Effects of treatment with 250 µM 2,2’-dipyridyl (DPD) and/or 0.8% bile salts on exponential growth of O157:H7 in LB medium.

<p>Four independent growth curve experiments were conducted and showed similar results. Expanded linear and semi-log plots of OD₆₀₀ for the first 3 hours 15 minutes of growth are provided for the same experimental data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074647#pone-0074647-g001" target="_blank">Figure 1</a>. Error bars represent standard deviations for triplicate platings.</p

Phevalin (aureusimine B)Production by <em>Staphylococcus aureus</em> Biofilm and Impacts on Human Keratinocyte Gene Expression

<div><p><em>Staphylococcus aureus</em> biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. <em>S. aureus</em> contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in <em>S. aureus</em>, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that <em>S. aureus</em> biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of <em>S. aureus</em> as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from <em>S. aureus</em> spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, <em>S. aureus</em> biofilm-based infections.</p> </div

<i>S. aureus</i> biofilms produce more phevalin than their planktonic counterparts.

<p>(A) HPLC-MS analysis of organic extracts from <i>S. aureus</i> biofilm, planktonic, and growth medium control revealed that biofilms produce more phevalin (aureusimine B) than planktonic cultures (arrow). A compound that is likely tyrvalin (aureusimine A) was also present at higher levels in the biofilm (*). (B) Phevalin production was detected directly in samples without prior organic extraction. Samples were normalized to cell density (optical density, 600 nm, OD₆₀₀) in biofilm (OD₆₀₀ 0.9), resuspended biofilm (OD₆₀₀ 1.4), and planktonic cultures (OD₆₀₀ 0.66). Data represent means ± SEM, n = 3, ***p<0.001.</p

Phevalin does not induce apoptosis in HKs.

<p>(A) Cell counts after 4 or 24 hours of exposure to phevalin, -PCM, or +PCM. (B) Percent cells staining positive for TUNEL after 4 or 24 hours of exposure to phevalin, -PCM, or +PCM. Data represent ± SEM, n = 6, *p<0.05, ** p<0.01, ns, not significant, p>0.05.</p

After the addition of 1 µM or 10 µM phevalin to HKs, only 24 genes were significantly regulated (±2 fold in any one condition, p<0.05) relative to control cells.

<p>After the addition of 1 µM or 10 µM phevalin to HKs, only 24 genes were significantly regulated (±2 fold in any one condition, p<0.05) relative to control cells.</p

Conditioned medium from <i>S. aureus</i> cultures with or without additional phevalin induces differential gene expression in HKs.

<p>Significant (p<0.05) transcripts regulated ±2 fold in any one condition relative to controls. Transcripts shared between HKs treated with BCM, +PCM, and –PCM are shown at ±2 and ±5 fold change cutoffs (A and B, respectively). HKs treated with BCM shared more transcripts with +PCM treated HKs than –PCM treated HKs (arrows). Transcripts shared between –PCM and BCM had modest fold changes as no transcripts were shared above the ±5 FC cutoff. (C) The top 20 upregulated and downregulated genes (p<0.05) in +PCM treated HKs relative to –PCM treated HKs are listed. For a complete list of significantly regulated genes, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040973#pone.0040973.s003" target="_blank">Table S1</a>. (D) Selected genes were confirmed by RT-qPCR. The fold change relative to a GAPDH normalizer is indicated (p<0.05 for all comparing DMSO to phevalin). (E) Functional annotation clustering of microarray data revealed significantly (Benjamini p<0.01) enriched biological processes in +PCM treated HKs.</p

Phevalin production in various strains of bacteria as detected by SRM HPLC-MS.

<p>Phevalin production in various strains of bacteria as detected by SRM HPLC-MS.</p