Analysis of Homotypic Interactions of Lactococcus lactis Pili Using Single-Cell Force Spectroscopy

Cell surface proteins of Gram-positive bacteria play crucial roles in their adhesion to abiotic and biotic surfaces. Pili are long and flexible proteinaceous filaments known to enhance bacterial initial adhesion. They promote surface colonization and are thus considered as essential factors in biofilm cohesion. Our hypothesis is that pili mediate interactions between cells and may thereby directly affect biofilm formation. In this study, we use single-cell force spectroscopy (SCFS) to quantify the force of the homotypic pili interactions between individual bacterial cells, using different Lactococcus lactis strains producing pili or not as model bacteria. Moreover the force-distance curves were analyzed to determine the physical and nanomechanical properties of L. lactis pili. The results for pili-devoided strains showed a weak adhesion between cells (adhesion forces and work in the range of 100 pN and 7 × 10^-18 J, respectively). On the contrary, the piliated strains showed high adhesion levels with adhesion forces and adhesion work over 200 pN and 50 × 10^-18 J, respectively. The force-extension curves showed multiple adhesion events, typical of the unfolding of macromolecules. These unfolding force peaks were fitted using the physical worm-like chain model to get fundamental knowledge on the pili nanomechanical properties. In addition, SCFS applied to a L. lactis isolate expressing both pili and mucus-binding protein at its surface and two derivative mutants revealed the capacity of pili to interact with other surface proteins including mucus-binding proteins. This study demonstrates that pili are involved in L. lactis homotypic interactions and thus can influence biofilm structuring.