Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.     

Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis

The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.     

Nonribosomal peptide synthetase genes pesL and pes1 are essential for Fumigaclavine C production in Aspergillus fumigatus

The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H(2)O(2), and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P < 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type and ΔpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.     

Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus

Calcineurin is implicated in a myriad of human diseases as well as homeostasis and virulence in several major human pathogenic microorganisms. The fungus Aspergillus fumigatus is a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive aspergillosis are often ineffective, and novel therapeutic approaches are urgently needed. We demonstrate that a mutant of A. fumigatus lacking the calcineurin A (cnaA) catalytic subunit exhibited defective hyphal morphology related to apical extension and polarized growth, which resulted in drastically decreased filamentation. The delta cnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the delta cnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the delta cnaA mutant in several distinct animal models with different types of immunosuppression and inoculum delivery led to a profound attenuation of pathogenicity compared to infection with the wild-type and complemented strains. Lung tissue from animals infected with the delta cnaA mutant showed a complete absence of hyphae, in contrast to tissue from animals infected with the wild-type and complemented strains. Quantitative fungal burden and pulmonary infarct scoring confirmed these findings. Our results support the clinical observation that substantially decreasing fungal growth can prevent disease establishment and decrease mortality. Our findings reveal that calcineurin appears to play a globally conserved role in the virulence of several pathogenic fungi and yet plays specialized roles in each and can be an excellent target for therapeutic intervention.     

Signature-tagged mutagenesis: barcoding mutants for genome-wide screens

DNA signature tags (molecular barcodes) facilitate functional screens by identifying mutants in mixed populations that have a reduced or increased adaptation to a particular environment. Many innovative adaptations and refinements in the technology have been described since its original use with Salmonella; they have yielded a wealth of information on a broad range of biological processes--mainly in bacteria, but also in yeast and other fungi, viruses, parasites and, most recently, in mammalian cells. By combining whole-genome microarrays and comprehensive ordered libraries of mutants, high-throughput functional screens can now be achieved on a genomic scale.     

压力应激影响烟曲霉致病力的研究进展

烟曲霉是环境中广泛存在的腐生真菌,是重要的机会致病菌。近年来随着免疫受损人群的增多,烟曲霉引起侵袭性曲霉病不断增加,在深部丝状真菌感染中居于首位。由于宿主体内环境与自然环境存在诸多差异,使得烟曲霉孢子适应体内环境条件是其能够生存并致病的前提。研究发现烟曲霉适应宿主环境的能力,如温度、氧化应激、渗透压、缺氧、营养缺乏等,与其致病力密切相关。对压力应激与致病力关系的研究有助于解析烟曲霉的致病机制,可为研发烟曲霉感染诊治的新途径提供理论基础。本文对烟曲霉的压力应激与其致病力的关系进行了综述。     

Disruption of a nonribosomal peptide synthetase in Aspergillus fumigatus eliminates gliotoxin production

The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in DeltagliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the DeltagliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the DeltagliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.     

Construction and pathogenicity of Aspergillus fumigatus mutants that do not produce the ribotoxin restrictocin

Aspergillus fumigatus, the most common cause of invasive pulmonary aspergillosis (IPA), produces a potent cytotoxjn called restrictocin. To investigate the role of restrictocin in (PA, we have constructed fungal strains in which the res gene has been inactivated by gene disruption. These disruptants lack the specific extracellular ribonucleolytic activity associated with restrictocin, as measured by an in vitro rabbit reticulocyte lysate assay. Western blot analysis of one drsruptant, using an anti-restrictocin monoclonal antibody, confirmed that the toxin is not produced. The growth characteristics of the disruptants could not be distinguished from those of their parental isolates on a variety of culture media. The pathogenicity of two disruptants was assessed in a murine model of IPA. There were no significant differences in mortality when these strains were compared with the parental isolates and an ectopic transformant. In addition, histological examination of infectediung tissue did not reveal any obvious differences in the number or size of fungal colonies or in the polymorphonuclearleucocyte response. Our results demonstrate that restrictocin is not an important virulence factor in this model of IPA.     

Signature-tagged mutagenesis of Edwardsiella ictaluri identifies virulence-related genes, including a salmonella pathogenicity island 2 class of type III secretion systems

Edwardsiella ictaluri is the leading cause of mortality in channel catfish culture, but little is known about its pathogenesis. The use of signature-tagged mutagenesis in a waterborne infection model resulted in the identification of 50 mutants that were unable to infect/survive in catfish. Nineteen had minitransposon insertions in miscellaneous genes in the chromosome, 10 were in genes that matched to hypothetical proteins, and 13 were in genes that had no significant matches in the NCBI databases. Eight insertions were in genes encoding proteins associated with virulence in other pathogens, including three in genes involved in lipopolysaccharide biosynthesis, three in genes involved in type III secretion systems (TTSS), and two in genes involved in urease activity. With the use of a sequence from a lambda clone carrying several TTSS genes, Blastn analysis of the partially completed E. ictaluri genome identified a 26,135-bp pathogenicity island containing 33 genes of a TTSS with similarity to the Salmonella pathogenicity island 2 class of TTSS. The characterization of a TTSS apparatus mutant indicated that it retained its ability to invade catfish cell lines and macrophages but was defective in intracellular replication. The mutant also invaded catfish tissues in numbers equal to those of invading wild-type E. ictaluri bacteria but replicated poorly and was slowly cleared from the tissues, while the wild type increased in number.     

Probing essential water in yeast pyrophosphatase by directed mutagenesis and fluoride inhibition measurements

The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed.     

Bidirectional gene transfer between Aspergillus fumigatus and Aspergillus nidulans

Abstract The rpmF-plsX-fabH gene cluster of Rhodobacter capsulatus homologous to that of Escherichia coli was identified. rpmF encodes ribosomal protein L32, plsX plays an undefined role in membrane lipid synthesis, and fabH encodes β-ketoacyl-acyl carrier protein synthase III. The R. capsulatus plsX gene complemented a defect in an E. coli strain with the plsX50 mutation. Overproduction of the fabH gene product of R. capsulatus in E. coli resulted in dramatically increased β-ketoacyl-acyl carrier protein synthase III activity. These results indicate that plsX and fabH apparently function the same in R. capsulatus as in E. coli .     

烟曲霉基因组与致病机制

烟曲霉(Aspergillus fumigatus)是环境中普遍存在的丝状腐生真菌,也是重要的机会致病菌。随着免疫受损人群的增多,由烟曲霉引起的系统性感染发病率不断升高,已成为重症免疫受损患者死亡的主要原因之一。文章结合烟曲霉全基因组序列信息对烟曲霉感染过程、致病机制等做一概述。     

The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus

Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.     

A nonribosomal peptide synthetase (Pes1) confers protection against oxidative stress in Aspergillus fumigatus

Aspergillus fumigatus is an important human fungal pathogen. The Aspergillus fumigatus genome contains 14 nonribosomal peptide synthetase genes, potentially responsible for generating metabolites that contribute to organismal virulence. Differential expression of the nonribosomal peptide synthetase gene, pes1, in four strains of Aspergillus fumigatus was observed. The pattern of pes1 expression differed from that of a putative siderophore synthetase gene, sidD, and so is unlikely to be involved in iron acquisition. The Pes1 protein (expected molecular mass 698 kDa) was partially purified and identified by immunoreactivity, peptide mass fingerprinting (36% sequence coverage) and MALDI LIFT-TOF/TOF MS (four internal peptides sequenced). A pes1 disruption mutant (delta pes1) of Aspergillus fumigatus strain 293.1 was generated and confirmed by Southern and western analysis, in addition to RT-PCR. The delta pes1 mutant also showed significantly reduced virulence in the Galleria mellonella model system (P < 0.001) and increased sensitivity to oxidative stress (P = 0.002) in culture and during neutrophil-mediated phagocytosis. In addition, the mutant exhibited altered conidial surface morphology and hydrophilicity, compared to Aspergillus fumigatus 293.1. It is concluded that pes1 contributes to improved fungal tolerance against oxidative stress, mediated by the conidial phenotype, during the infection process.     

Functional characterization of an Aspergillus fumigatus calcium transporter (PmcA) that is essential for fungal infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca(+2)-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca(+2)-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -B and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The ΔpmcA and ΔpmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the ΔcalA and ΔcrzA mutant strains. However, only the A. fumigatus ΔpmcA was avirulent in the murine model of invasive pulmonary aspergillosis.     

Screening method to identify inhibitors of siderophore biosynthesis in the opportunistic fungal pathogen, Aspergillus fumigatus

Aims:  Aspergillus fumigatus is the most common cause of airborne mould infections in immunocompromised patients worldwide. Our aim was to develop a method to identify agents that inhibit siderophore biosynthesis because this pathway is unique to the fungus and is essential for virulence.
Methods and Results:  A high-throughput two-step screening assay was developed using 96-well plates in which fungal growth and siderophore production is assessed spectrophotometrically. If a compound inhibits growth only in iron-limited medium (screen 1), its effect on siderophore production is then determined (screen 2). The proof of concept was demonstrated using a known antifungal agent, amphotericin B, and a strain of A. fumigatus deficient in siderophore production.
Conclusions:  The two-stage screening method clearly identified growth defects in A. fumigatus related specifically to siderophore biosynthesis.
Significance and Impact of the Study:  The increasing incidence of life-threatening fungal infections has produced an urgent need for novel antifungal agents. The method described in this report will facilitate the identification of novel antifungal compounds that inhibit a pathway critical for A. fumigatus virulence and have a reduced probability of affecting host metabolism.