Nox2 Modification of LDL Is Essential for Optimal Apolipoprotein B-mediated Control of <em>agr</em> Type III <em>Staphylococcus aureus</em> Quorum-sensing

<div><p><em>Staphylococcus aureus</em> contains an autoinducing quorum-sensing system encoded within the <em>agr</em> operon that coordinates expression of virulence genes required for invasive infection. Allelic variation within <em>agr</em> has generated four <em>agr</em> specific groups, <em>agr</em> I–IV, each of which secretes a distinct autoinducing peptide pheromone (AIP1-4) that drives <em>agr</em> signaling. Because <em>agr</em> signaling mediates a phenotypic change in this pathogen from an adherent colonizing phenotype to one associated with considerable tissue injury and invasiveness, we postulated that a significant contribution to host defense against tissue damaging and invasive infections could be provided by innate immune mechanisms that antagonize <em>agr</em> signaling. We determined whether two host defense factors that inhibit AIP1-induced <em>agr</em>I signaling, Nox2 and apolipoprotein B (apoB), also contribute to innate control of AIP3-induced <em>agr</em>III signaling. We hypothesized that apoB and Nox2 would function differently against AIP3, which differs from AIP1 in amino acid sequence and length. Here we show that unlike AIP1, AIP3 is resistant to direct oxidant inactivation by Nox2 characteristic ROS. Rather, the contribution of Nox2 to defense against <em>agr</em>III signaling is through oxidation of LDL. ApoB in the context of oxLDL, and not LDL, provides optimal host defense against <em>S. aureus agr</em>III infection by binding the secreted signaling peptide, AIP3, and preventing expression of the <em>agr</em>-driven virulence factors which mediate invasive infection. ApoB within the context of oxLDL also binds AIP 1-4 and oxLDL antagonizes <em>agr</em> signaling by all four <em>agr</em> alleles. Our results suggest that Nox2-mediated oxidation of LDL facilitates a conformational change in apoB to one sufficient for binding and sequestration of all four AIPs, demonstrating the interdependence of apoB and Nox2 in host defense against <em>agr</em> signaling. These data reveal a novel role for oxLDL in host defense against <em>S. aureus</em> quorum-sensing signaling.</p> </div

ROS-dependent oxidation of LDL but not of AIP3 mediates antagonism of <i>agr</i>III-dependent quorum sensing.

<p>(A, B) LDL, apoB or oxLDL at equimolar concentrations of apoB were treated as shown with (A) HOCl for 30 min at 37°C, with or without prior addition of the ROS scavenger N-acetylmethionine (NAM), or (B) singlet oxygen (5 min on ice). 10 nM ROS-treated lipoproteins or controls were cultured overnight with <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] plus 50 nM AIP. <i>agr</i>:P3 promoter activation was measured by flow cytometry. (C) Strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] was cultured overnight with 50 nM AIP3 plus LDL treated with increasing concentrations of singlet oxygen. (D) Measurement of LDL lipid oxidation before and after exposure to singlet oxygen or HOCl. Commercially available oxLDL and bovine serum albumin are given as controls. Data represent the mean ± SEM. (E) AIP1 and AIP3 were incubated with buffer or HOCl for 30 min at 37°C, treated with NAM to scavenge remaining ROS, and cultured at 50 nM for 2 h with <i>agr</i>I isolate LAC [<i>agr</i>::P3-yfp] or <i>agr</i>III MW2 [<i>agr</i>::P3-yfp], respectively. Data was measured by flow cytometry. Data points represent the mean ± SEM normalized to 100% activation by untreated AIP and statistics are in reference to this control. (F) AIP1 and AIP3 were exposed to singlet oxygen by incubation with rose bengal in the presence or absence of light, prior to culture with <i>agr</i>I isolate LAC [<i>agr</i>::P3-yfp] or <i>agr</i>III MW2 [<i>agr</i>::P3-yfp]. Data points represent the mean ± SEM normalized to 100% activation by untreated AIP and statistics are in reference to this control. (G) AIP1 was combined with buffer or a 10-fold molar excess of ethylthioacetate prior to exposure to singlet oxygen, followed by culture with <i>agr</i>I isolate LAC [<i>agr</i>::P3-yfp]. Data points represent the mean ± SEM of the mean channel fluorescence. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

Savirin inhibits AgrA-dependent transcription in clinical isolates.

<p>Effect of savirin (5 µg ml⁻¹) vs. vehicle on <i>psm alpha</i> transcripts determined by qRT-PCR in clinical <i>S. aureus</i> isolates of each <i>agr</i> allele after 5 hr of culture. Data are represented as the mean of 5 replicates. Significance determined by two-way repeated measures ANOVA.</p

Effect of savirin treatment on <i>in vitro</i> host-dependent killing of LAC <i>agr</i>+ and Δ<i>agr</i>.

<p>(<b>A</b>) Percent intracellular survival of LAC <i>agr</i>+ (plus AIP1) or Δ<i>agr</i> treated with savirin (5 µg ml⁻¹) vs. vehicle for 5 hr prior to opsonization and phagocytosis by mouse macrophages (MOI 1∶1). Viable intracellular CFU set at 100% after internalization for 1 hr. Mean ± s.e.m., n = 3 independent experiments performed in triplicate. (<b>B</b>) Log CFU remaining of 1.0×10⁸ LAC <i>agr</i>+ (plus AIP1) or Δ<i>agr</i> treated with savirin (5 µg ml⁻¹) vs. vehicle for 5 hr prior to incubation at pH 2.5 for 2 hr. Mean ± SEM, n = 6. ***p<0.001 **p<0.01, *p<0.05 by two-tailed Student's <i>t</i>-test.</p

Reduction of serum apoB levels in <i>Nox2</i> knockout mice but not wild-type mice significantly increases susceptibility to <i>agr</i>III-mediated <i>S. aur</i>eus virulence.

<p>(A) Blood was collected from wild-type or <i>Nox2^−/−</i> mice treated with 4APP or vehicle control. After clearing, the serum was heat inactivated and diluted to 10% in TSB for overnight culture with <i>agr</i>III MW2 [<i>agr</i>::P3-yfp]. <i>agr</i>::P3 promoter activation was measured by flow cytometry. Data reported are the mean ± SEM normalized to broth control at 100%. (B) Air-pouch schematic showing locations of lumen, epidermis, dermis and muscle. (C–E) Air-pouches were generated on the backs of 8 to 12 week old wild-type or <i>Nox2^−/−</i> mice. Where indicated 4APP treated mice were injected i.p. with 100 µl of 5 mg/ml 4APP or vehicle control at 48 h and 24 h prior to infection with MW2 or its <i>agr</i> deletion mutant (Δ<i>agr</i>) at the indicated concentrations. At time zero, air-pouches were injected with early exponential phase MW2. At 28 h post-infection, the following parameters were determined and data reported as the mean ± SEM: (Left to right) Representative confocal images of dermis from indicated pouches stained with TO-PRO-3 (Invitrogen; blue) and anti-<i>S. aureus</i> antibody (green fluorophore) (Scale bar = 20 µm); Quantification of bacterial density in dermis of pouches; Morbidity was scored on a 0–14 point scale and was based on weight loss, appearance, level of dehydration, mobility and responsiveness; Bacterial burden (Log CFU) in spleen. (C) Wild-type mice treated with 4APP or vehicle control. (D) <i>Nox2^−/−</i> mice treated with 4APP or vehicle control. (E) Wild-type and <i>Nox2^−/−</i> mice. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

Effect of oxLDL on antagonism of <i>agr</i>III dependent virulence factor production by MRSA and MSSA clinical isolates.

<p>(A–D) Bacteria were cultured overnight in the presence of media control or oxLDL (50 nM). (A) RNAIII and (B) <i>hla</i> transcription relative to 16S rRNA was determined by qRT-PCR (C) Alpha-hemolysin content of culture supernatants was measured by the ability to lyse rRBCs. (D) Supernatants from (C) were also assessed for lipase activity determined by rate of cleavage of the triglyceride substrate tributyrin. Data reported as the mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

Savirin inhibits RNA III levels in <i>S. aureus</i>, but not <i>S. epidermidis</i>, without affecting <i>agr</i>-independent growth.

<p>(<b>A</b>) Chemical structure of savirin (3-(4-propan-2-ylphenyl) sulfonyl-1H-triazolo [1,5-a] quinazolin-5-one). Effect of savirin (5 µg ml⁻¹) vs vehicle control on (<b>B</b>) RNAIII levels induced by 50 nM AIP1 at 1 hr in MRSA strain USA300 LAC; (<b>C</b>) RNAIII levels in LAC without exogenous AIP1 at 5 hrs; (<b>D</b>) growth of LAC compared to growth of LAC Δ<i>agr</i>; (<b>E</b>) RNAIII levels in <i>S. epidermidis</i> induced by overnight culture supernatant containing <i>S. epidermidis</i> AIP at 1 hr; and (<b>F</b>) growth of <i>S. epidermidis</i>. Data are represented as mean ± SEM, n = 3 experiments (<b>B</b>, <b>C</b>, <b>D</b>, & <b>F</b>) or n = 6 (<b>E</b>) performed in triplicate. ***p<0.001 **p<0.01, *p<0.05 by two-tailed Student's <i>t</i>-test.</p

Optimal apolipoprotein B-mediated antagonism of <i>agr</i>III-signaling requires oxidation of LDL via Nox2.

<p>(A) Schematic representation of AIP1 and AIP3. (B) Strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] was cultured overnight with broth control or 10 nM LDL, apoB or oxLDL, and <i>agr</i>::P3 activation was measured by flow cytometry. Colony forming units (cfus) were determined by plating on sheep blood agar. (C) Anti-apoB antibody reverses antagonism of <i>agr</i>::P3 promoter activation in MW2. Strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] was cultured overnight with broth control, 10 nM apoB or oxLDL, along with 30 nM control IgG or 30 nM apoB-specific IgG. (D) LDL, ApoB or oxLDL binding to immobilized AIP3 was measured by SPR following lipoprotein incubation with apoB-specific IgG or control IgG. Data were normalized to the mean ± SEM of LDL binding in the absence of antibody. (E) Blood was collected from wild-type or <i>Nox2^−/−</i> mice. After clearing, the serum was heat inactivated and diluted to 10% in TSB for overnight culture with strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp]. <i>agr</i>::P3 promoter activation was measured by flow cytometry. Data reported are the mean ± SEM normalized to broth control. (F) Strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] cultured 4 h with 100 nM AIP plus 10% sera from <i>Nox2^−/−</i> mice along with 50 nM LDL or 50 nM oxLDL. Data points represent the mean ± SEM normalized to broth control. (G) Immunoblot detection of apoB and oxidized LDL. Control LDL and oxLDL plus serum from wild-type and <i>Nox2^−/−</i> mice were vacuum transferred to nitrocellulose and stained for oxLDL using monoclonal antibody E06 or rabbit polyclonal antibody to apoB. A representative blot is shown. Band intensity was quantified using Carestream Molecular Imaging software (New Haven, Connecticut), and data normalized to oxLDL or wild-type sera with E06/apoB ratios equal to 1. (H) Strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] was cultured overnight with exogenous AIP3 (50 nM) and 10 nM of either human LDL, human oxLDL, LDL purified from wild-type mice, LDL purified from <i>Nox2^−/−</i> mice or broth control. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.</p

Exogenous oxLDL restores in vivo antagonism of <i>agr</i>III-signaling.

<p>(A) Control LDL and oxLDL plus air-pouch lavage from MW2 infected wild-type and <i>Nox2^−/−</i> mice was vacuum transferred to nitrocellulose and stained for oxLDL using monoclonal antibody E06 or rabbit polyclonal antibody to apoB. A representative blot is shown. Band intensity was quantified using Carestream Molecular Imaging software (New Haven, Connecticut), and data normalized to oxLDL or wild-type lavage with E06/apoB ratios equal to 1. (B) Air-pouches were generated on the backs of 8 to 12 week old <i>Nox2^−/−</i> mice treated with 4APP as previously described. At time zero, 4×10⁷ cfu of early exponential phase <i>agr</i>III isolate MW2 [<i>agr</i>::P3-yfp] were injected into the air-pouch along with saline control or the following as indicated: 100 nM AIP3 and either buffer control, 100 nM LDL or 100 nM oxLDL. After 4 h pouches were lavaged and <i>agr</i>::P3 promoter activation assessed by flow cytometry. Data reported are the mean ± SEM normalized to PBS/AIP3 control. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.</p

Savirin inhibits <i>agr</i>-regulated gene transcription and secreted virulence factor production.

<p>(<b>A</b>) Graphic representation by category of microarray results in LAC after 5 hrs of culture with 50 nM AIP1 and 5 µg ml⁻¹ of savirin. Of 205 <i>agr</i>-regulated transcripts, 122 were affected by savirin by 2 fold or greater and p<0.05, n = 3. All major virulence factors are represented; others shown are representative of the category. (<b>B</b>) Effect of savirin (5 µg ml⁻¹) on transcription induced by 50 nM AIP1 in LAC for <i>hla</i>, <i>psm alpha</i>, and <i>pvl</i> relative to 16S at 1 hr and for <i>agrA</i> and <i>agrC</i> at 5 hr. (<b>C</b>) Effect of savirin (5 µg ml⁻¹) on overnight alpha-hemolysin production by LAC. (<b>D</b>) Effect of savirin (5 µg ml⁻¹) vs. vehicle on the capacity of culture supernatant from a clinical <i>agr</i> I blood stream isolate to lyse human neutrophils as measured by LDH release after 2 hr. Data are represented as the mean ± SEM, n = 3 independent experiments. ***p<0.001 **p<0.01, *p<0.05 by two-tailed Student's <i>t</i>-test.</p