Two genes, designated chsC and chsG were isolated from DNA libraries of the opportunistic fungal pathogen, Aspergillus fumigatus. The genes were characterized with respect to their nucleotide sequences and mutant phenotypes. The complete deduced amino acid sequences of chsC and chsG show that the products of both genes are Class III zymogen-type enzymes. A mutant strain constructed by disruption of chsC is phenotypically indistinguishable from the wild-type strain, but chsG- and chsC- chsG- strains have reduced colony radial growth rate and chitin synthase activity, conidiate poorly and produce highly branched hyphae. Despite these defects, the double-mutant strain retained the ability to cause pulmonary disease in neutropenic mice. However, in comparison to the wild-type strain, there was a decrease in mortality and delay in the onset of illness in mice inoculated with the double-mutant strain, which was associated with smaller and more highly branched fungal colonies in lung tissue.

Invasive pulmonary aspergillosis (IPA) is an important cause of mortality and morbidity in the immunocompromised host. However, the diagnosis of this condition may be difficult, and it is sometimes missed because of the lack of sensitivity of available tests. Therefore, we used polymerase chain reaction (PCR)-based amplification of fragments of genes-encoding alkaline proteases from Aspergillus fumigatus and A. flavus to detect these organisms in bronchoalveolar lavage fluid specimens. The predicted size of the product (747 base pairs) after amplification of A. fumigatus was larger than that for A. flavus (690 base pairs). The reaction was highly sensitive (after amplification of 500 fg of A. fumigatus DNA, product could be detected by Southern analysis), and it was specific for A. fumigatus and A. flavus. Bronchoalveolar lavage fluid from four immunosuppressed patients with proved or probable IPA was positive by this assay (sensitivity, 100%); in addition, the sample from one patient with possible IPA was PCR-positive. Only one specimen from 18 immunosuppressed patients with no evidence of IPA was PCR-positive (specificity, 94.4%). Five of 28 bronchoalveolar lavage samples from nonimmunosuppressed patients were PCR-positive, probably representing colonization of the respiratory tract. PCR-based detection may prove useful in the diagnosis of IPA.

Forty-four oligonucleotide decamers were tested for their abilities to generate randomly amplified polymorphic DNA (RAPD) markers from genomic DNAs of three different isolates of Aspergillus fumigatus. Seven

Signature-tagged mutagenesis (STM) is a method that has been used to screen for genes required for in vivo survival of pathogenic bacteria, but has not been used to investigate a eukaryotic pathogen in an animal model of disease. We have adapted STM to identify genes required for in vivo growth of the opportunistic fungal pathogen Aspergillus fumigatus. Using a mouse model of invasive pulmonary aspergillosis, we have isolated several mutant strains with defects in their ability to replicate in vivo. One strain unable to cause lethal infection was further characterized and found to have an insertion into the promoter of a gene (pabaA) encoding para-aminobenzoic acid synthetase, an enzyme catalyzing a late step in the biosynthesis of folate. The complete inability of this strain, and other pabaA- strains constructed in this study by targeted gene deletion, to cause lethal infection in mice confirms the importance of the folate synthesis pathway for in vivo survival of this pathogen. The successful application of STM to A. fumigatus demonstrates that in vivo genetic analysis of eukaryotic pathogens is feasible and could result in the identification of potential targets, such as para-aminobenzoic acid synthetase, for novel antifungal therapies.

The areA gene of Aspergillus nidulans is a positive-acting transcriptional factor required for the expression of genes involved in the utilization of a broad range of nitrogen sources other than ammonium and glutamine. We have investigated the role in pathogenesis of the corresponding gene (AfareA) of Aspergillus fumigatus, a causative agent of invasive pulmonary aspergillosis. Stable and unstable AfareA- strains were constructed and tested for altered virulence in mice on the basis of host survival, mixed infection experiments and genetic reversion studies. These showed that the AfareA gene contributes to, but is not essential for, virulence. Strains carrying extragenic mutations that partially suppress the AfareA- phenotype for growth on different nitrogen sources were tested for altered virulence in mixed infection studies. One of these was found to be more virulent than the parental AfareA- strain.