Investigation of K1 bacterial capsular morphology and single molecules in capsular biosynthesis using super-resolution fluorescence microscopy (dSTORM)

Bacterial capsules are a virulence factor, especially in uropathogenic Escherichia coli (UPEC) that cause urinary tract infections (UTIs). The study of K1 capsule morphology and biosynthesis provide valuable information for the development of alternative treatments to problematic antibiotics. To resolve sub-diffraction limited details in the morphology of K1 bacterial capsule, direct stochastic optical reconstruction microscopy (dSTORM) with 50 nm lateral resolution was used to visualise the inhomogeneous thickness of the K1 bacterial capsules. The variation of capsule thicknesses (~49%) from polar and equatorial regions will affect bacterial adhesion and the infection of host cells. The investigation concluded the bimodal thickness was due to internal capsular biosynthesis in agreement with HPLC results. The heterogeneity of capsule thickness is independent of the bacterial curvature due to the dense grafting and strong polyelectrolyte properties of fully stretched K1capsular brushes. The steric stabilization due to the osmotic pressure of bacterial capsular brushes was confirmed using atomic force microscopy (AFM). The K1 biosynthesis of capsular polysaccharide was investigated via polysialyltransferase (NeuS) dynamics using a novel Halotag self-labelling method. The rapid dynamics of NeuS in capsular biosynthesis compared with a CPS mutant revealed a sub-diffusive regime with a 0.2-0.5 ms characteristic time for interaction using fluorescence correlation spectroscopy (FCS). The variation of NeuS interaction influences the polydispersity of capsular chains. Although densely grafted K1 capsular brushes fully cover the bacterial surface, positively charged amphiphilic peptides, G(IIKK)3I-NH2, could penetrate the thick layer of the bacterial capsule. This is similar to permeability in the nutrient consumption pathway. The inhomogeneous landscape of the bacterial surface mixture, which comprised proteins, lipid islets and other elements, is one of the factors. Thus, the K1 bacterial capsule is a permeable shield with a bimodal thickness that protects the bacterial surface from antimicrobial peptide treatment by introducing aggregation on its surface, through partial interaction with the bacterial membranes.