title: Sortase A localizes to distinct foci on the Streptococcus pyogenes membrane.
abstract: Cell wall peptidoglycan-anchored surface proteins are essential virulence factors in many gram-positive bacteria. The attachment of these proteins to the peptidoglycan is achieved through a transpeptidation reaction, whereby sortase cleaves a conserved C-terminal LPXTG motif and covalently attaches the protein to the peptidoglycan precursor lipid II. It is unclear how the sorting reaction is regulated spatially and what part sortase localization plays in determining the distribution of surface proteins. This is mainly the result of inadequate immunofluorescence techniques required to resolve these issues in certain bacterial pathogens. Here we describe the utilization of the phage lysin PlyC to permeabilize the cell wall of Streptococcus pyogenes to antibodies, thereby allowing the localization of sortase A using deconvolution immunofluorescence microscopy. We find that sortase localizes within distinct membranal foci, the majority of which are associated with the division septum and colocalize with areas of active M protein anchoring. Sortase distribution to the new septum begins at a very early stage, culminates during septation, and decays after division is completed. This implies that the sorting reaction is a dynamic, highly regulated process, intimately associated with cell division. The ability to study cytoplasmic and membrane antigens using deconvolution immunofluorescence microscopy will facilitate further study of cellular processes in S. pyogenes.
authors.affiliation: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. araz@rockefeller.edu
authors.forename: Assaf
authors.initials: A
authors.lastname: Raz
authors.forename: Vincent A
authors.initials: VA
authors.lastname: Fischetti
mesh_terms: D019881:Aminoacyltransferases / Q000276:immunology / Q000378:metabolism*; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D013297:Streptococcus pyogenes / Q000201:enzymology*
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases