Invasive pulmonary aspergillosis (IPA) occurs in immunocompromised patients after the inhalation of conidia of certain species of Aspergillus. The frequency of IPA is increasing in concert with the population of susceptible hosts. The pathogenesis of aspergillosis is not well understood, but there is strong evidence supporting a role for the elastinolytic proteinase of Aspergillus (Aspergillus elastase, AE) in the initiation of infection and progression of disease. Diagnosis of aspergillosis has been hampered by the ubiquity of the fungi which compromises the value of surveillance cultures, the lack of immune response in most susceptible patients, which devalues tests for antibodies, and the lack of a universally accepted, readily available test for Aspergillus antigen, which is due, in part, to the unavailability of standardized reagents. We propose to address these problems by cloning the gene for AE and using the gene probe and its recombinant protein product (rAE) as reagents for diagnostic testing. Our approach will be to determine the amino acid sequence of selected peptides of AE and from that sequence to deduce the corresponding nucleotide sequence. Oligonucleotide probes will be synthesized based on the predicted nucleotide sequence and will be used to screen a genomic DNA library which we will construct in bacteriophage lambda. The putative gene will be isolated and sequenced. The gene will be characterized for homology among other Aspergillus spp. and related fungi. An RNA copy of all or part of the gene will be used as a molecular reagent for tissue diagnosis of aspergillosis by in situ hybridization. The gene will be subcloned into expression vectors; rAE will be compared with authentic AE for identity. The recombinant protein will then be used to develop a competitive ELISA for circulating AE in body fluids of infected animals and humans. The rAE will also be used to further explore the role of AE in virulence. The use of molecular techniques to study virulence mechanisms and to develop new diagnostic tests allows us to increase our understanding of the pathogenesis of IPA and our ability to diagnose aspergillosis efficiently, and to contribute to the overall goal of this project, increasing our knowledge of the molecular and cell biology of fungal pathogens.
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