Conidial NADase - a novel potential target to treat fungal infections

Aspergilloses are among the most common human fungal infections and cause more than one million deaths every year. The airborne spores (conidia) of Aspergillus fumigatus are ubiquitously present in the environment and normally cleared from the lungs by the immune system. However, A. fumigatus infections represent a serious threat in immune-compromised patients. The spectrum of antifungal drugs is rather limited and development of resistance has become a major challenge for the treatment of these infections. We have recently identified the function of a protein on the surface of the conidia that may have a key role during the infection. Namely, the protein degrades a molecule (NAD) that is essential for the metabolism of the host organism. We hypothesize that this catalytic activity is critical for the fungus to evade the immune system. The main goal of the present study is to establish the structural and functional properties of this novel protein. Moreover, using a wide range of infection assays and molecular genetic tools, we will be able to determine its potential as a drug target. Finally, we will identify a first set of chemical compounds that may act as inhibitors of the protein activity. Thereby, we might be able to establish a new class of antifungal agents. During the first year of the project, we have achieved two critical goals that provide the basis for a successful continuation. First, we managed to produce large quantities of the A. fumigatus NAD-degrading enzyme, thereby enabling extensive structural and functional investigations. This step was challenging and we needed to resort to a backup solution to establish a suitable system. Second, based on the availability of the purified protein, we were able to obtain crystals that were of sufficiently high quality for high-resolution structural analyses using X-ray diffraction. These analyses established the first three-dimensional structure of a fungal NADase and identified its unique evolutionary occurrence. Now, we have started in-depth structural and functional studies of the homodimeric A. fumigatus enzyme. Unexpectedly, we identified a calcium binding site in each of the subunits. Using site-directed mutagenesis, enzymatic and structural assays, we established that calcium binding mediates conformational changes in the protein that promote catalysis. Thereby, calcium binding might be part of a mechanism regulating the catalytic activity of the NADase depending on environmental conditions. We have also identified and validated the genes encoding surface NADases in the plant pathogen Fusarium oxysporum and the red bread mold Neurospora crassa. While the catalytic and overall structural properties of these enzymes appear to resemble those of the A. fumigatus protein, there are individual traits that are distinct such as the absence of a calcium binding site. Therefore, while the biological function of these NADases may be similar in the different fungi, the mechanisms regulating their enzyme activity may differ. In the previous project period, we successfully established laboratory protocols to maintain protein stability and enzyme activity and validated suitable conditions for protein crystallization. Therefore, during the current project period, we could focus on three main aspects. First, we have identified the molecular mechanism of how the unique calcium binding site in the A. fumigatus protein acts to stabilize the overall protein structure and how changes in calcium binding can be transmitted to the catalytic center. These important insights were obtained from analyses of generated X-ray structures that represented the calcium-bound and -unbound protein forms. Moreover, various biophysical methods were used to consolidate the results. Second, we were able to crystallize the Fusarium enzyme and a slightly modified form of the Neurospora enzyme. Next, we will thoroughly analyze these structures to understand similarities and differences of these enzymes in the different clades. For example, it will be important to establish how similar the catalytic sites of these enzymes are, because this will have an immediate impact on the strategy to identify specific inhibitors. Third, we have developed biochemical and biophysical assays that enable accurate measurements of the enzyme stability and activity even at low substrate concentrations. Together with the above-mentioned protocols to produce stable proteins, we have generated the protein-chemical prerequisites to consider preliminary screening for modulators of enzyme activity, which will be a task in the upcoming project period.

Aspergilloses are among the most common human fungal infections and cause more than one million deaths every year. The airborne spores (conidia) of Aspergillus fumigatus are ubiquitously present in the environment and normally cleared from the lungs by the immune system. However, A. fumigatus infections represent a serious threat in immune-compromised patients with a lethality of up to 90%. The spectrum of antifungal drugs is rather limited and development of resistance has become a major challenge for the treatment of these infections. This proposal is based on our recent (unpublished) discovery of an NAD-cleaving enzyme (AfNADase) on the surface of conidia from Aspergillus fumigatus. Importantly, earlier virulence studies have indicated a prominent role of the encoding gene in the infection process; however, the function of the gene product has remained unknown. We hypothesize that the NADase activity may be critical for the adherence to the host cells or to evade the immune system. Our preliminary observations indicate that AfNADase represents a new class of enzymes with homologous representatives only in spore-forming fungi making it a promising drug target. In collaboration with internationally leading research groups, we will establish the molecular and catalytic properties of this new enzyme class including the X-ray structure. Moreover, we will use molecular genetics and a range of functional assays, including mouse models of A. fumigatus infection, to establish the biological function of AfNADase as well as its role in the infection process. Finally, we will produce a range of NAD analogs and test their inhibitory effect on AfNADase. Using effective analogs in biological assays, we intend to verify the suitability of AfNADase as a target for the treatment of aspergilloses and, potentially, other fungal infections.