Cell wall antibiotics provoke accumulation of anchored mCherry in the cross wall of Staphylococcus aureus.

Wenqi Yu,Friedrich Götz

doi:10.1371/journal.pone.0030076

Abstract

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.

Highlights

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 [1]
Immunofluorescence microscopy and immunoelectron microscopy have been used for surface proteins localization studies in the last decades
We aimed to develop a direct method for monitoring surface proteins’ subcellular distribution

Summary

Introduction

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 [1]. The process of anchoring surface proteins to the staphylococcal cell wall, termed the ‘sorting pathway’, includes three steps [3]: translocation, sorting and incorporation into mature peptidoglycan. Anchored proteins are distinguished by a C-terminal cell wall sorting signal (CWS). Sortase A (SrtA) [4], a membrane-bound transpeptidase, performs the sorting reaction by cleaving the amide bond between threonine and glycine within the LPXTG motif, which results in the acyl intermediate. The peptidoglycan precursor, Lipid II, serves as the substrate for the sorting reaction, which is the tethering of the C-terminal threonine of the surface protein to lipid II by an amide bond. Lipid II tethered with the surface proteins is incorporated into mature peptidoglycan [5]

Objectives

Methods

Results

Conclusion