Inibizione dell’acquisizione del ferro da parte di Staphylococcus aureus: un doppio approccio avente come bersaglio proteine coinvolte nei pathways mediati da emofori e siderofori

In the upcoming decades, one of the biggest risks to public health will be antimicrobial resistance (AMR), with Staphylococcus aureus being one of the most threatening multidrug resistant pathogens. Indeed, in 2017 the World Health Organization (WHO) drew up a Global Priority Pathogen list for antibiotic-resistant bacteria, indicating S. aureus methicillin and vancomycin resistant variants as a “high priority”. Furthermore, during the COVID-19 pandemic, the battle against AMR had faded into the background, contributing even more to its strengthening and diffusion. Iron acquisition has been demonstrated as a necessity for S. aureus pathogenicity in human infections. Indeed, the human host has evolved a defense mechanism, called "nutritional immunity", that regulates iron trafficking in an effort to lessen free iron toxicity and inhibit microbial growth. In turn, S. aureus has developed different strategies to overcome the host nutritional immunity, as the hemophore and siderophore-mediated systems for iron retrieval. The former bacterial strategy involves the Iron-regulated Surface Determinant (Isd) system enabling iron acquisition from the host hemoglobin (Hb), while the latter consists of small iron chelators that retrieve the iron bound to host proteins in the extracellular compartment. The Isd system consists of nine proteins, called hemophores, of which IsdB and IsdH are found on the bacterium surface and are involved in Hb binding and in the extraction and transfer of the heme to the downstream Isd effectors. In this context, only IsdB out of the two surface-exposed hemophores has been identified as an S. aureus virulence factor, essential for the bacterium pathogenesis. Despite different efforts have been reported in literature to develop antibacterials or vaccines targeting IsdB, no product has become commercially available yet, leaving ground for strategy improvements. At the same time, S. aureus has also evolved strategies for the retrieval of inorganic iron from the extracellular media of the host, mainly involving Staphyloferrin A (SA) and Staphyloferrin B (SB) siderophores. Both siderophores have been identified as S. aureus virulence factors, but only SB has been reported to be produced by the most invasive strains, and thus considered to be the greatest support of virulence. Within the SB biosynthetic pathway, the first step is catalysed by SbnA which, in concerted action with SbnB-catalysed reaction, produces two out of three building blocks needed for SB synthesis. The mutation of either sbnA or sbnB genes resulted in the abrogation of SB production, highlighting the key role of the two enzymes. A complete structural and functional characterization of SbnA has not been reported in literature yet, and no strategies have been described targeting the enzyme. In this context, the characterization of SbnA structural and functional properties and the identification of potential inhibitors would provide a potential combined strategy for the development of new antimicrobials targeting S. aureus iron acquisition pathways. Within the PhD project, an in-house developed immunoassay was exploited as a platform for the evaluation of molecules that interfere with IsdB-Hb complex formation. Notably, three commercial compounds, which had been previously identified, were re-synthesized by Prof. Lazzarato group (University of Turin) and evaluated in this thesis by the immunoassay. The group of Prof. Lazzarato synthesized also different analogues for all three compounds, designed to alleviate the solubility and/or stability issues that were previously encountered. No significant improvements in terms of potency were highlighted by the immunoassay when comparing the derivatives to their parent compounds, therefore further modifications will be required. The thesis project also encompassed the optimization of the conditions for the recombinant expression in E. coli and the purification of SaSbnA. The purified protein was characterized in its functional and regulatory properties by spectroscopic techniques, as UV-Vis and fluorescence spectrophotometry, as well as by an ad hoc optimized activity assay. Notably, the optimization of a continuous activity assay allowed to confirm that SaSbnA uses a ping-pong kinetic mechanism with substrate inhibition by L-glutamate. After the enzyme characterization, three assays were set up and validated for the evaluation of potential inhibitors of SaSbnA activity. The assays’ optimization allowed the assessment of both the effect of potential inhibitors on SaSbnA activity and their direct binding to the enzyme. For the activity evaluation, a discontinuous assay was optimized to provide a high-throughput evaluation of a broad number of molecules to have a first selection for compounds with a significative inhibitory effect. Subsequently, the optimized continuous assay was exploited for the characterization of the mechanism of action of the active molecules, with the determination of inhibition constants. At last, the binding of the active molecules to SaSbnA was evaluated by fluorescence spectroscopy. The defined testing platform was then exploited to study the effect of intermediates of the SB pathway on SaSbnA activity to potentially identify feedback mechanisms. Among the intermediates, citrate was analysed more carefully since previous studies highlighted its inhibitory effect on two other enzymes of the SB pathway, and its presence was identified in a SaSbnA structure (PDB ID 5D85). Indeed, the intermediate was identified as an active inhibitor, highlighting a more complex regulatory mechanism within the SB pathway. At last, the assays were exploited to test in vitro the first set of molecules, with the identification of one hit inhibitor, which was then further characterized, evaluating its mechanism of action, and measuring inhibition constants. A first derivative of the lead compound was synthesized by collaborators at the University of Turin and was tested using the same platform, highlighting a more potent molecule. The project also involved the production of a second construct for SaSbnA expression, which was designed for high throughput screening (HTS) experiments due to its improved yields. The purified protein was then exploited in an HTS of compound libraries to find potential inhibitors of SaSbnA activity. The discontinuous activity assay was optimized for HTS conditions and then exploited to screen compound libraries made available by the CEA Paris-Saclay centre. The HTS identified seven molecules that inhibited the activity of SaSbnA by 50% or more at 50 µM concentration. These selected hits were confirmed by further analyses and a preliminary characterization allowed to identify the best compounds for further tests and improvements.