Cané, Carolina (2024) Investigation of new antimicrobial compounds: identification of bacterial protein targets. [Tesi di dottorato]

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Abstract

The rapid emergence of resistant bacteria is occurring worldwide, compromising the efficacy of antibiotic-based therapies. In this context, the interest is focused on the development of new alternative therapeutic strategies effective against both Gram-positive and Gram-negative bacteria. The phenomenon of resistance is closely related to the mechanism of action of antimicrobial agents, and the identification of new protein targets involved in vital bacterial functions is central to the search for new pharmacological agents to fight resistant bacteria. Proteomics is a powerful tool to understand which biological functions are altered by an antimicrobial agent, with the goal of identifying key target proteins that might be addressed by new and more effective drugs to overcome the dramatic increase in MDR bacterial strains. In these perspectives, the present Ph.D. project has been focused to the investigation of the molecular mechanisms of AMPs and NPs using targeted and untargeted proteomics approaches to identify novel protein targets in the prokaryotic system. In chapter two, in vitro and ex-vivo strategies were employed to characterize the binding of Mag-2 to its target protein BamA in E. coli. BamA belongs to the Bam complex, previously identified as putative interactor of Mag-2 by functional proteomics. The recombinant form of BamA was expressed in E. coli, purified, and characterized; physico-chemical methodologies were then applied to demonstrate the peptide/protein interaction in vitro. Subsequently, the effective impairment of OMPs folding in the presence of Mag-2 was investigated using biochemical techniques and a targeted proteomic approach. Chapter three focused on evaluating the effect of Temp-L on S. aureus, a Gram-positive bacterium, one of the world's leading human pathogens. Previous morphological studies and differential proteomic approaches had suggested that Temp-L induces bacterial vesicle formation as a possible protective mechanism and impairs the expression of virulence factors. These hypotheses were confirmed by biological assays and spectroscopic analyses, suggesting that targeting virulence factors might be considered a novel strategy to replace conventional antimicrobial agents that can be used to treat the infections caused by the resistant pathogen S. aureus. In Chapter four, a comparative analysis of the inhibitory action of berberine derivatives on the recombinant FtsZ protein of E. coli has been performed. Medicinal plants belonging to the genus Berberis are considered an interesting source of drugs to counteract the MDR. Their important properties are due to the antimicrobial properties of berberine, which interacts with FtsZ protein of E. coli. It is a tubulin-like protein belonging to the cell division machinery that represents the most widely studied bacterial target for novel drug development. This knowledge led to the synthesis of new simplified analogues of berberine in an attempt to maximize their interaction with FtsZ. The recombinant form of FtsZ was expressed in E. coli, purified and characterized to study the inhibitory capabilities, both in silico and in vitro, of berberine derivatives on FtsZ. Lastly, in the Chapter five, the mechanism of action of Esc(1-21) diastereoisomer, named Esc(1-21)-1c, on P. aeruginosa PAO1 has been investigated. Differential proteomic and transcriptional analyses were performed to elucidate the impact of Esc(1-21)-1c on the expression profile of P. aeruginosa, revealing a reduction in the production of the MexAB-OprM efflux pump. Furthermore, LC-MS/MS in MRM scan mode validated the proteomic findings. The precise mechanism of action of Esc(1-21)-1c on P. aeruginosa was explored by functional proteomics, leading to the identification of a transcriptional regulator responsible for activating the mexAB-oprM operon.

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