Titolo della tesi: Proteins of Aspergillus fumigatus: their role in human diseases and their modulation as a therapeutic strategy for aspergillosis
Aspergillus fumigatus is an opportunistic human fungal pathogen that causes life-threatening disease in immunocompromised individuals. It is estimated that A. fumigatus may be responsible for over 600,000 deaths yearly, with a 25-90% mortality rate. Invasive aspergillosis commonly affects the lungs but can also disseminate to the central nervous system, increasing the mortality rate from 58% to 88%. It is known that astrocytes play a central role in regulating blood-brain barrier permeability and in the neuro repair process. It has been demonstrated that culture supernatants of A. fumigatus damage astrocytes. A. fumigatus produce extracellular proteins to adapt to different environments and to improve their arsenal of defenses against the host. Extracellular proteins of A. fumigatus, such as hexokinase, glucokinase, alcohol dehydrogenase, ribonucleases, chitinases, and superoxide dismutase, play a critical role in fungal-host interactions and pathogenesis.
This study has aimed to evaluate the virulence of extracellular proteins and their modulation using epigenetic modulators to identify new therapeutic strategies for aspergillosis.
In the first part of the study, the activity of A. fumigatus extracellular proteins on astrocytes has been analyzed. To date, central nervous system effects caused by exposure to A. fumigatus extracellular proteins have been poorly investigated. Our results have shown that exposure of astrocytes to extracellular proteins of A. fumigatus has led to IKKβ phosphorylation (p <0.001). It is known that IKKβ phosphorylation induces NF-κB translocation to the nucleus. NF-κB is a transcription factor that controls numerous physiological functions and is involved in various diseases' pathogenesis. In the nervous system, translocation to the nucleus NF-κB in glial and immune cells mediates pathological inflammatory processes. In this study, the translocation into the nucleus was observed by immunocytochemistry of NF-κB p100 and NF-κB p65 (p <0.001). NF-κB binds to its cognate DNA sequences and its coactivators to regulate gene expression ultimately. The results by immunocytochemistry have shown that exposure to extracellular proteins of A. fumigatus leads to IκB polyubiquitination and proteasomal degradation in astrocytes. Inhibitors of extracellular proteins implicated in the pathogenesis of aspergillosis could be a valuable aid for therapy.
In the second part of the study, the activity of epigenetic modulators on the modulation of A. fumigatus virulence and extracellular proteins has been evaluated. Bromo- and extra-terminal domain inhibitors, histone acetyltransferase, histone deacetylases inhibitors, demethylases, and hypermethylated inhibitors against A. fumigatus were tested. Extracellular proteins obtained after treatment of A. fumigatus with 100 μM of bromo- and extra-terminal domain inhibitor (JQ1) were 4.8 times lower than proteins obtained from cells without treatment. The results obtained on A. fumigatus virulence factors such as biofilm, hyphal growth, and melanin production showed that the bromo- and extra-terminal domain inhibitor JQ1 reduced biofilm formation and mature biofilm. JQ1 showed 55.2 % of inhibition of the radial growth rate (p <0.001). LC-MS/MS proteomic analysis of extracellular proteins identified 76 extracellular proteins. A comparison analysis on the variation in the relative abundance of proteins between the control group and the JQ1-treated group was established. Among them, 25 proteins were found to have significantly different abundances between the control group and the JQ1-treated group. The proteins that were dramatically downregulated in the JQ1-treated group included ribonuclease (Log2 fold change -0,58; p <0.01), chitinase (Log2 fold change -0,78; p <0.001), and superoxide dismutase (p <0.001), proteins involved in A. fumigatus virulence.
Moreover, with LC/MS analysis, a reduction of the intracellular protein Abr2 (p <0.001) has been demonstrated. The protein Abr2 is involved in the last step of the DHN-melanin formation pathway. Melanin is required for the correct assembly of the different layers of the conidial wall in A. fumigatus and, therefore, for the expression of adhesins and other virulence factors at the conidial surface. The reduction of A. fumigatus virulence after treatment with JQ1 has been demonstrated using Galleria mellonella larvae, a model for evaluating fungal virulence. The studies conducted aim to understand the effects of virulence factors but, above all, to identify new targets for the development of antifungals to fight infections without inducing resistance. The bromo- and extra-terminal domain inhibitors could represent new antifungal drugs. The use of epigenetic modulators in therapy allows to reduce virulence and therefore fight the disease without killing the microorganism and therefore without altering the microbiota.