Functional Studies Of Virulence-associated Proteins LeuB And MrsA In Aspergillus Fumigatus That Regulate Intracellular Iron Ion Balance

Over the years, with increases of immunosuppressed populations, the incidence of invasive fungal infection has risen simultaneously. To date, the widely concerned pathogenic fungi are Candida albicans, Cryptococcus and Aspergillus fumigatus.Among them, A. fumigatus is the most prevalent pathogen that responsible for invasive aspergillosis (IA). A. fumigatus is a wide spread saprophytic fungus with a large number of spores that distributed everywhere in the environment. Due to small diameter, the spores of A. fumigatus are easily inhaled by humans. For the healthy populations, the inhaled spores are been killed and eliminated timely by the immune system of host. However, for the immunocompromised individuals, if the inhaled spores can’t be eliminated by the host, the escaped spores are able to develop aspergillosis. At present, the primary used clinical antifungal drugs are itraconazole,voriconazole, amphotericin B and caspofungin. Nevertheless, many limitations of them have discovered: amphotericin B has bigger side effects on the host; the antifungal spectrum of caspofungin is relatively narrow and the widely used itraconazole and voriconazole is easy to develop azole resistance. Therefore, to develop a new drug with strong antifungal activity, wide antifungal spectrum and small side effects on host is a urgent issue and the most important point is to obtain a suitable drug target.Iron as an essential element is required for many biological processes. Because the host always tightly sequesters the available iron by hemoglobin and lactoferrin,the ability to obtain iron from the host is crucial for the virulence of A. fumigatus.Consequently, genes that related to iron regulation in pathogenic fungus frequently function as virulence factor, however, the mechanism of iron regulation in A.fumigatus is still limited。In this thesis, the function of LeuB and MrsA that involved in iron regulation and virulence of A. fumigatus are systematically studied using the method such as gene deletion and so on. The conclusion we obtained are as follows:1. We demonstrate that the transcript factor LeuB not only involved in leucine biosynthesis regulation, but also in iron acquisition. Deletion of leuB results in decreased gene transcription related to siderophore (sidG, sitl and mirB) and TAFC production. Consequently, ΔleuB exhibited severely growth defect under iron starvation. Double deletion of leuB and the major transcript factor hapX that stimulateiron uptake would exacerbates the growth defect of ΔleuB, while double deletion of leuB and the major transcript factor sreA that repress iron uptake would recover such defect of AleuB. Those results indicate that leucine biosynthesis transcription factor LeuB is essential for iron regulation.2. In the condition of iron depletion, leuB deficiency would lead to decreases of iron acquisition, which would results in decreased transcription of leuA, and thus inhibit leucine biosynthesis. The inhibition of leucine biosynthesis would lead to global protein degradation which was caused by proteasome with increased activity. Further,we found that the DNA binding domain (Zn(II)2Cys6) and C terminal of LeuB is required for the growth and protein stability of A. fumigatus under iron starvation.3. Here, we show that the mitochondrial iron transporter MrsA is critical for adaptation to iron-limited or iron-excess conditions in A. fumigatus. Deletion of mrsA leads to disruption of iron homeostasis with a decreased sreA expression, resulted in increased transcription of iron acquisition related genes that repressed by SreA, such as sidA, mirB, ftrA and fetC. Those results suggest that MrsA is required for the cellular iron homostasis of A. fumigatus.4. Further study shows that deletion of mrsA induces hyper-susceptibility to oxidative stresses and azole drugs which was resulted by abnormal ROS accumulation.Moreover, site-directed mutation experiments verified that three conserved histidine residues related to iron transport in MrsA are required for responses to oxidative and azole stresses.5. Importantly, leuB and mrsA deficiency attenuated the virulence of A. fumigatus in an immunocompromised murine model of aspergillosis.In conclusion, this study not only illustrates the mechanism of LeuB and MrsA-involved iron regulation, but also demonstrates that both LeuB and MrsA are essential for the virulence of A. fumigatus. This study provides a theoretical support for antifungal therapy in the future.