The bacterial cell envelope is a highly structured multi-layer meant to assure cell integrity and protection from environmental adversities while supporting in-out trafficking of nutrients and wastes. Surface proteins localized in the outer membrane participate in interactions with the environment, such as sensing the chemical and physical conditions of surroundings and transmitting appropriate signals to cytoplasm. These functions include adhesion to and, when possible, invasion of physical and biological supports (e.g. host cells for pathogens); as well as transport of nutrient molecules. Given these essential roles in bacterial life and in pathogenicity, identification and characterization of envelope proteins may lead to better explain the relation of bacteria with the surrounding environment. We here describe a new and specific technique for magneto-capturing of surface-exposed proteins in intact bacterial cells. This method is based on the use of carboxymethyl-dextran coated magnetic nanoparticles activated so to be able to establish covalent bonds with exposed lysine 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 approach greatly improves sensitivity and specificity of previous methods such as surface shaving with proteases. This techniques of 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 highly pathogenic bacteria.
Tuned magnetic nanoparticles for studying surface-exposed proteins in bacterial cells
M. Marengo;
2012-01-01
Abstract
The bacterial cell envelope is a highly structured multi-layer meant to assure cell integrity and protection from environmental adversities while supporting in-out trafficking of nutrients and wastes. Surface proteins localized in the outer membrane participate in interactions with the environment, such as sensing the chemical and physical conditions of surroundings and transmitting appropriate signals to cytoplasm. These functions include adhesion to and, when possible, invasion of physical and biological supports (e.g. host cells for pathogens); as well as transport of nutrient molecules. Given these essential roles in bacterial life and in pathogenicity, identification and characterization of envelope proteins may lead to better explain the relation of bacteria with the surrounding environment. We here describe a new and specific technique for magneto-capturing of surface-exposed proteins in intact bacterial cells. This method is based on the use of carboxymethyl-dextran coated magnetic nanoparticles activated so to be able to establish covalent bonds with exposed lysine 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 approach greatly improves sensitivity and specificity of previous methods such as surface shaving with proteases. This techniques of 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 highly pathogenic bacteria.
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