Date Published: January 10, 2012
Publisher: Public Library of Science
Author(s): Wenqi Yu, Friedrich Götz, Ben Adler. http://doi.org/10.1371/journal.pone.0030076
A fluorescence microscopy method to directly follow the localization of defined proteins in Staphylococcus was hampered by the unstable fluorescence of fluorescent proteins. Here, we constructed plasmid (pCX) encoded red fluorescence (RF) mCherry (mCh) hybrids, namely mCh-cyto (no signal peptide and no sorting sequence), mCh-sec (with signal peptide), and mCh-cw (with signal peptide and cell wall sorting sequence). The S. aureus clones targeted mCh-fusion proteins into the cytosol, the supernatant and the cell envelope respectively; in all cases mCherry exhibited bright fluorescence. In staphylococci two types of signal peptides (SP) can be distinguished: the +YSIRK motif SPlip and the −YSIRK motif SPsasF. mCh-hybrids supplied with the +YSIRK motif SPlip were always expressed higher than those with −YSIRK motif SPsasF. To study the location of the anchoring process and also the influence of SP type, mCh-cw was supplied on the one hand with +YSIRK motif (mCh-cw1) and the other hand with -YSIRK motif (mCh-cw2). MCh-cw1 preferentially localized at the cross wall, while mCh-cw2 preferentially localized at the peripheral wall. Interestingly, when treated with sub-lethal concentrations of penicillin or moenomycin, both mCh-cw1 and mCh-cw2 were concentrated at the cross wall. The shift from the peripheral wall to the cross wall required Sortase A (SrtA), as in the srtA mutant this effect was blunted. The effect is most likely due to antibiotic mediated increase of free anchoring sites (Lipid II) at the cross wall, the substrate of SrtA, leading to a preferential incorporation of anchored proteins at the cross wall.
Surface anchored proteins of Staphylococcus aureus represent a group of proteins that are exposed on the bacterial cell envelope and covalently anchored to the staphylococcal cell wall peptidoglycan . Many of the surface proteins belong to the MSCRAMM family (microbial surface components recognizing adhesive matrix molecules), which play key roles in colonization and adhesion of S. aureus.
So far, immunofluorescence microscopy and immunoelectron microscopy have been used for surface proteins localization studies in the last decades. To our knowledge, there is no direct visualization method to be applied in this field yet. In this study, we aimed to develop a direct method for monitoring surface proteins’ subcellular distribution. The recently developed fluorescent protein mCherry, the monomeric derivative of Discosoma sp. fluorescent protein ‘DsRed’ , provided us with an ideal tool. mCherry was found fully fluorescent after secretion through the Sec secretory pathway and was fluorescent in the membrane as well , . Here we show that mCherry can be secreted and anchored to staphylococcal cell wall while maintaining stable fluorescence.