| Popis: |
Pili are long and flexible proteinaceous filaments that project outwards from bacterial cell walls and are found both in Gram-negative and Gram-positive strains. These appendages play major roles in promoting adhesion of the bacteria to other bacteria, host tissue and abiotic substrates, thereby leading to host colonization and biofilm formation. Knowledge of the molecular mechanisms of pili-mediated adhesion is critical to our understanding of their functional roles, and offers the possibility of controlling the adhesion of pathogens and probiotics. While much progress has been made in the characterization of pili from Gram-negative bacteria, especially the pathogenic strains, the adhesive and mechanical properties of Gram-positive bacterial pili remain relatively unknown.The aim of this thesis was to gain detailed insights into the nanoscale surface properties of the clinically and commercially important probiotic bacterium Lactobacillus rhamnosus GG (LGG), with emphasis on cell surface pili. The methodology to reach this goal involved the improvement and use of advanced atomic force microscopy (AFM) methods, including bacterial sample preparation (pili and cell immobilization), AFM cantilever functionalization (with biomolecules or bacteria), preparation of chemically-modified model surfaces and imaging and force probing single-molecules and single-cells.Using these nanotechniques, we first showed that AFM is a powerful tool to image the nanoscale structure, organization and assembly of LGG pili. AFM images revealed intact pili on cells and also star-like assemblies formed by detached pili fragments. We demonstrated a simple two-step centrifugation procedure to separate large amounts of pili from cells, even though through their synthesis the pili are covalently anchored to the cell wall. We also found that the centrifuged pili assemble as long bundles, which we believe originate from a complex interplay of mechanical effects and biomolecular interactions involving the SpaC adhesin of LGG pili.Next, single-molecule force spectroscopy (SMFS) enabled us to unravel the binding mechanism of pili at the single-molecule level. The results showed that SpaC is a multifunctional adhesin with broad specificity. SpaC forms homophilic trans-interactions engaged in bacterial aggregation, and specifically binds mucin and collagen, two major extracellular components of host epithelial layers. Also, pulling experiments on living bacteria demonstrated that LGG pili exhibit two unique mechanical responses, i.e. zipper-like adhesion involving sequential binding between SpaC subunits, and nanospring properties that manifest as a change in stiffness in response to increasing force. These mechanical properties may represent a generic mechanism among Gram-positive bacterial pili for strengthening adhesion and withstanding shear stress in the natural environment.Finally, we used single-cell force spectroscopy (SCFS) to quantify the forces guiding the adhesion of whole LGG bacteria to hydrophobic surfaces, mucin and human epithelial (Caco-2) cells. On hydrophobic surfaces and mucin, bacterial pili were shown to strengthen adhesion through nanospring properties. This nanospring type mechanical response was no longer observed on Caco-2 cells. Rather, the force curves exhibited constant force plateaus with extended rupture lengths reflecting the extraction of membrane nanotethers from the Caco-2 cells.All together, our single-molecule and single-cell analyses provide novel insights into the molecular mechanisms by which piliated bacteria colonize surfaces, and offer exciting avenues in nanomedicine for understanding and controlling the adhesion of microbial cells of probiotic or pathogenic nature. status: published |
