We have cloned and characterized the Aspergillus fumigatus cpcA gene encoding the transcriptional activator of the cross-pathway control system of amino acid biosynthesis. cpcA encodes a functional orthologue of Saccharomyces cerevisiae Gcn4p. The coding sequence of the 2.2 kb transcript is preceded by two short upstream open reading frames, the larger one being well conserved among Aspergilli. Deletion strains in which either the coding sequence or the entire locus are replaced by a bifunctional dominant marker are impaired in their cross-pathway control response upon amino acid starvation, as demonstrated by analyses of selected reporter genes and specific enzymatic activities. In a murine model of pulmonary aspergillosis, cpcAdelta strains display attenuated virulence. Pathogenicity is restored to wild-type levels in strains with reconstitution of the genomic locus. Competitive mixed infection experiments additionally demonstrate that cpcAdelta strains are less able to survive in vivo than their wild-type progenitor. Our data suggest that specific stress conditions are encountered by A. fumigatus within the mammalian host and that the fungal cross-pathway control system plays a significant role in pulmonary aspergillosis.
"Fungal amino acid biosynthesis is vital to the pathogen metabolism and a conserved transduction cascade which links the environmental stimuli to amino acid homeostasis is the cross-pathway control (CPC) system. cpcA gene in A. fumigatus encodes the transcriptional activator of the CPC system of amino acid biosynthesis and the cpcA null mutant displayed attenuated virulence in a murine model of pulmonary aspergillosis . "
[Show abstract][Hide abstract] ABSTRACT: The interplay between Aspergillus fumigatus and the host immune response in lung infection has been subject of studies over the last years due to its importance in immunocompromised patients. The multifactorial virulence factors of A. fumigatus are related to the fungus biological characteristics, for example, structure, ability to grow and adapt to high temperatures and stress conditions, besides capability of evading the immune system and causing damage to the host. In this context, the fungus recognition by the host innate immunity occurs when the pathogen disrupts the natural and chemical barriers followed by the activation of acquired immunity. It seems clear that a Th1 response has a protective role, whereas Th2 reactions are often associated with higher fungal burden, and Th17 response is still controversial. Furthermore, a fine regulation of the effector immunity is required to avoid excessive tissue damage associated with fungal clearance, and this role could be attributed to regulatory T cells. Finally, in this work we reviewed the aspects involved in the complex interplay between the host immune response and the pathogen virulence factors, highlighting the immunological issues and the importance of its better understanding to the development of novel therapeutic approaches for invasive lung aspergillosis.
"CpcA binds to CPRE (cross-pathway control recognition elements) that locate not only within the promoter region of target genes but also within its own CpcA promoter, resulting in increased transcript levels for the activator (Hoffmann et al. 2001). A requirement of CpcA for pathogenicity of the human opportunistic pathogen A. fumigatus had been demonstrated (Krappmann et al. 2004) but the role of CpcA homologs in plant-pathogenic fungi is unclear. Transcript levels of the CpcA homolog CPC1 gene of the plant-pathogen V. longis porum increased when cultivated in the presence of plant xylem sap and suggested an imbalanced amino-acid supply (Singh et al. 2010). "
[Show abstract][Hide abstract] ABSTRACT: The plant pathogenic fungus Verticillium longisporum is a causal agent of early senescence and ripening in cruciferous crops like Brassica napus. Verticillium wilts have become serious agricultural threats during the last decades. Verticillium species infect host-plants through the roots and colonize xylem vessels of the host-plant. The xylem fluid provides an environment with limited carbon sources and imbalanced amino acid supply, which requires that V. longisporum induces the cross-pathway control of amino acid biosynthesis. RNA-mediated gene silencing reduced the expression of the two CPC1 isogenes (VlCPC1-1, VlCPC1-2) of the allodiploid V. longisporum up to 85%. VlCPC1 encodes the conserved transcription factor of the cross-pathway control. The silenced mutants were highly sensitive to amino acid starvation and the infected plants showed significantly less symptoms as stunting or early senescence in oilseed rape plant infection assays. Consistently, deletion of single CPC1 of the haploid V. dahliae resulted in strains, which are sensitive to amino acid starvation and cause strongly reduced symptoms in the plant-host tomato (Solanum lycopersicum). The allodiploid V. longisporum and the haploid V. dahliae are the first phytopathogenic fungi, which were shown to require CPC1 for infection and colonization of their respective host plants oilseed rape and tomato.
"These data raise the question of the responsible regulatory mechanism. Therefore, in a next approach we analyzed a potential involvement of the transcriptional activator CpcA, which mediates “cross pathway control” that is crucial for amino acid homeostasis and virulence of A. fumigatus
. The ΔcpcA mutant strain, which was generated in A. fumigatus strain D141 was described previously and kindly supplied by Dr. Sven Krappmann. "
[Show abstract][Hide abstract] ABSTRACT: The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants ΔargEF and ΔargB, which lack and possess mitochondrial ornithine production, respectively. Deficiency in argEF (encoding acetylglutamate kinase and acetylglutamyl-phosphate-reductase), but not argB (encoding ornithine transcarbamoyl transferase) decreased (i) the cellular ornithine content, (ii) extra- and intracellular SB, (iii) growth under harsh iron starvation, (iv) resistance to the ornithine decarboxylase inhibitor eflornithine, and (v) virulence in the Galleria mellonella larvae model. These lines of evidence indicate that SB is mainly fueled by mitochondrial rather than cytosolic ornithine production and underline the role of SB in virulence. Ornithine content and SB of ΔargB increased with declining arginine supplementation indicating feedback-inhibition of mitochondrial ornithine biosynthesis by arginine. In contrast to SB, the arginine and polyamine contents were only mildly affected in ΔargEF, indicating prioritization of the latter two ornithine-consuming pathways over SB. These data highlight the metabolic differences between the two arginine auxotrophic mutants ΔargEF and ΔargB and demonstrate that supplementation of an auxotrophic mutant does not restore the wild type metabolism at the molecular level, a fact to be considered when working with auxotrophic mutants. Moreover, cross pathway control-mediating CpcA was found to influence the ornithine pool as well as biosynthesis of siderophores and polyamines.
PLoS ONE 06/2013; 8(6):e67426. DOI:10.1371/journal.pone.0067426 · 3.23 Impact Factor
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