Functional categories of genes found by microarray to be differentially expressed in <i>E. faecalis</i> OG1RF <i>in</i><i>vivo</i> during subdermal abscess infection.

<p>The numbers of up- (right) and down-regulated (left) genes at two (top) or eight (bottom) hours post-inoculation are indicated on the horizontal axis.</p

Survival of <i>E. faecalis</i> OG1RF, Δ<i>rsh</i>, Δ<i>relQ</i>, and Δ<i>rsh</i>Δ<i>relQ</i> in subdermal abscesses at (A) early and (B) late time points post-inoculation.

<p>Subdermal abscess infections with the four strains were carried out as described in the text and in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115839#pone-0115839-g001" target="_blank">Fig. 1</a>. Results are reported as percent survival with the 0 hour time point set to 100%. Values and error bars represent the mean ± SEM of n = 2 rabbits. Data from the same rabbits were separated into panels (A) and (B) for clarity. (A) Student’s t-test comparing Δ<i>rsh</i> and Δ<i>rsh</i>Δ<i>relQ</i>, α = 0.1: red #, p = 0.08; red ∧, p = 0.06. (B) One-way ANOVA followed by Tukey’s Multiple Comparison post-hoc test: black *, p<0.05 for OG1RF versus Δ<i>relQ</i>; black **, p<0.01 for OG1RF versus Δ<i>rsh</i>Δ<i>relQ</i>; blue *, p<0.05 for Δ<i>rsh</i> versus Δ<i>rsh</i>Δ<i>relQ</i>.</p

Differentially regulated genes in subdermal abscesses identified by microarray that overlap with a previously conducted subdermal abscess RIVET screen [11].

1<p>Microarray UP and Microarray DOWN indicate that the locus was up- or down-regulated, respectively, in microarray analysis at the indicated time point.</p><p>Differentially regulated genes in subdermal abscesses identified by microarray that overlap with a previously conducted subdermal abscess RIVET screen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115839#pone.0115839-Frank1" target="_blank">[11]</a>.</p

Growth and survival of <i>E. faecalis</i> OG1RF, Δ<i>rsh</i>, Δ<i>relQ</i>, and Δ<i>rsh</i>Δ<i>relQ</i> in human (A) serum and (B) whole blood <i>ex vivo</i>.

<p>Overnight cultures of the four strains were diluted 1∶20 in serum or whole blood and incubated at 37°C. Dilutions of aliquots collected at 0, 3 (panel B), 6 (panel A), 9, 24, 48, and 72 hours were plated to assess bacterial survival. Results are reported as percent survival with the 0 hour time point set to 100%. Values and error bars represent the mean ± SEM of n = 3 independent replicates. Data were analyzed for statistical significance with two-way ANOVA followed by Dunnett’s multiple comparisons test. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Transcriptome Analysis of <i>Enterococcus faecalis</i> during Mammalian Infection Shows Cells Undergo Adaptation and Exist in a Stringent Response State

<div><p>As both a commensal and a major cause of healthcare-associated infections in humans, <i>Enterococcus faecalis</i> is a remarkably adaptable organism. We investigated how <i>E. faecalis</i> adapts in a mammalian host as a pathogen by characterizing changes in the transcriptome during infection in a rabbit model of subdermal abscess formation using transcriptional microarrays. The microarray experiments detected 222 and 291 differentially regulated genes in <i>E. faecalis</i> OG1RF at two and eight hours after subdermal chamber inoculation, respectively. The profile of significantly regulated genes at two hours post-inoculation included genes involved in stress response, metabolism, nutrient acquisition, and cell surface components, suggesting genome-wide adaptation to growth in an altered environment. At eight hours post-inoculation, 88% of the differentially expressed genes were down-regulated and matched a transcriptional profile consistent with a (p)ppGpp-mediated stringent response. Subsequent subdermal abscess infections with <i>E. faecalis</i> mutants lacking the (p)ppGpp synthetase/hydrolase RSH, the small synthetase RelQ, or both enzymes, suggest that intracellular (p)ppGpp levels, but not stringent response activation, influence persistence in the model. The ability of cells to synthesize (p)ppGpp was also found to be important for growth in human serum and whole blood. The data presented in this report provide the first genome-wide insights on <i>E. faecalis in</i><i>vivo</i> gene expression and regulation measured by transcriptional profiling during infection in a mammalian host and show that (p)ppGpp levels affect viability of <i>E. faecalis</i> in multiple conditions relevant to mammalian infection. The subdermal abscess model can serve as a novel experimental system for studying the <i>E. faecalis</i> stringent response in the context of the mammalian immune system.</p></div

Overlap of differential gene expression between microarray and <i>in</i><i>vivo</i>-activated transcripts identified by RIVET screen in the <i>E. faecalis</i> subdermal abscess model [11].

<p>(A) Up-regulated genes identified by microarray compared with RIVET sense direction clones. (B) Down-regulated genes identified by microarray compared with sense strand ORFs that are complementary to RIVET antisense clones. See text and reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115839#pone.0115839-Frank1" target="_blank">[11]</a> for additional details on the subdermal abscess RIVET screen that was previously conducted. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115839#pone-0115839-t001" target="_blank">Table 1</a> for the identities of the genes from microarray analyses that overlap with RIVET analysis genes. See column H of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115839#pone.0115839.s003" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115839#pone.0115839.s004" target="_blank">S2 Tables</a> for the identities of genes found in both microarray analyses.</p

Recovery of Sephadex G100 fractions of S. sanguinis 133-79 tryptic digest.

a<p>PRP was preincubated with the indicated fraction at a final concentration of 0.1 mg/ml.</p>b<p>Proteins were incubated with 50 µM of AMP, ADP or ATP for 15 minutes at 37°C at a final concentration of 10 µg/ml.</p

Characterization of Nt5e activity on <i>S. sanguinis</i> 133-79 whole cells.

<p>Nt5e activity was measured by the release of inorganic phosphate (Pi) from adenine nucleotides. For (A), (B), and (C), the Michaelis-Menten curves were showed as enzyme velocity (represented as nmole/min/10⁶ cells) vs. concentration of ATP, ADP and AMP substrates. (D) Effect of Nt5e inhibitor APCP on AMPase activity of <i>S. sanguinis</i> 133-79. The curve was fitted to a sigmoidal inhibitory dose-response curve and the inhibitory concentration 50% (IC₅₀) value derived from the curve fit was shown. (E) Michaelis-Menten curves of AMPase activity vs. substrate concentration in the absence and presence of APCP. (F) pH dependence of AMPase activity of Nt5e. Statistical analysis was performed using non-linear regression. The results were represented as mean±SE, n = 3; *significantly decreased compared to no inhibitor (P<0.05).</p

<i>nt5e</i> confers Nt5e activity on <i>S. sanguinis</i> SK36 whole cells.

<p>Nt5e activity was measured by the release of inorganic phosphate (Pi) from adenine nucleotides. (A), (B), and (C) were showed as enzyme velocity vs. concentration of ATP, ADP and AMP substrates, where the results were represented as mean±SE, n = 3. Statistical analysis was performed by one-way ANOVA with Dunnett’s post-test for multiple comparisons. *significantly decreased compared to wild-type strain SK36 (P<0.05). Δ<i>nt5e</i>: 5′-nucleotidase deletion mutant; Δ<i>nucH</i>: extracellular nuclease deletion mutant; Δ<i>cnp</i>: cyclo-nucleotide phosphodiesterase deletion mutant; and Δ<i>rad3:</i> DNA repair ATPase deletion mutant.</p

Proteins in G100-3 identified by mass spectrometry.

<p>Proteins in G100-3 identified by mass spectrometry.</p