Staphylococcus aureus uses the ArlRS and MgrA cascade to regulate immune evasion during skin infection

Skin is one of the most common sites of host immune response against Staphylococcus aureus infection. Here, through a combination of in vitro assays, mouse models, and intravital imaging, we find that S. aureus immune evasion in skin is controlled by a cascade composed of the ArlRS two-component regulatory system and its downstream effector, MgrA. S. aureus lacking either ArlRS or MgrA is less virulent and unable to form correct abscess structure due to de-repression of a giant surface protein, Ebh. These S. aureus mutants also have decreased expression of immune evasion factors (leukocidins, chemotaxis-inhibitory protein of S. aureus [CHIPS], staphylococcal complement inhibitor [SCIN], and nuclease) and are unable to kill neutrophils, block their chemotaxis, degrade neutrophil extracellular traps, and survive direct neutrophil attack. The combination of disrupted abscess structure and reduced immune evasion factors makes S. aureus susceptible to host defenses. ArlRS and MgrA are therefore the main regulators of S. aureus immune evasion and promising treatment targets

PSM associate with serum lipoproteins.

<p>(A) Serum pull down assay with PSMα1 and PSMα3 coupled to CNBr beads. Beads were extensively washed with PBS or PBS with tween (PBST). Serum proteins bound and eluted from the beads were visualized by SDS-page followed by instant blue staining. Protein bands specifically appearing in the PSMα1 and PSMα3 lane were identified by MALDI-TOF mass spectrometry as ApoA1. (B) Gel filtration association assay. Comparison of absorption (OD492 nm) profiles of 100 µg/ml PSMα3-FITC pre-incubated with PBS, 10% human serum, 1 mg/ml HDL, 1 mg/ml LDL or 1 mg/mL VLDL for 30 min, before separation on a gel filtration column. For monomerization of PSMα3-FITC, the gel filtration column was equilibrated with PBS containing 0.1% sodium deoxycholate (DOC). Representative figures of two independent experiments.</p

Human serum inhibits PSM-mediated neutrophil activation.

<p>(A) Calcium mobilization of human neutrophils. Neutrophils were stimulated with 10⁻⁶ M PSMα1, 10⁻⁷ M PSMα2, 10⁻⁷ M PSMα3, 10⁻⁶ M PSMα4, 3×10⁻⁶ M δ-toxin, 10⁻⁵ M PSMβ1, 10⁻⁵ M PSMβ2, 10⁻⁹ M fMLP, 10⁻¹⁰ M C5a and 10⁻¹⁰ M IL-8, all preincubated with or without 0.1% heat-inactivated human serum, before calcium mobilization was measured by flow cytometry. *, p<0.001; N.S., not significant. (B) Time-dependent inhibition of PSMα3-mediated calcium mobilization of HL60/FPR2 cells. PSMα3, 100 nM or 500 nM, was preincubated with 0.1% human serum and calcium mobilization was measured at different time-points by flow cytometry. (C) Dose-response curves for calcium mobilization in HL-60/FPR2 cells induced by PSMα3 or serum-treated PSMα3. Data represent means ± SEM of at least three independent experiments.</p

Human serum inhibits the activity of PSM in culture supernatants.

<p>(A) Dose-dependent calcium mobilization of HL-60/FPR2 cells by culture supernatants of <i>S. aureus</i> strains MW2 and MW2 <i>agr</i> KO, with or without preincubation with 1% heat-inactivated human serum. (B) Dose-dependent neutrophil lysis by <i>S. aureus</i> culture supernatants with or without preincubation with 5% heat-inactivated human serum. Neutrophil lysis was measured through LDH release. Data represent means ± SEM of three independent experiments.</p

Identifying the most potent inhibitor of PSM in serum.

<p>Functional screening of serum fractions, isolated by gel filtration, for the inhibition of neutrophil lysis. Serum fractions were incubated with (A) 50 µM or (B) 10 µM of PSMα3 before addition to neutrophils and neutrophil lysis was measured via LDH release. Data represent means ± SEM of three independent experiments. (C) PSM concentration measured in isolated lipoprotein fractions after spiking human serum with 0.5 mg/ml PSMα2. PSM concentration was measured by reverse phase-HPLC and represents the mean of the area under the curve of PSMα2 of 3 independent experiments. (D) Measurement of the concentration PSM by HPLC in isolated HDL fraction from an overnight whole blood culture of the <i>S. aureus</i> MW2 strain (black line) or control (no bacteria; gray line). HDL fractions were subjected to HPLC and absorbance at 214 nm was obtained. Respective PSM were identified by LC/MS, δ-toxin and PSMα2 were contained in the same peak in this assay condition.</p

Serum lipoproteins inhibit PSM-mediated neutrophil lysis and activation.

<p>(A) Dose-dependent neutrophil lysis by synthetic PSMα3 preincubated with 1% and 10% human serum or human lipid-free (LF) serum or preincubated with (B) 5 and 50 µg/ml HDL or 10 and 100 µg/ml LDL (concentrations are based on protein content). PBS was used as buffer control. Neutrophil lysis was measured via LDH release. (C) Calcium mobilization of human neutrophils. Neutrophils were stimulated with 10⁻⁶ M PSMα1, 10⁻⁷ M PSMα2, 10⁻⁷ M PSMα3, 10⁻⁶ M PSMα4, 3×10⁻⁶ M δ-toxin, 10⁻⁵ M PSMβ1, 10⁻⁵ M PSMβ2, 10⁻⁹ M fMLP, 10⁻¹⁰ M C5a and 10⁻¹⁰ M IL-8, all preincubated with or without 5 µg/ml HDL or LDL, before calcium mobilization was measured by flow cytometry. *, p<0.001; N.S., not significant.</p

Rapid upregulation of PSMα expression after phagocytosis.

<p><i>S. aureus</i> MW2 containing the PSMα promoter-GFP construct was incubated with neutrophils, and fluorescence as a measure for PSMα expression was monitored over time (0–3 hours). <i>Arrows</i> indicate neutrophils which have phagocytozed <i>S. aureus</i>. (*) Indicate growing reporter bacteria outside of the cells. Bars: 50 µm.</p