1,721,027 research outputs found
Tools to study molecular mechanisms of Aspergillus pathogenicity
No full textThe unique nature of Aspergillus fungi represents a challenge for scrutinizing the attributes that render these saprophytic microorganisms pathogenic or allergenic under certain environmental circumstances. Recent publication of the genomic sequence from an isolate of the major pathogen Aspergillus fumigatus denotes enormous progress in aiming at cellular features and gene products that contribute to its pathogenicity. Latest developments to study virulence-related characteristics comprise profiling techniques, conditional gene inactivation and precise manipulation of the genome by means of gene targeting. Advances in assessing the virulence potential of particular mutant strains in alternative test systems complement these approaches
The Asexual Pathogen Aspergillus fumigatus Expresses Functional Determinants of Aspergillus nidulans Sexual Development
No full textThe major fungal pathogen of humans, Aspergillus fumigatus, lacks a defined sexual cycle, although the presence of genes encoding putative mating type idiomorphs and regulators of Aspergillus sexual development heightens the potential for cryptic sexuality in this deuteromycete. To test the functionality of these genetic determinants, we transferred the alpha box-encoding mat1-1 idiomorph from an A. fumigatus isolate to the homothallic fertile species Aspergillus nidulans. Abundant formation of fruiting bodies (cleistothecia) containing viable ascospores establishes functionality of this mating type gene product in the transgenic strain. Using a similar approach, we also established that the conserved transcriptional regulator from A. fumigatus, the nsdD gene product, can act as a functional, positively acting factor for A. nidulans cleistothecium development; moreover, high-level expression of NsdD in the endogenous host A. fumigatus profoundly alters hyphal development by triggering the formation of coiled hyphae. Our findings demonstrate that the presumably asexual pathogen A. fumigatus encodes functional regulators of mating and sexual development, thereby potentiating the case for cryptic sexuality in this fungal pathogen
Deletion of Aspergillus nidulans aroC using a novel blaster module that combines ET cloning and marker rescue
No full textBlaster cassettes are of significant value in functional genomics, as they represent tools with which to inactivate duplicated or homologous genes in an individual organism. We have constructed a novel blaster module which allows repeated gene deletion in the filamentous fungus Aspergillus nidulans. Because bacterial resistance marker cassettes are employed as flanking repeats in direct orientation, the blaster cassette is suited for recombinogenic engineering by ET cloning in Escherichia coli. The functionality of the blaster module was demonstrated by deleting the chorismate mutase-encoding gene aroC of A. nidulans, followed by marker rescue based on mitotic recombination. The resulting aroCDelta strains are auxotrophic for phenylalanine but not tyrosine, and display a limited capacity for fruit body formation and ascosporogenesis, which depends on the phenylalanine/tyrosine supply. The data support the notion that amino acid status has a strong impact on cleistothecium development in A. nidulans
Amino acid acquisition, cross-pathway control, and virulence in Aspergillus
No full textSupply of all amino acids required for translation is crucial for the synthesis of new proteins. Fungal amino acid biosynthesis has to be coordinated with amino acid uptake as well as protein degradation. A global regulator that connects amino acid biosynthesis and developmental programs is the transcription factor CpcA/Gcn4p. This transcriptional activator is conserved within the fungal kingdom and the cellular levels of this protein are carefully regulated. Deletion of the encoding cpcA gene in the opportunistic pathogen Aspergillus fumigatus results in impaired virulence in immuno-compromised mice, suggesting a role of the cross-pathway control system in fungal pathogenicity
Deletion and allelic exchange of the Aspergillus fumigatus veA locus via a novel recyclable marker module
No full textDetailed evaluation of gene functions in an asexual fungus requires advanced methods of molecular biology. For the generation of targeted gene deletions in the opportunistic pathogen Aspergillus fumigatus we designed a novel blaster module allowing dominant selection of transformants due to resistance to phleomycin as well as dominant (counter) selection of a Cre recombinase-mediated marker excision event. For validation purposes we have deleted the A. fumigatus pabaA gene in a wild-type isolate by making use of this cassette. The resulting pabaA::loxP strain served as the recipient for subsequent targeting of the velvet locus. Homologous reconstitution of the deleted gene was performed by an allele whose expression is driven in a nitrogen source-dependent manner, as validated by Northern analyses. Overexpression of the veA locus in A. fumigatus does not result in any obvious phenotype, whereas the sporulation capacities of the veA null mutant are reduced on nitrate-containing medium, a phenotype that is completely restored in the reconstituted strain
Allosteric regulation of catalytic activity: Escherichia coli aspartate transcarbamoylase versus yeast chorismate mutase
No full textAllosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins. In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypetides, and the less complicated chorismate mutase derived from baker's yeast, which functions as a homodimer. Chorismate mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors. Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and chorismate mutase shed more and more light on allostery as an important regulatory mechanism of any living cell. The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail
Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end-joining-deficient genetic background
No full textThe akuA gene encoding the Ku70 component of the nonhomologous end-joining machinery was deleted in the opportunistic pathogen Aspergillus fumigatus. No obvious phenotype could be assessed for the corresponding mutant strain but relative frequencies of homologous recombination were increased as deduced from targeting the laccase-encoding abr2 gene
Nitrogen metabolism ofAspergillusand its role in pathogenicity
No full textAspergilli represent unique pathogens. Based on their saprophytic life style they are able to colonize a variety of ecological niches, among them the immunocompromised individual. Distinct fungal attributes that play a role in pathogenicity of aspergilli have been described, and primary metabolism indisputably has to be taken into account for contributing to the virulence potential of this fungal genus. Here we present an overview of studies that focus on this aspect of nutritional versatility In the predominant pathogenic representative Aspergillus fumigatus regulation of nitrogen utilization and sensing of nitrogen sources have been scrutinized with respect to pathogenicity. The impact of distinct metabolic pathways on virulence capacities could be evaluated by inspection of auxotrophic mutant strains. Among them, para-aminobenzoic acid-requiring mutants revealed that this biosynthetic route is strictly required for pathogenicity. For amino acid anabolism only lysine biosynthesis has been investigated in this regard. Fungal amino acid biosynthesis is generally subject to strict regulation mediated by the Cross-Pathway Control system, a conserved regulatory circuit evolved to counteract conditions of nutritional stress. A clear influence of the system on pathogenicity could be observed by targeting its transcriptional activator CpcA. However, additional metabolic characteristics as well as regulatory instruments that compensate environmental challenges need to be addressed in future research with the aim to assess the significance of fungal primary metabolism for pathogenicity of aspergillus species
Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae
No full textControl of transcription and enzyme activities are two interwoven regulatory systems essential for the function of a metabolic node. Saccharomyces cerevisiae strains differing in enzyme activities at the chorismate branch point of aromatic amino acid biosynthesis were constructed by recombinant DNA technology. Expression of an allosterically unregulated, constitutively activated chorismate mutase encoded by the ARO7T(226I) (ARO7(c)) allele depleted the chorismate pool. The resulting tryptophan limitation caused growth defects, which could be counteracted only by transcriptional induction of TRP2 encoding the competing enzyme anthranilate synthase. ARO7 expression is not transcriptionally regulated by amino acids. Transcriptional activation of the ARO7(c) allele led to stronger growth retardation upon tryptophan limitation. The same effect was achieved by removing the competing enzyme anthranilate synthase, which is encoded by the TRP2 gene, from the transcriptional control. The allelic situation of ARO7(c) being under general control instead of TRP2 resulted in severe growth defects when cells were starved for tryptophan. In conclusion, the specific regulatory pattern acting on enzymatic activities at the first metabolic node of aromatic amino acid biosynthesis is necessary to maintain proper flux distribution. Therefore, the evolution of the sophisticated allosteric regulation of yeast chorismate mutase requires as prerequisite (i) that the encoding ARO7 gene is not transcriptionally regulated, whereas (ii) the transcription of the competing feedback-regulated anthranilate synthase-encoding gene is controlled by availability of amino acids.NIGMS NIH HHS [R01 GM006920
Sexual diploids of Aspergillus nidulans do not form by random fusion of nuclei in the heterokaryon
No full textThe sexual stage of Aspergillus (Emericella) nidulans consists of cleistothecia containing asci, each with eight ascospores. The fungus completes the sexual cycle in a homokaryotic or a heterokaryotic mycelium, respectively. The common assumption for the last 50 years was that different nuclear types are not distinguishable,lc when sexual development is initiated. When cultured on a medium limited for glucose supplemented with 2% sorbitol, sexual development of A. nidulans is slowed and intact tetrads call be isolated. Through tetrad analysis we found that unlike haploid nuclei fuse preferentially to the prezygotic diploid nucleaus. When heterokaryons are formed between nuclei of different genetic backgrounds, then recombinant asci derived from opposite nuclei are formed exclusively. Strains in the same heterokaryon compatibility group with moderate differences in their genetic backgrounds can discriminate between the nuclei of a heterokaryon and preferentially form a hybrid diploid nucleus, resulting in 85% recombinant tetrads. A. nidulans strains that differ at only a single genetic marker fuse the haploid nuclei at random for formation of diploid nuclei during meiosis. These results argue for a genetically determined "relative heterothallism'' of nuclear recognition within a heterokaryon and a specific recruitment of different nuclei for karyogamy when available
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