Abstract
This PhD thesis describes the structural, biophysical, and functional characterizations of proteins involved in siderophore-mediated iron uptake in Erwinia amylovora, the causal agent of fire blight in apple and pear. South Tyrol, with its largest apple orchards (18,538 hectares) in Europe, contributes to up to 50% of Italy's apple production, 15% of Europe's, and 2% of the world's apple production. One of the major challenges in apple cultivation is the bacterial infestation in the pre-harvest stage. This problem becomes particularly severe when the pathogen has a history of causing outbreaks and lacks effective chemical control measures. E. amylovora, a destructive pathogen, fits this category and poses a potential threat to apple production. As the use of antibiotics is forbidden in Italy and the current protocols of chemical controls are not efficient, it is crucial to develop novel chemical control against fire blight. For this, studying virulence-related proteins at the structural level is essential. E. amylovora primarily relies on three pathogenicity factors; amylovoran, the Type III secretion system (T3SS), and siderophore-mediated iron uptake (Chapter 1) This work primarily focuses on the siderophore iron uptake pathway, selecting two target proteins, ViuB (the oxidoreductase) and FhuD (periplasmic binding protein), which are exclusively present in Erwinia species infecting rosaceous plants. The methods for obtaining ViuB, from expression to crystallization and subcloning of FhuD are comprehensively described in (Chapter 2). ViuB, being a soluble cytoplasmatic protein, was easier to purify. However, crystallization posed a significant challenge. After numerous unsuccessful crystallization trials at UNIBZ, biophysical characterizations and crystallization trials for ViuB was performed at three different laboratories across Europe; BIOCEV, Prague, Robotein at University of Liege, and EMBL in Hamburg (Chapter 3). Additionally, nanobodies targeting the full-length ViuB protein were produced to stabilize ViuB by binding to its epitopes present in the disordered regions. (Chapter 4). After several optimizations, particularly adjusting the crystallization temperature and removing tags, ViuB crystals were formed using the Classic and Morpheus screens. Notably, crystals only grew at 4°C. Attempts to incubate ViuB without the His-tag at 20°C with the same batch of protein did not result in crystal formation. The ViuB structure was solved by Small-angle X-ray scattering (SAXS) and X-Ray crystallography at 2.3 Å (Chapter 5). For another target, the periplasmic binding protein FhuD, soluble protein was not obtained when cloned in vector pMCSG49 and expressed in the cytoplasm of E. coli cells, leading to its subcloning into two EMBL vectors (pETM-41 and pETM-50). pETM-50 encodes His-tagged DsbA, which leads to periplasmic expression, whereas pETM-41 houses a His-tagged MBP solubility tag, increasing solubility in the cytoplasm. Given the crucial role of FhuD in iron uptake, modeling and bioinformatics analysis were performed with its homologs and orthologs proteins to compare the ligand binding pockets (Chapter 6). Overall, the PhD work adhered strictly to the initial plan, successfully solving the structure of ViuB by X-Ray crystallography. The structure could aid in designing the inhibitors to disrupt bacterial iron utilization, providing a foundation for developing sustainable chemical controls against fire blight. Determining the structure of ViuB with its substrate bound in the active site and performing the enzymatic assays could be the future perspective of this work.