Staphylococcus aureus–Derived PSMα Peptides Activate Neutrophil FPR2 but Lack the Ability to Mediate β-Arrestin Recruitment and Chemotaxis

Abstract

Formyl peptide receptor 2 (FPR2) is a G protein-coupled pattern recognition receptor sensing both mitochondrial-and bacterialderived formylated peptides, including the PSMa toxins secreted by community-associated methicillin-resistant Staphylococcus aureus strains. Similar to many other FPR2 agonistic peptides, nanomolar concentrations of both PSMa2 and PSMa3 activate neutrophils to increase the cytosolic concentration of Ca 2+ and release NADPH oxidase-derived reactive oxygen species. In addition, the PSMa peptides induce FPR2 homologous desensitization, actin polymerization, and neutrophil reactivation through a receptor cross-talk mechanism. However, in contrast to conventional FPR2 agonistic peptides, including the host-derived formyl peptide MCT-ND4, we found that the PSMa peptides lacked the ability to recruit b-arrestin and induce neutrophil chemotaxis, supporting the previous notion that b-arrestin translocation is of importance for cell migration. Despite the lack of b-arrestin recruitment, the PSMa peptides induced an FPR2-dependent ERK1/2 phosphorylation and internalization. Furthermore, structure-activity relationship analysis with PSMa2 derivatives revealed critical roles of the first 3 aa linked to N-fMet as well as the C terminus of PSMa2 in promoting FPR2 to recruit b-arrestin. In summary, our data demonstrate a novel neutrophil activation pattern upon FPR2 sensing of PSMa peptides, signified by the ability to induce increased intracellular Ca 2+ , ERK1/2 phosphorylation, internalization, and NADPH oxidase activity, yet lack of b-arrestin recruitment and neutrophil chemoattraction. These novel features adopted by the PSMa peptides could be of importance for S. aureus virulence and might facilitate identification of new therapeutic strategies for treating S. aureus infections.

Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis

“…Similarly, Barbadin also significantly primed the ROS production induced by F2Pal10 and Cmp 14 ( Fig 3F). The fact that F2Pal10 and Cmp 14 cannot induce -arrestin recruitment at these concentrations ( [9][10][11], Fig 2A), strongly suggests that the priming effect of Barbadin on FPR2-mediated ROS production is independent of the ability of the activating agonist to recruit -arrestin. In addition, the ROS release following activation with PMA, a compound that bypasses receptors and directly activate protein kinase C (PKC), was unaffected by Barbadin ( Fig 3G, H), which suggests that Barbadin lacks a direct effect on the ROS-producing NADPH-oxidase machinery.…”

“…Barbadin was present in both upper and lower chambers to avoid any gradient effect. The migrated cells were quantified by measuring myeloperoxidase activity of cell lysates [11]. All migration (myeloperoxidase activity) values were subtracted from the level of migration without any attractant in the lower compartment (negative control), and the resulting data are presented as the number of cells recovered in the lower compartment, relative to the total number of cells applied to the migration system (neutrophils were added directly to the bottom well of the chemotaxis plate).…”

“…To study the effect of Barbadin on agonist-induced FPR2 internalization, we used the peptide agonist WKYMVM and two lipopeptides, the peptidomimetic Cmp 14 and the pepducin F2Pal10 that are functionally biased (structures are shown in Fig 1). All three agonists (WKYMVM, Cmp 14 and F2Pal10) are potent in inducing an FPR2-mediated transient rise in [Ca 2+ ]i, ERK1/2 phosphorylation, and ROS production in human neutrophils, but Cmp 14 and F2Pal10 are biased away from -arrestin recruitment and chemotaxis [9][10][11]. By using an enhanced bystander bioluminescence resonance energy transfer (ebBRET) assay system, we here confirmed that Cmp 14 and F2Pal10 are poor inducers of arrestin recruitment (Fig 2A).…”

“…a large number of ligands belonging to different chemical classes [5][6][7]. Depending on the agonists examined, the signals generated by FPR2 mediate primarily pro-inflammatoryactivities, yet anti-inflammatory activities have also been described [8][9][10][11]. Based on the important roles of FPR2 in host defense and regulation of inflammation, this GPCR has been considered as a promising drug target for inflammatory conditions including cardiovascular diseases [12,13].…”

“…The precise role of -arrestin in regulating FPR2 signaling and function is not known, but it is clear that some FPR2 agonists, but not all, are capable of promoting recruitment of -arrestin [5]. We have previously proposed that the dynamic transfer of agonist-occupied FPR2 from an active signaling state to a non-signaling state (i.e., receptor desensitization) is not directly associated with the ability of the receptoragonist complex to recruit -arrestin [9], but appears to involve receptor coupling to the actin 5 cytoskeleton [11,[15][16][17][18]. Nevertheless, the ability to recruit -arrestin is of functional importance also in neutrophils as demonstrated by the finding that FPR2 agonists that are biased away from -arrestin recruitment induce ROS production without concomitant induction of chemotactic migration [9][10][11].In the present study, we investigated the role of β-arrestin in regulating FPR2 endocytosis and the NADPH-oxidase activation in neutrophils by using Barbadin as a tool compound.…”