The virulence regulator Agr controls the staphylococcal capacity to activate human neutrophils via the formyl peptide receptor 2

Abstract

The Agr quorum-sensing system represents the master regulator for staphylococcal virulence factors and is known to have a strong impact on the release of pathogen-associated molecular pattern (PAMP) molecules. Among the various staphylococcal PAMPs, phenol-soluble modulin (PSM) peptides have attracted increasing interest because they are crucial for staphylococcal virulence and have neutrophil-recruiting properties. The latter depend on recognition of PSMs by the neutrophil formyl peptide receptor 2 (FPR2/ALX), for which PSMs are highly efficient agonists. We demonstrate that Agr inactivation in Staphylococcus aureus or S. epidermidis leads to strongly reduced neutrophil responses, which is in agreement with the previously reported strict control of PSM expression by Agr. Agr had a distinct and profound impact on activation of FPR2/ALX but not of the related FPR1 receptor that senses bacterial formylated peptides. S. epidermidis PSMs had similar FPR2/ALX-activating properties but differed in their dependence on N-terminal formylation compared to S. aureus PSMs. Moreover, S. aureus and S. epidermidis PSMs upregulated the neutrophil complement receptor CD11b via FPR2/ALX stimulation in an Agr-dependent fashion. Hence, Agr controls the capacity of staphylococcal pathogens to activate FPR2/ALX-dependent neutrophil responses, underscoring the crucial role of FPR2/ALX and PSMs in staphylococcus-host interaction.

Fig. 1

S. epidermidis PSM peptides induce chemotaxis and calcium ion fluxes in human neutrophils. a Formylated PSMs and nonformylated PSMε, PSMβ1 and PSMβ2 induce Ca²⁺ flux in human neutrophils (values normalized to 1 μM). b, c Neutrophil stimulation by PSMs (formylated PSMα 300 nM, PSMδ 200 nM, δ-toxin 500 nM, and PSMε 200 nM, formylated and nonformylated PSMβ1 1.5 μM, PSMβ2 1.8 μM and nonformylated PSMε 300 nM) is sensitive to PTX and the FPR2/ALX-specific inhibitor FLIPr, but not the FPR1-specific inhibitor CHIPS. fMLF (10 nM) and MMK1 (20 nM) are synthetic control ligands of FPR1 and FPR2/ALX, respectively. d Formylated PSMα (250 nm), PSMδ (250 nm), PSMε (100 nm), δ-toxin (1 μm) and nonformylated PSMε (100 nm) induce chemotaxis in human neutrophils. e Formylated and nonformylated PSMε stimulates chemotaxis in human neutrophils at nanomolar concentrations with a typical bell-shaped dose-response curve. Data represent means ± SEM of 3 independent experiments. * p < 0.05; ** p < 0.005; *** p < 0.001 versus no inhibition (b, c). a–c All values were significant versus buffer control in (a). δ = δ-Toxin; fl = fluorescence; inhib = inhibitor; n.s. = not significant.

Fig. 2

S. epidermidis PSMs specifically activate FPR2/ALX-transfected HL60 cells. a–c PSMs (formylated PSMα 300 nM, PSMδ 200 nM, δ-toxin 500 nM, PSMε 200 nM and nonformylated PSMε 300 nM) stimulated strong calcium fluxes in FPR2/ALX-transfected (b) but not in FPR1-transfected cells (a) and only slightly in FPR3-transfected cells (c). Nontransfected HL60 cells exhibited no significant responses (mean fluorescence values below 1; data not shown). fMLF (10 nM) and MMK1 (10 nM) are synthetic control ligands of FPR1 and FPR2/ALX, respectively. d Diluted culture filtrates (0.375%) of 11 clinical (catheter) isolates and 4 nasal isolates from healthy volunteers induce calcium fluxes in FPR2-transfected cells. Data represent means ± SEM of at least 3 independent experiments. * p < 0.05; *** p < 0.001 versus nontransfected cells. δ = δ-Toxin; fl = fluorescence; n.s. = not significant.

Fig. 3

IL-8 secretion and calcium fluxes of neutrophils stimulated by staphylococcal culture filtrates are largely Agr and FPR2/ALX-dependent. IL-8 release from neutrophils induced by culture filtrates from S. aureus USA300 and the isogenic Agr mutant (a), S. aureus RN6390 and the isogenic Agr mutant RN6911 (b) and S. epidermidis and the isogenic Agr mutant (c) in the presence or absence of FLIPr. d Calcium influx in neutrophils stimulated by S. aureus or S. epidermidis culture filtrates in the presence or absence of FLIPr. Data represent means ± SEM of 3 independent experiments with neutrophils of different persons and at least 3 different culture filtrates. * p < 0.05; ** p < 0.005; *** p < 0.001. n.s. = Not significant.

Fig. 4

Calcium influx stimulated by S. aureus and S. epidermidis culture filtrates are FPR2/ALX-dependent. FPR2/ALX-transfected HL60 cells respond strongly to S. epidermidis 1457 (a), S. aureus RN6390 (b) and USA300 (c) wild-type strains but only moderately to the corresponding isogenic Agr mutants, while FPR1- or FPR3-mediated responses differ only slightly between the various S. epidermidis (d, g) and S. aureus (e–h) strains. Nontransfected HL60 cells exhibited no significant responses (mean fluorescence values below 1; data not shown). Data represent means ± SEM of 3 independent experiments and at least 3 different culture filtrates.

Fig. 5

PSM-induced CD11b upregulation is inhibited by the FPR2/ALX antagonist FLIPr. CD11b upregulation of neutrophils induced by formylated PSM peptides of S. aureus (PSMα2 1 μM, PSMα3 500 nM, δ-toxin 250 nM) or S. epidermidis (PSMα 500 nM, PSMδ 500 nM, and PSMε 500 nM) with and without FLIPr preincubation (a) and culture filtrates from S. epidermidis 1457 (0.75%), S. aureus RN6390 (0.75%), and USA300 (0.09%) or the isogenic Agr mutants (b). CD11b upregulation of neutrophils with and without FLIPr preincubation induced by culture filtrates from S. aureus RN6390 and the isogenic Agr mutant (0.75%) (c) or USA300 and USA300 ∆agr (0.09%) (d). Data represent means ± SEM of 3 independent experiments with neutrophils of different individuals and at least 3 different culture filtrates. * p < 0.05; *** p < 0.001. HSA = Human serum albumin; n.s. = not significant; S.a. = S. aureus; S.e. = S. epidermidis; TSB = tryptic soy broth.