Transcriptomes of AH- and VH-grown <i>S. aureus</i> SA564.

a<p> ORFs were identified by BLAST analysis of Affymetrix array target sequences, as described in the materials and methods. If a corresponding ORF was identified in <i>S. aureus</i> COL, that strain's ORF identifiers were used as default. SACOL####, <i>S. aureus</i> COL (GenBank accession number CP000046.1); SAV####, <i>S. aureus</i> Mu50 (BA000017.4); SA####, <i>S. aureus</i> N315 (BA000018.3); SAOUHSC_####, <i>S. aureus</i> NCTC 8325 (CP000253.1). Vertical lines indicate genes computationally predicted to be in the same transcriptional unit. Gene names in brackets were assigned in this study using data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110872#pone.0110872.s001" target="_blank">Table S1</a>.</p>b<p> Fold change in expression of genes during <i>S. aureus</i> SA564 growth in AH as compared to growth in glucose-supplemented CDM; a positive number indicates an up-regulation of the gene during growth in AH. Standard deviation is shown in parentheses. Fold changes ≥3 and <10 are shown and italicized.</p>c<p> Fold change in expression of genes during <i>S. aureus</i> SA564 growth in VH as compared to growth in glucose-supplemented CDM; a positive number indicates an up-regulation of the gene during growth in VH. Standard deviation is shown in parentheses. Fold changes ≥3 and <10 are shown and italicized.</p>d<p> Fold change in expression of genes during <i>S. aureus</i> SA564 growth in VH as compared to growth in AH; a positive number indicates an up-regulation of the gene during growth in VH. Standard deviation is shown in parentheses. Fold changes ≥3 and <10 are shown and italicized.</p>e<p> At least two differentially expressed probe sets were assigned to these ORFs. Data for all probe sets are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110872#pone.0110872.s001" target="_blank">Table S1</a>.</p>f<p> On genomic island <i>v</i>SA4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110872#pone.0110872-Gill1" target="_blank">[48]</a>.</p>g<p> On φCOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110872#pone.0110872-Gill1" target="_blank">[48]</a>.</p>h<p> On φSa1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110872#pone.0110872-Gill1" target="_blank">[48]</a>.</p>i<p> On φSa3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110872#pone.0110872-Gill1" target="_blank">[48]</a>.</p><p>Transcriptomes of AH- and VH-grown <i>S. aureus</i> SA564.</p

<i>S. aureus</i> growth in CDM, AH and VH.

<p><i>S. aureus</i> SA564 (black circles), CDM7 (white squares), and MS7 (grey triangles) were grown in CDM, AH, or VH, as described in the text. Growth was monitored by optical density at 600 nm (OD_{600 nm}). Representative growth curves are shown. Arrows indicate time points where microarray sampling occurred.</p

<i>In Vitro</i> and <i>In Vivo</i> Models of <i>Staphylococcus aureus</i> Endophthalmitis Implicate Specific Nutrients in Ocular Infection

<div><p>Purpose</p><p>To define global transcriptional responses of <i>Staphylococcus aureus</i> and its <i>codY</i> mutant (CodY is a transcription regulator of virulence and metabolic genes in response to branched-chain amino acids) when growing in bovine aqueous (AH) and vitreous humor (VH) <i>in vitro</i>, and to investigate the impact of <i>codY</i> deletion on <i>S. aureus</i> virulence in a novel murine anterior chamber (AC) infection model.</p><p>Methods</p><p>For the <i>in vitro</i> model, differential transcriptomic gene expression of <i>S. aureus</i> and its <i>codY</i> mutant grown in chemically defined medium (CDM), AH, and VH was analyzed. Furthermore, the strains were inoculated into the AC of mice. Changes in bacterial growth, electroretinography and inflammation scores were monitored.</p><p>Results</p><p>Bovine AH and VH provide sufficient nutrition for <i>S. aureus</i> growth <i>in vitro</i>. Transcriptome analysis identified 72 unique open reading frames differentially regulated ≥10-fold between CDM, AH, and VH. In the AC model, we found comparable growth of the <i>codY</i> mutant and wild type strains <i>in vivo</i>. Average inflammation scores and retinal function were significantly worse for <i>codY</i> mutant-infected eyes at 24 h post-infection.</p><p>Conclusion</p><p>Our <i>in vitro</i> bovine AH and VH models identified likely nutrient sources for <i>S. aureus</i> in the ocular milieu. The <i>in vivo</i> model suggests that control of branched-chain amino acid availability has therapeutic potential in limiting <i>S. aureus</i> endophthalmitis severity.</p></div

Genes differentially expressed by the <i>S. aureus</i> SA564 <i>codY</i> mutant during growth in CDM, AH and VH.

a<p> ORFs were identified by BLAST analysis of Affymetrix array target sequences, as described in the materials and methods. If a corresponding ORF was identified in <i>S. aureus</i> COL, that strain's ORF identifiers were used as default. SACOL####, <i>S. aureus</i> COL (GenBank accession number CP000046.1); SAV####, <i>S. aureus</i> Mu50 (BA000017.4); SA####, <i>S. aureus</i> N315 (BA000018.3); SAOUHSC_####, <i>S. aureus</i> NCTC 8325 (CP000253.1).</p>b<p> Fold change in expression of genes by <i>S. aureus</i> CDM7 as compared to the wild-type strain during growth in the indicated medium; a positive number indicates an up-regulation of the gene by the <i>codY</i> mutant. Standard deviation is shown in parentheses.</p>c<p> At least two differentially expressed probe sets were assigned to these ORFs. Data for all probes sets are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110872#pone.0110872.s002" target="_blank">Table S2</a>.</p><p>Genes differentially expressed by the <i>S. aureus</i> SA564 <i>codY</i> mutant during growth in CDM, AH and VH.</p

Semi-quantitative RT-PCR.

<p>Differential expression of <i>tst</i>, <i>nanA</i> and <i>cidA</i> in AH, VH and CDM. <i>clpX</i> was used as a constitutively expressed control gene.</p

Inflammation and retinal responsiveness in <i>S. aureus</i> infected eyes.

<p>Inflammation scores (A) and % retinal responsiveness (B) for murine eyes infected with SA564, CDM7, or MS7, assessed 24 h post-inoculation. Average values are indicated by heavy black horizontal lines. *, p≤0.001, Mann-Whitney test.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

<i>S. aureus</i> SA564, CDM7 and MS7 <i>in vivo</i> growth yields.

a<p> Number of CFU recovered per homogenized eye is shown. Each entry represents one eye.</p>b<p> ND, Not detected. The limit of detection for these experiments was 1×10² CFU.</p><p><i>S. aureus</i> SA564, CDM7 and MS7 <i>in vivo</i> growth yields.</p

Identification of a Functionally Unique Family of Penicillin-Binding Proteins

Penicillin-binding proteins (PBPs) are enzymes involved in the assembly of the bacterial cell wall, a major target for antibiotics. These proteins are classified by mass into high-molecular-weight PBPs, which are transpeptidases that form peptidoglycan cross-links, and low-molecular-weight PBPs, which are typically hydrolases. We report a functionally unique family of low-molecular-weight PBPs that act as transpeptidases rather than hydrolases, but they do not cross-link peptidoglycan. We show that these PBPs can exchange d-amino acids bearing chemical tags or affinity handles into peptidoglycan precursors, including Lipid II, enabling biochemical studies of proteins involved in cell wall assembly. We report that, in two organisms, the PBPs incorporate lysine into cellular peptidoglycan and that, further, the PBPs have the unprecedented ability to transfer the primary ε-amine of lysine to peptidoglycan

Apical cell surface abundance of MUC16 is reduced following treatment of epithelia with SP168 growth culture filtrate.

<p>Apical membrane levels of MUC16 were determined by cell surface biotinylation. <b>A</b>) Western blot analyses (using M11 antibody) of the amount of released and residual biotin-labeled surface MUC16 after exposure to medium only control or SP168 growth culture filtrate. <b>B</b>) Quantitative analyses of the western blot in (A) showing a reduction in the abundance of surface MUC16 following treatment with the SP168 culture filtrate (p<0.0001, Student's t-test, n = 3). Data for MUC16 surface abundance is expressed as a percentage of the total released plus surface MUC16 ± SEM.</p