Deciphering the nanometer-scale organization and assembly of Lactobacillus rhamnosus GG pili using atomic force microscopy.

In living cells, sophisticated functional interfaces are generated through the self-assembly of bioactive building blocks. Prominent examples of such biofunctional surfaces are bacterial nanostructures referred to as pili. Although these proteinaceous filaments exhibit remarkable structure and functions, their potential to design bioinspired self-assembled systems has been overlooked. Here, we used atomic force microscopy (AFM) to explore the supramolecular organization and self-assembly of pili from the Gram-positive probiotic bacterium Lactobacillus rhamnosus GG (LGG). High-resolution AFM imaging of cell preparations adsorbed on mica revealed pili not only all around the cells, but also in the form of remarkable star-like structures assembled on the mica surface. Next, we showed that two-step centrifugation is a simple procedure to separate large amounts of pili, even though through their synthesis they are covalently anchored to the cell wall. We also found that the centrifuged pili assemble as long bundles. We suggest that these bundles originate from a complex interplay of mechanical effects (centrifugal force) and biomolecular interactions involving the SpaC cell adhesion pilin subunit (lectin-glycan bonds, hydrophobic bonds). Supporting this view, we found that pili isolated from an LGG mutant lacking hydrophilic exopolysaccharides show an increased tendency to form tight bundles. These experiments demonstrate that AFM is a powerful platform for visualizing individual pili on bacterial surfaces and for unravelling their two-dimensional assembly on solid surfaces. Our data suggest that bacterial pili may provide a generic approach in nanobiotechnology for elaborating functional supramolecular interfaces assembled from bioactive building blocks.