Sortase a Localizes to Distinct Foci on the Streptococcus Pyogenes Membrane
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Sortase A localizes to distinct foci on the Streptococcus pyogenes membrane Assaf Raz1 and Vincent A. Fischetti Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, 1230 York Avenue, New York, NY 10021 Edited by Emil C. Gotschlich, The Rockefeller University, New York, NY, and approved October 8, 2008 (received for review August 21, 2008) Cell wall peptidoglycan-anchored surface proteins are essential that the sorting reaction is spatially controlled came from studies virulence factors in many gram-positive bacteria. The attachment conducted in the 1960s that examined the regeneration of M of these proteins to the peptidoglycan is achieved through a protein on cells treated with trypsin (11, 12). These studies transpeptidation reaction, whereby sortase cleaves a conserved showed that M protein is actively anchored to the streptococcal C-terminal LPXTG motif and covalently attaches the protein to the cell wall solely at the division septum. On the other hand, protein peptidoglycan precursor lipid II. It is unclear how the sorting F, a major fibronectin-binding protein, was subsequently found reaction is regulated spatially and what part sortase localization to be localized mainly at the old pole (13). A recent study showed plays in determining the distribution of surface proteins. This is that the signal sequence directs these two proteins to their mainly the result of inadequate immunofluorescence techniques respective positions on the cell surface and that switching the required to resolve these issues in certain bacterial pathogens. signal sequence between them results in altered localization (14). Here we describe the utilization of the phage lysin PlyC to perme- Our current knowledge regarding the spatial organization of abilize the cell wall of Streptococcus pyogenes to antibodies, protein sorting in S. pyogenes is mainly derived from the study of thereby allowing the localization of sortase A using deconvolution anchored surface proteins, while the actual distribution of sortase immunofluorescence microscopy. We find that sortase localizes remains unknown. Localization of this protein by immunofluores- within distinct membranal foci, the majority of which are associ- cence has so far been hindered by the fact that lysozyme, a muralytic enzyme commonly used to permeabilize bacterial cell walls to ated with the division septum and colocalize with areas of active antibodies, has only a marginal effect on S. pyogenes (15, 16). In this M protein anchoring. Sortase distribution to the new septum report, we introduce the use of the phage lysin PlyC (17) as a tool begins at a very early stage, culminates during septation, and to overcome this problem. We found that low-dose treatment of decays after division is completed. This implies that the sorting fixed cells with this highly efficient cell wall hydrolase, permeabilizes reaction is a dynamic, highly regulated process, intimately associ- the bacterial cell wall to antibodies without causing adverse effects ated with cell division. The ability to study cytoplasmic and mem- to the cellular morphology. Preservation of cellular morphology is brane antigens using deconvolution immunofluorescence micros- largely dependent on the presence of M protein anchored to the cell copy will facilitate further study of cellular processes in S. wall. Using this method, we were able to determine the localization pyogenes. of sortase by deconvolution immunofluorescence microscopy. immunofluorescence ͉ microscopy ͉ protein sorting ͉ phage lysin ͉ Results M protein Production and Validation of Anti-Sortase A Antibodies. Hexahisti- dine-tagged sortase A, lacking its N-terminal transmembrane ell wall anchored surface proteins play a vital role in the domain, was purified (supporting information (SI) Fig. S1A) and Cpathogenic process of many gram-positive bacteria (1). determined to be catalytically active (data not shown). This These proteins all have a conserved C-terminal anchor domain protein was used for immunization of mice and affinity purifi- comprised of an LPXTG motif followed by a hydrophobic region cation of resulting anti-sortase antibodies. We deleted sortase and a few positively charged amino acids at the C terminus (2, from D471 and found that the resulting strain, AR01, missorted MICROBIOLOGY 3). During protein export, the C-terminal anchor domain is M protein to the cytoplasm and supernatant in agreement with stalled in the secretion channel, leaving the LPXTG motif previous results (8). Complementation of AR01 with pAR107, exposed on the outer surface of the membrane. The transpep- a plasmid expressing sortase, restored the wild-type phenotype (Fig. S1B). We then analyzed the specificity of the anti-sortase tidase sortase then cleaves this motif between the threonine and antibodies by Western blot (Fig. S1C). These antibodies reacted glycine residues (4) and attaches the freed threonine to the with a band of the correct size in the wild-type strain D471, but peptidoglycan precursor, lipid II (5). Lipid II then serves as not with the sortase mutant AR01. Recombinant sortase ex- substrate for peptidoglycan synthesis, leading to the covalent pressed from a plasmid restored reactivity to the antibodies. An attachment of the protein to the cell wall. additional faint band, present in both D471 and plasmid- The important human pathogen Streptococcus pyogenes (6) complemented AR01, is likely to be a sortase cleavage product, employs an impressive array of wall-anchored virulence factors as it is completely absent from AR01. that function in immune evasion, adherence, and invasion, among other roles (7). In S. pyogenes, sortase A is the house- The Effect of Mild PlyC Treatment on the Morphology of Fixed S. keeping sortase and was shown experimentally to anchor M pyogenes Cells. Permeabilization of the cell wall to antibodies is protein, protein F, C5a peptidase (ScpA), and protein G-related a prerequisite for the use of immunofluorescence microscopy for ␣2-macroglobulin-binding protein (GRAB) to the cell wall (8). In addition to sortase A, S. pyogenes harbors two specialized sortases, associated with the FCT (fibronectin-binding, collagen- Author contributions: A.R. and V.A.F. designed research; A.R. performed research; A.R. and binding T antigen) region. Of these, one is specific for T antigen, V.A.F. analyzed data; and A.R. and V.A.F. wrote the paper. a major component of the streptococcal pili (8, 9), while the The authors declare no conflict of interest. other recognizes an altered consensus sequence, QVPTGV and This article is a PNAS Direct Submission. anchors a protein of unknown function (10). 1To whom correspondence should be addressed. E-mail: [email protected]. While the biochemical aspects of the sorting reaction have This article contains supporting information online at www.pnas.org/cgi/content/full/ been studied in detail in various organisms (1), much less is 0808301105/DCSupplemental. known about the spatial organization of this process. First clues © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808301105 PNAS ͉ November 25, 2008 ͉ vol. 105 ͉ no. 47 ͉ 18549–18554 Downloaded by guest on September 28, 2021 in preserving cell wall integrity following PlyC treatment. Even A B mild PlyC treatment of the isogenic M protein knockout strain JRS75, causes the removal of the cell wall and the creation of spheroplasts (Fig. 1, compare D, untreated and E, treated). Furthermore, removal of M protein from D471 by trypsin digestion similarly predisposes the cell wall to removal by PlyC and formation of spheroplasts (Fig. 1F). In addition, PlyC treatment of the sortase mutant, AR01, which does not anchor M protein to the cell wall (Fig. S1B), leads to a similar phenotype (Fig. 1G), while complementation of AR01 with pAR107 restores near-WT phenotype (Fig. 1H). We C D therefore propose that M protein may be forming a cross-linked mesh around the cell following fixation, and that this mesh preserves the gross overall integrity of the cell wall following gentle PlyC treatment, despite the partial degradation of the peptidoglycan. Localization of Sortase in S. pyogenes. The application of mild PlyC treatment allowed us to study the localization of sortase A in S. pyogenes using deconvolution microscopy. The images in Fig. 2 are presented as serial Z-stack captures and represent a typical EF distribution of sortase in a short chain of streptococcal cells. Sortase was found to localize to a number of foci in D471 cells (Fig. 2A, Movie S1), while the sortase mutant AR01 did not react with the antibodies (Fig. 2C). Sortase foci were predom- inantly associated with the division septum, but were not always strictly confined to the division plane. Sortase foci could also be found at the equatorial rings (discussed below) and, to a lesser extent, at the poles. In these experiments, wheat germ agglutinin (WGA) was used to visualize the cell wall peptidoglycan and carbohydrate, and glyceraldehyde-3-phosphate dehydrogenase G H (GAPDH) was used as a cytoplasmic marker. Cytoplasmic GAPDH labeling was used to rule out the possibility that sortase localization in foci results from incom- plete permeabilization of the cell wall. GAPDH is a glycolytic enzyme, which has also been found on the surface of several S. pyogenes strains, where it plays a role in binding various host factors (18). We found that log-phase D471 cells however, express very little surface GAPDH in comparison to the vast cytoplasmic pool (data not shown). This fact enabled us to use it as a cytoplasmic marker, applying labeling conditions under Fig. 1. Morphology of S. pyogenes following PlyC treatment. Fixed cells which surface GAPDH fluorescence is negligible. Effective were attached to glass slides, dipped in methanol, and washed with PBS. labeling of cytoplasmic GAPDH demonstrates that antibodies Unless otherwise noted, the cells were treated with 3 U/ml PlyC in PBS for 10 have free access into the cell. Cytoplasmic areas from which min at room temperature, before processing for scanning electron microscopy GAPDH is excluded, coincide with the location of the nucleoid examination.