The outer membrane of bacteria acts as a permeability barrier that separate cells from its extracellular milieu. Thus, this surface must facilitate essential biological processes such as nutrient uptake, secretion of various factors, as well as interaction with the extracellular environment and potentially a host organism (and its immune system). This complex surface is composed of a mixture of lipids, proteins and carbohydrates, all relevant to the survival of bacteria in different environments. Traditional Structural and molecular biological means have elucidated their functional role from many aspects in the outer membrane. However, only a handful of membrane proteins have been characterised in terms of their spatial organization and distribution on the cell surface. Recent advances in Single Molecule Localization super-resolution microscopy have allowed us to explore cellular processes occurring at bacterial cell surface in an unprecedented level of sensitivity, specificity and spatial resolution.
Many outer membrane proteins are considered to be virulent determinants in bacterial pathogenicity. Autotransporters (AT) are a sub set of surface exposed membrane proteins known to display a wide array of functionalities such as cellular auto-aggregation which in turn promotes bio-film formation and aids bacterial colonization in the host environment. Antigen 43 (Ag43) is a well characterised AT that confers cell-cell aggregation in most pathogenic and non-pathogenic Escherichia coli strains. It consists of two subunits: a functional surface displayed N-terminal passenger domain (α) and a C-terminal β-barrel membrane domain. A numbers of diffraction limited microscopy based studies carried out most notably using K-12 strains have demonstrated the phase variability of Ag43 in E.coli. Here we optimise the use of direct Stochastic Optical Reconstruction Microscopy (dSTORM); a form of Single Molecule Localization microscopy methods to examine surface of bacteria and in particular the expression and nanoscopic localization/distribution features of Ag43.