D'Amico, Raffaele (2025) Structure-function relationships of bacterial glycans in biofilm-associated infections: LPS remodelling in Pseudomonas aeruginosa and antibiofilm strategies from Psychrobacter sp. TAE2020. [Tesi di dottorato]

Documento PDF PhD_thesis_DAmico_Raffaele.pdf Download (4MB)

Abstract

This project investigated extracellular glycans produced by different bacterial species to understand how their structures influence physiological adaptations and biological activities, including antimicrobial resistance, immunomodulation, and potential biotechnological applications. Two complementary research lines were pursued: (i) the characterization of extracellular glycans, particularly lipopolysaccharides (LPS), from Pseudomonas aeruginosa strains isolated from cystic fibrosis (CF) patients, to assess how structural variations contribute to antibiotic resistance and inflammation; and (ii) the analysis of surface-associated glycans from the non-pathogenic Antarctic bacterium Psychrobacter sp. TAE2020 as a source of molecules with antibiofilm activity and potential nanocarrier applications. For P. aeruginosa, six clinical isolates (WT2, WT4, MDR1, MDR5, PDR5, PDR7) and the reference strain PA14 were analyzed under planktonic and biofilm conditions reproducing key features of the CF lung environment. Structural analyses of LPS and lipid A by DOC-PAGE, NMR, HR-MAS NMR, GC-MS, MALDI-TOF, and ESI-MS/MS revealed a conserved lipid A backbone undergoing strain- and condition-specific remodelling, with increased acyl heterogeneity in biofilm-grown and multidrug-resistant strains. Functional assays showed that extracellular molecules produced under biofilm conditions, particularly from WT4 and PDR7, triggered stronger activation of TLR-4, NF-κB, and COL1A1, key mediators of the persistent inflammatory and fibrotic processes typical of CF lungs. qRT-PCR, ELISA, and Western blot analyses confirmed elevated expression of IL-6, TLR-4, and COL1A1 together with NF-κB activation, especially in lipid A and LPS from biofilm-derived PDR7, without inducing cytotoxicity. These findings indicate that specific lipid A remodelling events contribute to sustained inflammatory and fibrotic responses, promoting infection persistence and antibiotic tolerance. The second research line focused on extracellular glycans from Psychrobacter sp. TAE2020. Psychrobacter sp. TAE2020 is a Gram-negative bacterium that produces extracellular membrane vesicles (EMVs) and a bioactive complex named CATASAN. The capsular polysaccharide (CPS), the major bioactive component of the CATASAN complex, together with LPS and the outer membrane protein PsyOmp38, shows an unusual tetrasaccharide repeating unit composed of α-D-galactosamine, 2,4-diacetamido-2,4,6-trideoxy-glucose (α-D-bacillosamine), and the rare α-L-gulosamine. This amino sugar-rich composition confers distinctive physicochemical properties, including strong antibiofilm and emulsifying activities. These properties were further enhanced when CPS was combined with LPS or used to coat liposomes, while depolymerization completely abolished its activity, highlighting the requirement for structural integrity. Confocal laser scanning microscopy and COMSTAT analysis, a software tool for the quantitative evaluation of three-dimensional biofilm structures, showed a marked reduction in biofilm biomass and disruption of biofilm architecture without cytotoxic effects. SAXS and ζ-potential analyses confirmed CPS adhesion to lipid membranes, forming multilayer coatings with physicochemical features suitable for nanocarrier development. LPS from Psychrobacter sp. TAE2020 exhibited a rough-type profile containing rhamnose, glucose, glucosamine, and Kdo, with lipid A variably acylated by C10:0, C12:0(3-OH), and C14:0(3-OH). Overall, this work highlights clear structure-function relationships within extracellular glycans: in P. aeruginosa, lipid A remodelling modulates inflammatory and fibrotic responses associated with chronic infection, whereas in Psychrobacter sp. TAE2020, amino sugar-rich extracellular glycans emerge as multifunctional biopolymers with antibiofilm, emulsifying, and nanocarrier potential.

Downloads

Actions (login required)

Modifica documento