In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.

Peptidoglycans provide bacterial cell walls with mechanical strength. The spatial organization of peptidoglycan has previously been difficult to study. Here, atomic force microscopy, together with cells carrying mutations in cell-wall polysaccharides, has allowed an in-depth study of these molecules.

Although teichoic acids are major constituents of bacterial cell walls, little is known about the relationships between their spatial localization and their functional roles. Here, we used single-molecule atomic force microscopy (AFM) combined with fluorescence microscopy to image the distribution of wall teichoic acids (WTAs) in Lactobacillus plantarum, in relation with their physiological roles. Phenotype analysis of the wild-type strain and of mutant strains deficient for the synthesis of WTAs (ΔtagO) or cell wall polysaccharides (Δcps1-4) revealed that WTAs are required for proper cell elongation and cell division. Nanoscale imaging by AFM showed that strains expressing WTAs have a highly polarized surface morphology, the poles being much smoother than the side walls. AFM and fluorescence imaging with specific lectin probes demonstrated that the polarized surface structure correlates with a heterogeneous distribution of WTAs, the latter being absent from the surface of the poles. These observations indicate that the polarized distribution of WTAs in L. plantarum plays a key role in controlling cell morphogenesis (surface roughness, cell shape, elongation, and division).

The ability of Staphylococcus aureus to colonize the human nares is a crucial prerequisite for disease. IsdA is a major S. aureus surface protein that is expressed during human infection and required for nasal colonization and survival on human skin. In this work, we show that IsdA binds to involucrin, loricrin, and cytokeratin K10, proteins that are present in the cornified envelope of human desquamated epithelial cells. To measure the forces and dynamics of the interaction between IsdA and loricrin (the most abundant protein of the cornified envelope), single-molecule force spectroscopy was used, demonstrating high-specificity binding. IsdA acts as a cellular adhesin to the human ligands, promoting whole-cell binding to immobilized proteins, even in the absence of other S. aureus components (as shown by heterologous expression in Lactococcus lactis).Inhibition experiments revealed the binding of the human ligands to the same IsdA region. This region was mapped to the NEAT domain of IsdA. The NEAT domain also was found to be required for S. aureus whole-cell binding to the ligands as well as to human nasal cells. Thus, IsdA is an important adhesin to human ligands, which predominate in its primary ecological niche.

Oligomers of β-amino acids ("β-peptides") can be designed to fold into stable helices that display side chains with a diverse range of chemical functionality in precise arrangements. We sought to determine whether the predictable, three-dimensional side-chain patterns generated by β-peptides could be used in combination with single-molecule force spectroscopy to quantify how changes in nanometer-scale chemical patterns affect intermolecular interactions. To this end, we synthesized β-peptides that were designed to be either globally amphiphilic (GA), i.e., display a global segregation of side chains bearing hydrophobic and cationic functional groups, or non-globally amphiphilic (iso-GA), i.e., display a more uniform distribution of hydrophobic and cationic functional groups in three-dimensions. Single-molecule force measurements of β-peptide interactions with hydrophobic surfaces through aqueous solution (triethanolamine buffer, pH 7.2) reveal that the GA and iso-GA isomers give rise to qualitatively different adhesion force histograms. The data are consistent with the display of a substantial nonpolar domain by the GA oligomer, which leads to strong hydrophobic interactions, and the absence of a comparable domain on the iso-GA oligomer. This interpretation is supported by force measurements in the presence of methanol, which is known to disrupt hydrophobic interactions. Our ability to associate changes in measured forces with changes in three-dimensional chemical nanopatterns projected from conformationally stable β-peptide helices highlights a contrast between this system and conventional peptides (α-amino acid residues): conventional peptides are more conformationally flexible, which leads to uncertainty in the three-dimensional nanoscopic chemical patterns that underlie measured forces. Overall, we conclude that β-peptide oligomers provide a versatile platform for quantifying intermolecular interactions that arise from specific functional group nanopatterns.