Summary: The interaction of bacterial exopolysaccharides, produced by opportunistic lung pathogens, with antimicrobial peptides of the innate primate immune system was investigated. The exopolysaccharides were produced by Pseudomonas aeruginosa, Inquilinus limosus and clinical isolates of the Burkholderia cepacia complex, bacteria that are all involved in lung infections of cystic fibrosis patients. The effects of the biological activities of three orthologous cathelicidins from Homo sapiens sapiens, Pongo pygmaeus (orangutan) and Presbitys obscurus (dusky leaf monkey) were examined. Inhibition of the antimicrobial activity of peptides was assessed using minimum inhibitory concentration assays on a reference Escherichia coli strain in the presence and absence of exopolysaccharides, whereas complex formation between peptides and exopolysaccharides was investigated by means of circular dichroism, fluorescence spectroscopy and atomic force microscopy. Biological assays revealed that the higher the negative charge of exopolysaccharides the stronger was their inhibiting effect. Spectroscopic studies indicated the formation of molecular complexes of varying stability between peptides and exopolysaccharides, explaining the inhibition. Atomic force microscopy provided a direct visualization of the molecular complexes. A model is proposed where peptides with an α‐helical conformation interact with exopolysaccharides through electrostatic and other non‐covalent interactions.

In the last years, outer membrane vesicles have attracted a lot of attention for the development of vaccines against bacterial pathogens. Extracellular vesicles can be obtained in high yields by genetic mutations, resulting in generalized modules for membrane antigens (GMMA). Methods to check the quality, consistency of production, and stability of GMMA vaccines are of fundamental importance. In this context, analytical methods for size distribution determination and verifying the integrity and possible aggregation of GMMA particles are strongly needed. Herein, GMMA particle size distribution has been evaluated by means of three different techniques. Dynamic light scattering (DLS), multiangle light scattering (MALS) coupled with high-performance liquid chromatography–size exclusion chromatography (SEC), and nanoparticle tracking analysis (NTA) have been compared to characterize GMMA from different mutants of Salmonella typhimurium and Salmonella enteritidis strains. We found that the presence of O-antigen chains on GMMA determined higher Z-average diameters by DLS compared to size estimation by MALS and that the hydrodynamic diameter increased with the number of O-antigen chains per GMMA particle. In the case of SEC-MALS, the size of the whole population better reflects the size of the most abundant particles, whereas DLS diameter is more influenced by the presence of larger particles in the sample. SEC-MALS and NTA are preferable to DLS for the analysis of bimodal samples, as they better distinguish populations of different size. MALS coupled to a size exclusion chromatography module also allows checking the purity of GMMA preparations, allowing determination of generally occurring contaminants such as soluble proteins and DNA. NTA permits real-time visualization with simultaneous tracking and counting of individual particles, but it is deeply dependent on the choice of data analysis parameters. All of the three techniques have provided complementary information leading to a more complete characterization of GMMA particles.

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1–35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation.

Biofilms are complex systems produced by bacteria and constituted by macromolecular matrix embedding cells. They provide advantages to bacteria including protection against antimicrobials. The protection given by biofilms produced by Klebsiella pneumoniae strains towards antimicrobial peptides of the innate immune system was investigated. In particular, the role of matrix bacterial exopolysaccharides was explored. Three clinical strains producing exopolysaccharides with different chemistry were selected and the interaction of purified biofilm polysaccharides with two bovine cathelicidins was studied by circular dichroism spectroscopy and microbiological assays to establish their influence on the peptide’s antimicrobial activity. The spectroscopic data indicated a different extent of interaction with the two peptides, in a manner dependent on their sugar composition, and in particular the presence of rhamnose residues correlated with a lower interaction. The extent of interaction was then related to the protection towards antimicrobial peptides, conferred by the addition of the different exopolysaccharides, in minimum inhibitory concentration (MIC) assays against a reference Escherichia coli strain. Microbiological results were in very good agreement with spectroscopic data, confirming the active role of matrix polysaccharides in determining a biofilm’s protective capacity and indicating lower protection levels afforded by rhamnose containing exopolysaccharides.