ApoB48-LP binds AIP and antagonizes <i>agr</i>-signaling.

<p>(A) <i>S</i>. <i>aureus agr</i>-I strain AH1677 (LAC) (2 x 10⁷ CFUs ml^-1) was cultured for 2 hrs with 10 nM exogenous AIP1 and serum from <i>apoE</i>^<i>-/-</i> or wild type (C57BL/6) mice (0.3%). <i>agr</i>::P3 promoter activation was measured by flow cytometry as mean channel fluorescence (MCF) and the MCF of the no serum control was normalized to 100%. (B) <i>S</i>. <i>aureus</i> was cultured as in (A) along with different concentrations of apoB48-LP or 50 nM human LDL (apoB100). <i>agr</i>::P3 promoter activation was measured by flow cytometry and the MCF of the no apoB control was normalized to 100%. Results shown are means ± SEM from three independent experiments performed in triplicate. (C-D) qRT-PCR analysis of (C) RNAIII and (D) <i>hla</i> expression relative to 16S rRNA under conditions described above. Results are means ± SEM from three independent experiments. (E) (Left) SDS-PAGE analysis of supernatants from 5 h cultures of LAC grown alone or with 50 nM AIP ± 50 nM apoB48-LP. Arrowhead indicates migration of Hla. (Right) Relative Hla concentration was determined by Western blot followed by quantification of band intensity compared to the + AIP control. Data are mean ± SEM of three experiments performed in triplicate. (F) Hla expression in supernatants grown as for (E), assessed via the rabbit red blood cell lysis assay. HA₅₀ is the bacterial supernatant dilution factor required for lysis of 50% of the RBCs. Data are the mean ± SEM of triplicate experiments performed in duplicate. (G) Surface plasmon resonance (SPR) analysis of apoB48-LP binding to immobilized biotinylated AIP1. Binding was measured in resonance units (RU). (H) Anti-apoB antibody at 5-, 10- and 30-fold molar excess, but not IgG control, blocks apoB48-LP binding to AIP1. Results are the mean ± SEM of N = 3 to 5. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p≤0.0001.</p

ApoB48- and apoB100-LP demonstrate equivalent inhibition of agr.

<p>AH1677 (2x10⁷ CFUs ml^-1) was cultured with 50 nM AIP and increasing concentrations of mouse apoB48-LP or human LDL (apoB100). Data from three independent experiments are shown as normalized MCF versus the log of LP concentration.</p

Model of lipoprotein access to sites of infection.

<p>(1) In a healthy subject, the liver releases HDL and VLDL, the latter of which is reduced to LDL by lipase activity. (2) Following oral feeding, enterocytes package dietary lipids and apoB48 into chylomicrons (CM). (3) Lipoproteins are available for host innate defense in the circulation or upon serum extravasation to sites of peripheral infection or inflammation. In a critically ill patient, LP release from the liver is limited as part of the APR and oral feeding may not be possible. The resulting reductions in serum LP levels may negatively impact both peripheral and systemic host innate defense against bacterial pathogens.</p

Depiction of apolipoprotein B.

<p>Schematic of human apoB100 (UniProt P04114) indicating the region aligning with vitellogenin, for which a crystal structure is available [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125027#pone.0125027.ref057" target="_blank">57</a>], the C-terminus of apoB48 and the low density lipoprotein receptor (LDLR) recognition site which facilitates uptake and clearance of apoB100 by the LDLR.</p

Regulatory Role of Glu546 in Flavin Mononucleotide  Heme Electron Transfer in Human Inducible Nitric Oxide Synthase

Nitric oxide (NO) production by mammalian NO synthase (NOS) is believed to be regulated by the docking of the flavin mononucleotide (FMN) domain in one subunit of the dimer onto the heme domain of the adjacent subunit. Glu546, a conserved charged surface residue of the FMN domain in human inducible NOS (iNOS), is proposed to participate in the interdomain FMN/heme interactions [Sempombe et al. <i>Inorg. Chem.</i> <b>2011</b>, <i>50</i>, 6869–6861]. In the present work, we further investigated the role of the E546 residue in the FMN–heme interdomain electron transfer (IET), a catalytically essential step in the NOS enzymes. Laser flash photolysis was employed to directly measure the FMN–heme IET kinetics for the E546N mutant of human iNOS oxygenase/FMN (oxyFMN) construct. The temperature dependence of the IET kinetics was also measured over the temperature range of 283–304 K to determine changes in the IET activation parameters. The E546N mutation was found to retard the IET by significantly raising the activation entropic barrier. Moreover, pulsed electron paramagnetic resonance data showed that the geometry of the docked FMN/heme complex in the mutant is basically the same as in the wild type construct, whereas the probability of formation of such a complex is about twice lower. These results indicate that the retarded IET in the E546N mutant is not caused by an altered conformation of the docked FMN/heme complex, but by a lower population of the IET-active conformation. In addition, the negative activation entropy of the mutant is still substantially lower than that of the holoenzyme. This supports a mechanism by which the FMN domain can modify the IET through altering probability of the docked state formation

Probing the Hydrogen Bonding of the Ferrous–NO Heme Center of nNOS by Pulsed Electron Paramagnetic Resonance

Oxidation of l-arginine (l-Arg) to nitric oxide (NO) by NO synthase (NOS) takes place at the heme active site. It is of current interest to study structures of the heme species that activates O₂ and transforms the substrate. The NOS ferrous–NO complex is a close mimic of the obligatory ferric (hydro)­peroxo intermediate in NOS catalysis. In this work, pulsed electron–nuclear double resonance (ENDOR) spectroscopy was used to probe the hydrogen bonding of the NO ligand in the ferrous–NO heme center of neuronal NOS (nNOS) without a substrate and with l-Arg or <i>N</i>-hydroxy-l-arginine (NOHA) substrates. Unexpectedly, no H-bonding interaction connecting the NO ligand to the active site water molecule or the Arg substrate was detected, in contrast to the results obtained by X-ray crystallography for the Arg-bound nNOS heme domain [Li et al. J. Biol. Inorg. Chem. 2006, 11, 753−768]. The nearby exchangeable proton in both the no-substrate and Arg-containing nNOS samples is located outside the H-bonding range and, on the basis of the obtained structural constraints, can belong to the active site water (or OH). On the contrary, in the NOHA-bound sample, the nearby exchangeable hydrogen forms an H-bond with the NO ligand (on the basis of its distance from the NO ligand and a nonzero isotropic <i>hfi</i> constant), but it does not belong to the active site water molecule because the water oxygen atom (detected by ¹⁷O ENDOR) is too far. This hydrogen should therefore come from the NOHA substrate, which is in agreement with the X-ray crystallography work [Li et al. Biochemistry 2009, 48, 10246−10254]. The nearby nonexchangeable hydrogen atom assigned as H_ε of Phe584 was detected in all three samples. This hydrogen atom may have a stabilizing effect on the NO ligand and probably determines its position

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

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

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