Hunting for bacterial interactors: activated magnetic nanoparticles for exploring membrane proteomics in bacteria

Introduction: Proteomics studies on the envelope(s) of different type of bacterial cells are typically performed through the separation of the different envelope layers or through the use of proteases to directly remove surface-exposed protein domain from intact bacterial cells. This approach has been successfully used for Gram positive bacteria but protease shaving of Gram-negative bacteria surface appears less straightforward, as with contamination with cytoplasmic peptides is likely to occur in Gram-negative bacteria. Results: A new and specific technique for magneto-capturing of surface-exposed proteins from intact bacterial cells was developed. This method is based on the use of carboxymethyl-dextran coated magnetic nanoparticles specifically activated to establish covalent bonds with exposed lysine amino groups in proteins. Given their chemical composition, size (average diameter = 80-90 nm), and negative charge, these NPs were expected to be atoxic for bacterial cells, as they are for eukaryotic cells. This work investigated in detail the outer-membrane composition of P. aeruginosa, a highly adaptable Gram negative bacterium which lives in a huge number of ecosystems and can infect multiple hosts from plants to humans, where it represents an important opportunistic pathogen. By using this technique we identified a total of 63 proteins that were captured directly by the activated nanoparticles, along with 67 proteins that were co-captured, being likely embedded in neighboring regions of the cell envelope. The envelope localization of most of the identified proteins had been only predicted, or had been unknown so far. Conclusions: This approach developed in this study improves sensitivity and specificity of previous methods, such as surface shaving with proteases. Magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction also allows the identification of the captured proteins, and that of neighboring ones. The magneto-capture procedure is simple, safe, and rapid, and appears well-suited for envelope studies in many proteomics studies, including those regarding highly pathogenic bacteria.