<i>Staphylococcus aureus</i> Surface Proteins Involved in Adaptation to Oxacillin Identified Using a Novel Cell Shaving Approach

Staphylococcus aureus is a Gram-positive pathogen responsible for a variety of infections, and some strains are resistant to virtually all classes of antibiotics. Cell shaving proteomics using a novel probability scoring algorithm to compare the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted to high levels of oxacillin (APT). APT displayed altered cell morphology compared with COL and increased aggregation in biofilm assays. Increased resistance to β-lactam antibiotics was observed, but adaptation to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150 proteins at a threshold determined by the scoring algorithm. Proteins unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR and SACOL2302. Quantitative RT-PCR showed increased expression of <i>sacol2302</i> in APT grown with oxacillin (>6-fold compared with COL). Overexpression of <i>sacol2302</i> in COL to levels consistent with APT (+ oxacillin) did not influence biofilm formation or β-lactam resistance. Proteomics using iTRAQ and LC–MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated elevated APT abundances of LCP proteins, capsule and peptidoglycan biosynthesis proteins, and proteins involved in wall remodelling. Adaptation to oxacillin also induced urease proteins, which maintained culture pH compared to COL. These results show that S. aureus modifies surface architecture in response to antibiotic adaptation

<i>Staphylococcus aureus</i> Surface Proteins Involved in Adaptation to Oxacillin Identified Using a Novel Cell Shaving Approach

Staphylococcus aureus is a Gram-positive pathogen responsible for a variety of infections, and some strains are resistant to virtually all classes of antibiotics. Cell shaving proteomics using a novel probability scoring algorithm to compare the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted to high levels of oxacillin (APT). APT displayed altered cell morphology compared with COL and increased aggregation in biofilm assays. Increased resistance to β-lactam antibiotics was observed, but adaptation to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150 proteins at a threshold determined by the scoring algorithm. Proteins unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR and SACOL2302. Quantitative RT-PCR showed increased expression of <i>sacol2302</i> in APT grown with oxacillin (>6-fold compared with COL). Overexpression of <i>sacol2302</i> in COL to levels consistent with APT (+ oxacillin) did not influence biofilm formation or β-lactam resistance. Proteomics using iTRAQ and LC–MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated elevated APT abundances of LCP proteins, capsule and peptidoglycan biosynthesis proteins, and proteins involved in wall remodelling. Adaptation to oxacillin also induced urease proteins, which maintained culture pH compared to COL. These results show that S. aureus modifies surface architecture in response to antibiotic adaptation

ParM-YFP filaments are dynamic in the presence of <i>parC</i> and ParR.

<p><i>E</i>. <i>coli</i> cells expressing either ParM-YFP in isolation (A) or ParM-YFP in the presence of <i>parC</i> and ParR were grown to mid-logarithmic phase and selective photobleaching experiments were undertaken. Fluorescence recovery was monitored by imaging cells every 7.6 seconds over the course of three minutes. The first image in the series depicts a pre-bleached cell. Phase contrast/fluorescence overlay images of cells expressing ParM-YFP are shown to the left of the photobleaching montage. Boxes indicate regions of laser photobleaching. Time units (seconds; s) are shown. Scale bars represent 2 μm.</p

Segregational stability of <i>parC</i>-containing pSK7780 or pSK7780-derived test plasmids in the presence or absence of ParM or ParM-YFP in the <i>E</i>. <i>coli</i> DH5α cells.

<p>The retention of pSK7780, containing the <i>parC</i> centromere and <i>parR</i> transcribed from P_<i>par</i> was determined in the presence of pSK9026 (squares; expressing ParM-YFP), pSK9034 (triangles; expressing ParM which had been uncoupled from YFP), or pSG1193 (diamonds; expressing YFP only). The retention of pSK9113 (stars), containing <i>parR</i> transcribed from P_<i>par</i>, but with <i>parC</i> cloned downstream from the <i>parR</i> ORF, was determined in the presence of pSK9026. Five days of serial subculture represents approximately 50 generations of growth. Each data point is the mean of three biological replicates. Standard error is shown.</p

ParM-YFP filament formation in the presence or absence of <i>parC</i> and ParR.

<p><i>E</i>. <i>coli</i> cells expressing ParM-YFP in isolation (A) or expressing ParM-YFP in the presence of <i>parC</i> and ParR (B) were visualized by fluorescence microscopy. ParM-YFP filaments exhibit a dramatic shift in morphology in the presence of <i>parC</i> and ParR. Hook shaped filaments are indicated with an arrow-head, and ParM-YFP foci are shown with a chevron. Left to right, both panels: fluorescence image, phase contrast image; merge of phase contrast and fluorescent images. Scale bars represent 1 μm.</p