Molecular regulatory mechanisms of iron assimilation in Aspergillus and their links to fungal pathogenicity

The genus Aspergillus is one of the most ubiquitous microorganisms worldwide and various Aspergillus species are responsible for the clinical syndromes of allergie bronchopulmonary aspergillosis, aspergilloma and pulmonary aspergillosis. In particular, pulmonary aspergillosis represents a life-threatening disease of increasing incidence in immunocompromised patients which has been attributed to increasing iatrogenic immunosuppression and successful life-support measures for neutropenic patients. Most virulence factors of microorganisms are strictly regulated, with their expression linked to various environmental signals. Since iron is tightly sequestered in mammalian hosts by high-affinity iron-binding proteins, microbes require efficient iron-scavenging systems to survive and proliferate within the host. Under iron starvation, most fungi synthesize and excrete low-molecular- weight, iron speciflc chelators, called siderophores, which have therefore often been suggested to function as virulence factors. The aim of this study is to evaluate the impact of siderophores and other iron scavenging mechanisms of Aspergillus species on pathogenicity. The molecular cloning and analysis of genes involved in iron metabolism will be undertaken in A. nidulans since this species is more suitable for genetic and molecular genetic techniques; the clinical studies will be done partly employing the close relative A. fumigatus because this fungus is the most common pathogenic Aspergillus species. Recently, we have isolated a gene encoding a putative repressor (SREA) of siderophore biosynthesis from A. nidulans. In the course of this work the precise function of SREA shall be characterized by constructing a sreA loss of function Aspergillus strain. The role of siderophore biosynthesis in pathogenicity will be investigated by constitutive overproduction of SREA in Aspergillus, thereby repressing synthesis of siderophores, and testing the strains in murine models. Additionally, genes encoding siderophore biosynthesis enzymes and components of alternative iron transport systems will be identified and isolated due to their expected different expression under iron replete and deplete conditions. The function of the isolated genes will again be characterized by construction of loss of function strains. Subsequently, these strains will then be utilized to evaluate their importance in pathogenicity in murine models. Excretion of siderophores will be investigated immunohistochemically in thin- layer sections of human aspergilloma specimens, using antiserum which will be raised against purified siderophores of A. fumigatus, as well as in sputum of cystic fibrosis patients with Aspergillus infections. The purified Aspergillus siderophores will also be employed to isolate the gene(s) encoding the siderophore receptor(s) utilizing a phage display approach. This project will lead to a better insight into iron metabolism of Aspergillus and possibly also to the identification of putative virulence factors. Better understanding of the multifaceted virulence strategies of pathogenic fungi may suggest new methods for treatment and prevention that intercede at different phases of infection.