Other iron chelators also reduce ROS production by PMNs.

<p>Along with yersiniabactin, aerobactin, deferoxamine, and deferiprone also reduce ROS production by PMNs. To determine the effect of other iron-binding molecules on ROS production by PMNs, PMNs were pre-incubated with aerobactin, deferoxamine, deferiprone, or yersiniabactin. The pre-treated PMNs were then activated using PMA, and the production of ROS was measured using luminol. Red line: the concentration-dependent decrease in ROS production following yersiniabactin treatment. Gray line: the decrease in ROS production following treatment with aerobactin. Yellow line: the concentration-dependent decrease in ROS production following deferiprone treatment. Green line: the concentration-dependent decrease in ROS production following deferoxamine treatment. The level of ROS produced by stimulated PMNs without additives was set as 100%. All experiments were replicated three times, and each sample was analyzed in duplicate.</p

Growth curves of the EHOS E4 isolate and its corresponding <i>irp2</i> knockout strain.

<p>The results are presented as the mean of three experiments performed in duplicate on different days. Cells were grown in: A) nutrient broth treated with 1% Chelex; B) nutrient broth treated with 1% Chelex and supplemented with 80% iron-saturated lactoferrin (∼6.2 µM, Fe-LF) or unsaturated lactoferrin (∼6.4 µM,0-LF); C) nutrient broth treated with 1% Chelex and supplemented with 17 µM holo-transferrin or 17 µM apo-transferrin; D) nutrient broth treated with 1% Chelex and supplemented with 10 µM or 30 µM hemin.</p

In the <i>irp2</i> knockout strain the production of yersiniabactin is blocked.

<p>Yersiniabactin production was tested using a GFP reporter assay with the knockout strain WA-CS <i>irp1</i>::Kan^r containing the pCJG3.3N plasmid as the reporter strain to detect yersiniabactin in supernatants. The number of bacteria versus the measured arbitrary amount of fluorescence from 50,000 counted bacteria is shown in panel A. A) Supernatants from the <i>irp2</i> knockout (E4Δ<i>irp2</i>) cultures (grey) induced a lower amount of GFP compared to supernatants from the wild-type (WT) cultures (white). This indicates that production of yersiniabactin was blocked in the <i>irp2</i> knockout. The experiments were performed in duplicate. B) The GFP reporter assay is dependent on yersiniabactin production, and production of yersiniabactin in the <i>irp2</i> knockout is blocked. Yersiniabactin production is only seen in the wild-type (WT) cells cultured in iron-depleted medium (NBD), while the <i>irp2</i> knockout (E4Δ<i>irp2</i>) strain cultured in NBD and iron-containing medium (NB), as well as the wild-type strain grown in NB, did not produce yersiniabactin. Three different experiments were performed, with each sample analyzed in duplicate. C) The expression of HMWP1 and HMWP2 is disrupted by the insertion of a kanamycin resistance gene into <i>irp2</i> using the Tagetron Knockout System. The <i>irp2</i> knockout (E4Δ<i>irp2</i>) strain was not able to produce HMWP1 and HMWP2 when cultured in NBD. M: marker; lane 1: wild-type strain E4; lane 2–5: <i>irp2</i> gene knockouts created using the wild-type strain E4; lane 6 wild-type EHOS strain 03-702; lane 7: wild-type EHOS strain 03-819 served as HPI-positive control because the HMWP2 of this strain was confirmed by Edman degradation.</p

ROS production by PMNs is reduced by yersiniabactin.

<p>To determine the effect of yersiniabactin on the ROS production of PMNs, PMNs were pre-incubated with yersiniabactin. PMNs were then activated by PMA, and the production of ROS was measured using luminol. A) Absolute ROS production measured in arbitrary fluorescence units (A.U.) of PMNs after pre-incubation with yersiniabactin or iron-saturated yersiniabactin or the controls (PMNs that were only pre-incubated in RPMI/HSA medium and PMNs that were pre-incubated with yersiniabactin but not stimulated with PMA). B) The yersiniabactin-mediated inhibition of ROS production by PMNs is concentration dependent. Red line: the concentration-dependent decrease in production of ROS following treatment with yersiniabactin. Blue line: no decrease in ROS production following treatment with different concentrations of iron-saturated yersiniabactin. Black dotted line: negative control (PMNs incubated with yersiniabactin, but not stimulated with PMA); ***<i>p</i> value<0.0005, **<i>p</i> value<0.005, *<i>p</i> value<0.05. C) Treatment of PMNs with unsaturated lactoferrin or holo-transferrin partly inhibits the yersiniabactin-mediated inhibition of ROS production. Black bars: relative ROS production following treatment with yersiniabactin; gray bars: relative ROS production following treatment with yersiniabactin and unsaturated lactoferrin; white bars: relative ROS production following treatment with yersiniabactin and holo-transferrin. ^aConcentration of the iron chelator. D) Treatment of PMNs with saturated lactoferrin or apo-transferrin partly inhibits the yersiniabactin-mediated inhibition of ROS production. Black bars: relative ROS production following treatment with yersiniabactin; gray bars: relative ROS production following treatment with yersiniabactin and saturated lactoferrin; white bars: relative ROS production following treatment with yersiniabactin and apo-transferrin. All experiments were replicated three times, and each sample was analyzed in duplicate.</p

Peaks identified by Unsupervised Feature Selection to be determining the subgroups found by Spectral Clustering. Peaks are listed in decreasing order of their effect on the clustering.

<p>Peaks identified by Unsupervised Feature Selection to be determining the subgroups found by Spectral Clustering. Peaks are listed in decreasing order of their effect on the clustering.</p

Differences between Spectral Clustering clusters in salivary lysozyme and chitinase activity compared using one-way ANOVA and Games-Howell post-hoc tests.

<p>The magnitude of the inter-cluster differences is quantified using Cohen’s <i>d</i> measure of effect size (<i>i</i>.<i>e</i>. the difference between the two means divided by the standard deviation of the data).</p

Boxplots stratified on the 4 clusters showing peak intensities across the sample population for the 9 peaks determining the clustering structure.

<p>X-axis contains the 4 clusters. Y-axis contains the peak intensity, scaled from 0 (minimum detection level) to 100 (maximum detected intensity of any peak across all samples and peaks). Peaks are listed in decreasing order of their importance for the clustering, same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156707#pone.0156707.t002" target="_blank">Table 2</a>. (A) P-C peptide, Fr. 15–44. (B) P-C peptide, Fr. 1–25. (C) unidentified peak (m/z = 5980). (D) P-C peptide (1+ charge). (E) unidentified peak (m/z = 2725). (F) Cystatin B, Fr. 1–53. (G) P-C peptide, Fr. 15–35. (H) P-C peptide (2+ charge). (I) II-2 basic proline-rich protein, phosphorylated.</p

Spectral Clustering co-occurrence plot.

<p>Participants are ordered along both X- and Y-axis according to the co-occurrence score (i.e. the more similar the peptide profiles of any two participants, the higher their tendency to cluster together and the closer they are placed on the axis). Co-occurrence score values range from 0 (for participants who never cluster together) to 1.0 (for participants who always cluster together). The horizontal bar delimits the four clusters.</p

Putative identities for peaks in the MALDI-TOF MS spectra of saliva from healthy volunteers.

<p>Putative identities for peaks in the MALDI-TOF MS spectra of saliva from healthy volunteers.</p

PCA scores plot.

<p><b>(A) PCA based on all 80 peaks compared to (B) PCA scores plot based solely on the 9 peaks resulting from the Feature Selection procedure.</b> Individuals are color labeled according to their cluster. The proportion of variance explained was as follows: (A) PC1–17.4%, PC2–8.8% (26.2% cumulative). (B) PC1–39.5%, PC2–26.3% (65.8% cumulative).</p