysteine and methionine reflect intracellular sulphur content and modulate sulphur uptake via sulphur metabolite repression involving the positive-acting
“…For further details, see text. Abbreviations: Dox, doxycycline; VP16, transcriptional activator domain from herpes simplex virus tc, tetracycline; TetR, tetracycline-dependent Tet repressor; tetO, tetracycline operator sequence; tetO7, seven copies of tetracycline operator sequence; tTA, tetracyclin-dependent transactivator; tTA2 S , mammalian tetracyclin-dependent transactivator; T crgA , crgA terminator from Aspergillus fumigatus ; T trpC , trpC terminator from Aspergillus nidulans ; P min , minimal promoter (adopted from Brockamp et al 2002, Wanka et al 2016)Fig. 2Tet-On expression system.…”
Section: Introductionmentioning
confidence: 99%
“…1b) (Wanka et al 2016). Strains with a single copy of the modulated Tet-Off showed a strong luciferase activity, whereas the addition of different Dox concentrations resulted in a downregulation of the luciferase gene expression.…”
In industry, filamentous fungi have a prominent position as producers of economically relevant primary or secondary metabolites. Particularly, the advent of genetic engineering of filamentous fungi has led to a growing number of molecular tools to adopt filamentous fungi for biotechnical applications. Here, we summarize recent developments in fungal biology, where fungal host systems were genetically manipulated for optimal industrial applications. Firstly, available inducible promoter systems depending on carbon sources are mentioned together with various adaptations of the Tet-Off and Tet-On systems for use in different industrial fungal host systems. Subsequently, we summarize representative examples, where diverse expression systems were used for the production of heterologous products, including proteins from mammalian systems. In addition, the progressing usage of genomics and functional genomics data for strain improvement strategies are addressed, for the identification of biosynthesis genes and their related metabolic pathways. Functional genomic data are further used to decipher genomic differences between wild-type and high-production strains, in order to optimize endogenous metabolic pathways that lead to the synthesis of pharmaceutically relevant end products. Lastly, we discuss how molecular data sets can be used to modify products for optimized applications.
“…in vivo presents a promising tool in this fungus to explore the in vivo impact of presumed essential genes on disease. 17,34 The importance of probing gene function in vivo really cannot be over-stated, as the in vivo environment still remains a vast frontier with many hidden nuances and complexities. Recent in vivo gene expression studies in Candida albicans and A. fumigatus highlight how different fungal genetic programs are in vivo vs. favorite in vitro culture conditions.…”
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