Abstract
The aim of this study is to use different approaches, both in-silico and in-vitro, to characterize proteins involved in iron uptake systems in Aspergillus fumigatus. The in-silico approaches comprise of alignment analysis, transcriptomic analysis and in-silico folding, the in-vitro approaches comprise of protein expression and purification coupled with biophysics analysis. After an initial literature review on Aspergillus fumigatus (Reported in Chapter 1), a first exploratory analysis was performed on the pathogenicity genes of Aspergillus fumigatus. The main objectives were 1) to confirm the relevance of iron uptake genes, the focus of the project this PhD was part of, and 2) to explore the use of computational tools to identify interesting targets for the biochemical and biophysics experiments. This preliminary analysis is reported in Chapter 2 of this manuscript, with the identification of nutrients uptake genes, especially the ones involved in iron uptake, as interesting targets for downstream analysis. The two main approaches (in-silico and in-vitro) used in this thesis were then employed in two parallel projects during the entirety of the second year and the start of the third year, correlated by the focus on a specific step in the biosynthesis of siderophores in Aspergillus fumigatus. In Chapter 4, purification attempts were performed on SidL, and in Chapter 3, bioinformatic tools were used to try to identify SidL hypothetical companion protein. The biochemistry approach led to a result which complements and concludes the bioinformatic approach, and the two chapters are therefore presented to the reader in the most logical order, to build an easier to follow narrative. Chapter 3 reports the analysis of available mRNA-seq datasets to identify a hypothetical protein able to compensate for the activity of SidL. While this method proved to be ineffective, the project remained open in the laboratory, leading to the results in Chapter 4, and the understanding of mRNA-seq datasets analysis was one of the starting points of the work in Chapter 5. In Chapter 4, a purification protocol was established for SidL, using a denaturation/refolding strategy to extract the protein from the inclusion bodies, a common problem in protein expression. The purified protein was unstable and an investigation on the mRNA data indicated the sequence was wrongly annotated in online databases. In the meanwhile, AlphaFold2 was made available for use and the comparison of the predicted SidL structure (with the corrected aminoacidic sequence) and the experimental structure of SidF (another protein involved in the siderophore production) led to a new hypothesis. Since SidL and SidF presented very similar folding, we propose SidF may be able to compensate SidL, being the missing protein subject of Chapter 3 analysis. Interaction between SidF and different substrates was tested using Microscale Thermophoresis, showing the ability to act on SidL’s substrates. In the bigger picture of the development of this PhD project, the inconsistency of the annotation for SidL’s gene model was one of the starting point of the work on Chapter 5. In Chapter 5, the Fre protein family was analyzed using transcriptomic data to update the available gene models. The new sequences were used to obtain 3D models using AlphaFold2. The structural comparison allowed to identify a new Domain of Unknown Function. This new domain seems to be present in a subfamily of the Fre protein across different species. The differential expression of the proteins containing the new domain was analyzed to propose a possible function of the subfamily. A schematic representation of the content of this PhD thesis is presented in the next page, with the chapter presented in blue, the conclusion and results presented in red, and the key decisions that led to the use of different techniques or the start of new project in green.