Figure 1.
S. aureus biofilms produce more phevalin than their planktonic counterparts.
(A) HPLC-MS analysis of organic extracts from S. aureus 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.
Table 1.
Phevalin production in various strains of bacteria as detected by SRM HPLC-MS.
Table 2.
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.
Figure 2.
Conditioned medium from S. aureus cultures with or without additional phevalin induces differential gene expression in HKs.
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 Table S1. (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.
Figure 3.
Phevalin does not induce apoptosis in HKs.
(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.
Figure 4.
Increased amounts of phevalin did not impact the extracellular metabolome of S. aureus as detected by HPLC-MS.
(A) Overlays of representative HPLC-MS base-peak chromatograms of +PCM (red) and –PCM (blue) revealed no substantial differences with the exception of phevalin (arrow). (B) XCMS analysis identified 22 features significantly (p<0.05) regulated ±2 fold in +PCM relative to –PCM. Those features grouped by RT into four groups (numbered bars). Group 2 contained phevalin, its fragment ions, and various adducts. Groups 1, 3, and 4 contained trace features associated with compounds used during the synthesis of phevalin. Data represent means ± SEM, n = 3.
Figure 5.
Analysis of +PCM and –PCM by NMR revealed minimal differences in metabolite composition.
(A) Representative raw NMR spectra of +PCM (red) and –PCM (blue) did not show any major differences in metabolite compositions. (B) Analysis of NMR spectra by Chenomx revealed only two compounds, ethanol and formate, that were produced in higher quantities in –PCM relative to +PCM. DSS is the chemical shape indicator. Data represent means ± SEM, n = 3, *p<0.02. ns, not significant, nd, not detected.