<i>Staphylococcus aureus sarA</i> Regulates Inflammation and Colonization during Central Nervous System Biofilm Formation

<div><p>Infection is a frequent and serious complication following the treatment of hydrocephalus with CSF shunts, with limited therapeutic options because of biofilm formation along the catheter surface. Here we evaluated the possibility that the <i>sarA</i> regulatory locus engenders <i>S. aureus</i> more resistant to immune recognition in the central nervous system (CNS) based on its reported ability to regulate biofilm formation. We utilized our established model of CNS catheter-associated infection, similar to CSF shunt infections seen in humans, to compare the kinetics of bacterial titers, cytokine production and inflammatory cell influx elicited by wild type <i>S. aureus</i> versus an isogenic <i>sarA</i> mutant. The <i>sarA</i> mutant was more rapidly cleared from infected catheters compared to its isogenic wild type strain. Consistent with this finding, several pro-inflammatory cytokines and chemokines, including IL-17, CXCL1, and IL-1β were significantly increased in the brain following infection with the <i>sarA</i> mutant versus wild type <i>S. aureus</i>, in agreement with the fact that the <i>sarA</i> mutant displayed impaired biofilm growth and favored a planktonic state. Neutrophil influx into the infected hemisphere was also increased in the animals infected with the <i>sarA</i> mutant compared to wild type bacteria. These changes were not attributable to extracellular protease activity, which is increased in the context of SarA mutation, since similar responses were observed between <i>sarA</i> and a <i>sarA/</i>protease mutant. Overall, these results demonstrate that <i>sarA</i> plays an important role in attenuating the inflammatory response during staphylococcal biofilm infection in the CNS via a mechanism that remains to be determined.</p></div

<i>sarA</i> is critical for catheter-associated biofilm formation in a CNS catheter infection model.

<p>Infected catheters were removed, rinsed and sonicated for quantification of viable bacteria associated with wild type ACH1719 (<b>WT</b>) or ACH1719Δ<i>sarA</i> (<b>Δ</b><b><i>sarA</i></b>) <i>S. aureus</i>. At all time points, bacterial burdens in brain parenchyma were lower than the corresponding catheter cultures (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084089#pone.0084089.s002" target="_blank">Figure S2</a>). <b>*</b> = p<0.05 WT catheter vs Δ<i>sarA</i> catheter; (n = 11–21 mice/group/time point).</p

CNS catheter-associated biofilm infection is associated with attenuated inflammation compared to parenchymal abscesses.

<p>The tissues encompassing the brain abscess and the tissues surrounding the infected catheters were homogenized and the resulting supernatants analyzed for levels of the pro-inflammatory mediators CXCL1 (A, B) and IL-17 (C, D). Raw cytokine/chemokine levels are reported (A, C) as well as correction for disparate bacterial burdens (B, D) * = p<0.05 (n = 12–18 mice/group/time point).</p

<i>sarA</i> dictates the extent of proinflammatory mediator expression during CNS catheter-associated infection.

<p>Catheter-associated infections were generated with either wild type ACH1719 (<b>WT</b>) or ACH1719Δ<i>sarA</i> (<b>Δ</b><b><i>sarA</i></b>). The tissue surrounding the infected catheters was homogenized and the resulting supernatant analyzed for levels of the pro-inflammatory mediators CXCL1 (A, B) and IL-1β (C, D). Results are presented as raw data (A and C) and after adjustment for divergent bacterial burdens (B and D). * = p<0.05 (n = 11–21 mice/group/time point).</p

<i>sarA</i> influences peripheral immune cell influx during CNS catheter-associated infection.

<p>Catheter associated cells were recovered from mice at days 3, 7 and 14-infection, whereupon pooled tissue from 4–5 mice per group (wild type ACH1719– <b>WT</b> vs <i>ACH1719</i>Δ<i>sarA</i> – <b>Δ</b><b><i>sarA</i></b>) was analyzed for neutrophil (A), macrophage (B) and T cell (C) infiltrates, with three independent replicates. Data was corrected for bacterial burdens at each time point. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084089#pone.0084089.s002" target="_blank">Figure S2</a> for representative histograms.</p

<i>sarA</i> and extracellular proteases significantly affect biofilm formation in a CNS catheter infection model.

<p>Infected catheters were removed, rinsed and sonicated for quantification of viable bacteria associated with wild type USA300 LAC (<b>WT catheter</b>), USA300 LACΔ<i>sarA</i> (<b>SarA-deficient catheter</b>), extracellular protease deficient USA300 LAC (<b>Protease-deficient catheter</b>), or SarA and extracellular protease deficient (<b>SarA-Protease-deficient catheter</b>) <i>S. aureus</i>. At all time points, bacterial burdens in brain parenchyma were 3–5 log lower than the corresponding catheter cultures (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084089#pone.0084089.s004" target="_blank">Figure S4</a>). <b>a</b> = p<0.05 WT vs SarA-deficient catheter; <b>b</b> = p<0.05 WT vs Protease-deficient catheter; <b>c</b> = p<0.05 WT vs SarA-Protease-deficient catheter; <b>d</b> = p<0.05 SarA-deficient vs Protease-deficient catheter; <b>e</b> = SarA-deficient vs SarA-Protease-deficient catheter; <b>f</b> = SarA-Protease-deficient vs Protease-deficient catheter (n = 13–15 mice/group/time point).</p

MSSA CNS catheter-associated biofilm infection persists longer than a parenchymal brain abscess.

<p>The infected tissue was removed, homogenized and cultured to enumerate bacterial burdens in the tissue containing the brain abscess or surrounding the infected catheter. Catheters were also removed, rinsed and sonicated for quantification of viable bacteria. * = p<0.05 (n = 12–18 mice/group/time point).</p

α-toxin levels are reduced during CNS catheter-associated infection with a <i>sarA</i> mutant.

<p>Alpha-toxin levels were measured in brain homogenates of animals infected with the ACH1719 <i>sarA</i> mutant (<b>Δ</b><b><i>sarA</i></b>) or wild type ACH1719 strain (<b>WT</b>) at the indicated time points post-infection using a custom-designed ELISA. * = p<0.05 (n = 13–15 mice/group/time point).</p

Inflammatory mediators are elevated in response to infection with <i>sarA</i> and <i>sarA-</i>protease mutants independently of protease activity.

<p>Catheter-associated infections were generated with wild type USA300 LAC (<b>WT catheter</b>), USA300 LACΔ<i>sarA</i> (<b>SarA-deficient catheter</b>), extracellular protease deficient USA300 LAC (<b>Protease-deficient catheter</b>), or SarA and extracellular protease deficient (<b>SarA-Protease-deficient catheter</b>) <i>S. aureus</i>. The tissue surrounding the infected catheters was homogenized and the resulting supernatant analyzed for levels of the pro-inflammatory mediators CXCL1 (A, B) and IL-1β (C, D). Results are presented as raw data (A and C) and after adjustment for divergent bacterial burdens (B and D). * = p<0.05 (n = 13–15 mice/group/time point).</p