Marseglia, Angela (2024) Analysis of the molecular basis behind the recognition mechanisms of prokaryotic glycans by the host immune system. [Tesi di dottorato]

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Abstract

The present PhD project, funded by PON Ricerca e Innovazione 2014 – 2020, Azione I.1 “Dottorati Innovativi con Caratterizzazione Industriale”, aims to investigate the recognition mechanisms underlying host-guest interactions. These interactions are fundamental molecular relationships that underpin many essential processes within living organisms, especially at eukaryotes-bacteria interface. The interactions between microbial guest and human host can be pathogenic or symbiotic, including, as examples, the recognition of microbial pathogens by the host immune system or their beneficial interaction with the gut microbiota, a collection of microorganisms which colonizes the human gastrointestinal tract, developing a mutually beneficial relationship with the host. On the microbe side, these interactions are mediated by complex glycans exposed on bacterial cells, termed Microbe Associated Molecular Patterns (MAMPs). These are recognized by specific host receptors, known as Pathogen Recognition Receptors (PRRs), that, as part of the innate immune system, detect potentially harmful pathogens, triggering an inflammatory reaction. However, some of them may also be exploited by bacteria to escape immune responses; several feared pathogens have indeed developed the capability to cover their surfaces with glycans mimicking eukaryotic Self Associated Molecular Patterns (SAMPs) structures which can interact with inhibitory host receptors, thereby eluding host immune responses and facilitating the infection. Over the course of the PhD, the molecular processes governing host-guest interactions have been unraveled, focusing on the comprehensive analysis of molecular recognition events. Bacterial glycans including capsular polysaccharides (CPS), as the one extracted and characterized by an Acinetobacter baumannii clinical isolate, and lipopolysaccharides (LPS), like those isolated from Bacteroides vulgatus and Escherichia coli, have been studied in their interactions with human proteins. A special focus has been pointed out on the C-type lectin DC-SIGN, expressed and purified during my secondment at Institut de Biologie Structurale, Grenoble, France. Additionally, interactions of eukaryotic N- and O-glycans with bacterial adhesins, such as P110 and P40/P90 from Mycoplasma genitalium and Mycoplasma pneumoniae, respectively, have been investigated. These interactions were explored using different wet lab procedures, NMR spectroscopy, Mass Spectrometry and other different biophysical techniques like Fluorescence and Surface Plasmon Resonance together with computational approaches. In vivo studies, conducted at GSK – Siena, Italy, allowed to evaluate the immunogenic activity of characterized CPS on mice. Understanding the molecular mechanisms of microbial glycans recognition is critical for designing glycomimetics capable of modulating the function of host immune system proteins. Moreover, unraveling the molecular details of these interactions can provide crucial insights for the development of novel therapeutic strategies and vaccines essential for combating microbial infections.

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