<i>Stenotrophomonas maltophilia</i> Virulence and Specific Variations in Trace Elements during Acute Lung Infection: Implications in Cystic Fibrosis

<div><p>Metal ions are necessary for the proper functioning of the immune system, and, therefore, they might have a significant influence on the interaction between bacteria and host. Ionic dyshomeostasis has been recently observed also in cystic fibrosis (CF) patients, whose respiratory tract is frequently colonized by <i>Stenotrophomonas maltophilia</i>. For the first time, here we used an inductively mass spectrometry method to perform a spatial and temporal analysis of the pattern of changes in a broad range of major trace elements in response to pulmonary infection by <i>S. maltophilia</i>. To this, DBA/2 mouse lungs were comparatively infected by a CF strain and by an environmental one. Our results showed that pulmonary ionomic profile was significantly affected during infection. Infected mice showed increased lung levels of Mg, P, S, K, Zn, Se, and Rb. To the contrary, Mn, Fe, Co, and Cu levels resulted significantly decreased. Changes of element concentrations were correlated with pulmonary bacterial load and markers of inflammation, and occurred mostly on day 3 post-exposure, when severity of infection culminated. Interestingly, CF strain – significantly more virulent than the environmental one in our murine model - provoked a more significant impact in perturbing pulmonary metal homeostasis. Particularly, exposure to CF strain exclusively increased P and K levels, while decreased Fe and Mn ones. Overall, our data clearly indicate that <i>S. maltophilia</i> modulates pulmonary metal balance in a concerted and virulence-dependent manner highlighting the potential role of the element dyshomeostasis during the progression of <i>S. maltophilia</i> infection, probably exacerbating the harmful effects of the loss of CF transmembrane conductance regulator function. Further investigations are required to understand the biological significance of these alterations and to confirm they are specifically caused by <i>S. maltophilia</i>.</p></div

Assessment of lung damage in DBA/2 lung following infection with <i>S. maltophilia</i>.

<p>Lung damage was macroscopically and histologically assessed in DBA/2 mice (n = 18) on days 1, 3, and 7 p.e. to <i>S. maltophilia</i> (Sm111 CF strain, red line; C39 environmental strain, green line), or PBS only (control; black line). A–C) Macroscopic examination. A) Photographs of control and infected mouse lungs are representative for lung damage observed at each time point (days 1, 3, and 7 p.e.) in 8 mice per group. C) Lung damage was assessed by using “four-point scoring system” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088769#pone.0088769-Johansen1" target="_blank">[24]</a>, and results are shown as median values. B–D) Histological assessment. B) Lung sections were stained with hematoxylin-eosin and ten fields/lung were observed at low (10×), and high (63×) magnification. For each group, only one representative microscopic field was reported. Since there were no significant differences in the degree of inflammation at any of the time point when evaluating the left and the right lung separately, the data were pooled and considered representative for both lungs. Mean percentage of tissue area characterized by the presence of neutrophilic infiltrate: 60% (Sm111-infected mice) vs 20% (C39-infected mice) (p<0.001, chi-square test.). D) Lung damage was quantified by using the “five-point scoring system” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088769#pone.0088769-Dubin1" target="_blank">[25]</a>, and results are shown as median histopatological index. *** p<0.001, ** p<0.01, * p<0.05 vs ctrl; °° p<0.01, ° p<0.05 Sm111 vs C39; Kruskall-Wallis followed by Dunn's multiple comparison <i>post-hoc</i> test.</p

Correlations among trace elements, cytokines, and bacterial load observed in lung tissue from control and <i>S. maltophilia</i>-exposed DBA/2N mice.

<p>Spearman coefficients calculated for significant correlations are shown in bold: ^* p<0.05, ^** p<0.01, ^*** p<0.001. Correlations between pulmonary bacterial load (CFU/lung) and element concentrations were assessed by combining data sets from lung collected on days 1, 3, and 7 p.e. to PBS or <i>S. maltophilia</i> (Sm111 and C39 strains). Correlations between element concentrations and cytokines levels were calculated by combining data sets from lung collected on days 1 and 3 p.e. to PBS or <i>S. maltophilia</i> (Sm111 and C39 strains).</p

Trace elements, bacterial load, and cytokine levels observed in lung tissue of <i>S. maltophilia</i>-infected DBA/2N mice.

<p>A–C) Score and loading plots obtained by Principal Component Analysis. These plots show PC1 vs PC2summarizing 56%, 76%, and 59% of the variation among lung samples collected on days 1 (A), 3 (B), and 7 (C) p.e. to <i>S. maltophilia</i> (Sm111 CF strain, red circles; C39 environmental strain, green triangles) or PBS only (controls, black rhombi). The amounts of variance explained by each PC are shown in parentheses. D–E) Correlation maps. The heatmaps show the unsupervised hierarchical clustering of lung elements, bacterial counts in lung homogenate (CFU), and cytokines assessed using Spearman's correlation in response to <i>S. maltophilia</i> infection by Sm111 CF (D) or C39 (E) strain. Direct and inverse correlations are shown in red and green, respectively. Correlation and cluster analysis were performed using MetaboAnalyst statistical analysis module <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088769#pone.0088769-Xia1" target="_blank">[29]</a>.</p

Trace elements, bacterial load, and cytokine levels observed in BAL samples of <i>S. maltophilia</i>-infected DBA/2N mice.

<p>A–C) Score and loading plots obtained by Principal Component Analysis. These plots show PC1 vs PC2 summarizing 57%, 69%, and 53% of the variation among BAL samples collected on days 1 (A), 3 (B), and 7 (C) p.e. to <i>S. maltophilia</i> (Sm111 CF strain, red circles; C39 environmental strain, green triangles) or PBS only (controls, black rhombi). The amounts of variance explained by each PC are shown in parentheses. D–E) Correlation maps. The heatmaps show the unsupervised hierarchical clustering of BAL elements, bacterial counts in lung homogenate (CFU), and pulmonary cytokines assessed using Spearman's correlation in response to <i>S. maltophilia</i> infection by Sm111 CF (D) or C39 (E) strain. Direct and inverse correlations are shown in red and green, respectively. Correlation and cluster analysis were performed using MetaboAnalyst statistical analysis module <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088769#pone.0088769-Xia1" target="_blank">[29]</a>.</p

Correlations among trace elements found in BAL from control and <i>S. maltophilia</i>-exposed DBA/2N mice.

<p>Spearman rank correlation coefficients were calculated and significant correlations are shown in bold: ^* p<0.05, ^** p<0.01, ^*** p<0.001. Correlations between pulmonary bacterial load (CFU/lung) and element concentrations were assessed by combining data sets from lung collected on days 1, 3, and 7 p.e. to PBS or <i>S. maltophilia</i> (Sm111 and C39 strains). Correlations between element concentrations and cytokines levels were calculated by combining data sets from BAL collected on days 1 and 3 p.e. to PBS or <i>S. maltophilia</i> (Sm111 and C39 strains).</p

Correlations between elements in lung tissue and BAL from control and <i>S. maltophilia</i>-exposed DBA/2N mice.

<p>Spearman correlation coefficients were calculated and significant correlations are shown in bold: ^* p<0.05, ^** p<0.01, ^*** p<0.001.</p><p>° Significant correlation between lung tissue and BAL levels for the same element.</p

Changes in element profiles observed in BAL samples of control and <i>S. maltophilia</i>-infected DBA/2N mice.

<p>Amount of each element was assessed - on days 1, 3, and 7 p.e. to <i>S. maltophilia</i> (Sm111 CF strain, red bars; C39 environmental strain, green bars)or PBS only (controls, black bars) - by ICP-MS analysis in BAL from DBA/2 mice (n = 10 each group). Results are expressed as µg of element for liter of BAL, and graphed as means + SDs. ^*<i>p</i><0.05, vs control mice. ^∧<i>p</i><0.05, between days post-exposure; two-way ANOVA followed by Tukey's post-hoc test.</p

Changes in element profiles observed in lung tissues of control and <i>S. maltophilia</i>-infected DBA/2N mice.

<p>Amount of each element was assessed - on days 1, 3, and 7 p.e. to <i>S. maltophilia</i> (Sm111 CF strain, red bars; C39 environmental strain, green bars)or PBS only (controls, black bars) - by ICP-MS analysis in lung homogenates from DBA/2 mice (n = 10 each group). Results are expressed as ng of element for mg of lung tissue (dry weight), and graphed as means + SDs. ^* p<0.05, vs control mice; ° p<0.05, vs C39 strain; ^∧ p<0.05, between days p.e.; two-way ANOVA followed by Tukey's post-hoc test.</p