Receptor-dependent and -independent immunomodulatory effects of phenol-soluble modulin peptides from Staphylococcus aureus on human neutrophils are abrogated through peptide inactivation by reactive oxygen species.
ABSTRACT: The virulence and pathogenesis mechanisms of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains depend on a newly described group of phenol-soluble modulin (PSM) peptides (the PSM? peptides) with cytolytic activity. These toxins are ?-helical peptides with a formyl group at the N terminus, and they activate neutrophils through formyl peptide receptor 2 (FPR2), a function closely correlated to the capacity of staphylococcal species to cause invasive infections. The effects of two synthetic PSM? peptides were investigated, and we show that they utilize FPR2 and promote neutrophils to produce reactive oxygen species (ROS) which in turn trigger inactivation of the peptides. Independently of FPR2, the PSM? peptides also downregulate the neutrophil response to other stimuli and exert a cytolytic effect to which apoptotic neutrophils are more sensitive than viable cells. The novel immunomodulatory functions of the PSM? peptides were sensitive to ROS generated by the neutrophil myeloperoxidase (MPO)-H(2)O(2) system, suggesting a role for this enzyme system in counteracting bacterial virulence.
Project description:The mechanisms used by the immune system to discriminate between pathogenic and commensal bacteria have remained largely unclear. Recently, we have shown that virulence of Staphylococcus aureus depends on secretion of phenol-soluble modulin (PSM) peptides that disrupt neutrophils at micromolar concentrations. Moreover, all S. aureus PSMs stimulate and attract neutrophils at nanomolar concentrations via interaction with the formyl-peptide receptor 2 (FPR2). Here, we demonstrate that FPR2 allows neutrophils to adjust their responses in relation to the aggressiveness of staphylococcal species, which differ largely in their capacity to infect or colonize humans and animals. PSM-related peptides were detected in all human and animal pathogenic staphylococci, but were absent from most commensal species. Three PSM?-like peptides produced by the serious human pathogen Staphylococcus lugdunensis were identified as the previously described S. lugdunensis-synergistic hemolysins (SLUSHs). SLUSHs attracted and stimulated human leukocytes in a FPR2-dependent manner, indicating that FPR2 is a general receptor for all PSM-like peptide toxins. Remarkably, the release of PSMs correlated closely with the apparent capacity of staphylococcal species to cause invasive infections and with their ability to activate FPR2. These findings suggest that the innate immune system may be able to respond in different ways to pathogenic or innocuous staphylococci by monitoring the presence of PSMs via FPR2.
Project description:Neutrophil granulocytes act as a first line of defense against pathogenic staphylococci. However, <i>Staphylococcus aureus</i> has a remarkable capacity to survive neutrophil killing, which distinguishes it from the less-pathogenic <i>Staphylococcus epidermidis.</i> Both species release phenol-soluble modulin (PSM) toxins, which activate the neutrophil formyl-peptide receptor 2 (FPR2) to promote neutrophil influx and phagocytosis, and which disrupt neutrophils or their phagosomal membranes at high concentrations. We show here that the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin G and proteinase 3, which are released into the extracellular space or the phagosome upon neutrophil FPR2 stimulation, effectively degrade PSMs thereby preventing their capacity to activate and destroy neutrophils. Notably, <i>S. aureus</i>, but not <i>S. epidermidis</i>, secretes potent NSP-inhibitory proteins, Eap, EapH1, EapH2, which prevented the degradation of PSMs by NSPs. Accordingly, a <i>S. aureus</i> mutant lacking all three NSP inhibitory proteins was less effective in activating and destroying neutrophils and it survived less well in the presence of neutrophils than the parental strain. We show that Eap proteins promote pathology <i>via</i> PSM-mediated FPR2 activation since murine intraperitoneal infection with the <i>S. aureus</i> parental but not with the NSP inhibitors mutant strain, led to a significantly higher bacterial load in the peritoneum and kidneys of mFpr2<sup>-/-</sup> compared to wild-type mice. These data demonstrate that NSPs can very effectively detoxify some of the most potent staphylococcal toxins and that the prominent human pathogen <i>S. aureus</i> has developed efficient inhibitors to preserve PSM functions. Preventing PSM degradation during infection represents an important survival strategy to ensure FPR2 activation.
Project description:Vascular leakage frequently occurs in patients with severe Staphylococcus aureus infection. However, the mechanism underlying S. aureus infection-induced vascular leakage remains unclear. Here, we identified the S. aureus virulence factor phenol-soluble modulin (PSM)α4 from the culture supernatant of strain USA300 as a stimulator of heparin-binding protein (HBP) release from polymorphonuclear neutrophils (PMNs) and demonstrated that PSMα4-induced HBP release from PMNs leads to vascular leakage. PSMα4 appeared less cytolytic than PSMα1-3 and was insensitive to lipoproteins; it significantly increased myeloperoxidase and elastase release from PMNs and cell surface CD63 expression in PMNs. PSMα4-induced HBP release required formyl peptide receptor 2 (FPR2) and phosphoinositide 3-kinase (PI3K) and depended on Ca(2+) influx and cytoskeleton rearrangement. Thus, PSMα4 may stimulate HBP release by activating FPR2 and PI3K to initiate PMN degranulation. PSMα4-induced HBP release from PMNs increased endothelial cell monolayer permeability in vitro and induced vascular leakage in mice. This novel function of PSMα4 may contribute to the pathogenesis of S. aureus and may be a potential therapeutic target.
Project description:Virulence of emerging community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and other highly pathogenic S. aureus strains depends on their production of phenol-soluble modulin (PSM) peptide toxins, which combine the capacities to attract and lyse neutrophils. The molecular basis of PSM-stimulated neutrophil recruitment has remained unclear. Here, we demonstrate that the human formyl peptide receptor 2 (FPR2/ALX), which has previously been implicated in control of endogenous inflammatory processes, senses PSMs at nanomolar concentrations and initiates proinflammatory neutrophil responses to CA-MRSA. Specific blocking of FPR2/ALX or deletion of PSM genes in CA-MRSA severely diminished neutrophil detection of CA-MRSA. Furthermore, a specific inhibitor of FPR2/ALX and of its functional mouse counterpart blocked PSM-mediated leukocyte infiltration in vivo in a mouse model. Thus, the innate immune system uses a distinct FPR2/ALX-dependent mechanism to specifically sense bacterial peptide toxins and detect highly virulent bacterial pathogens. FPR2/ALX represents an attractive target for new anti-infective or anti-inflammatory strategies.
Project description:Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin ? (PSM?) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSM?-induced NETs contained the typical protein markers and were able to capture microbes. The PSM?-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSM?-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSM? peptides, a process that may be of importance for CA-MRSA virulence.
Project description:Formyl peptide receptor 1 (FPR1) mediates bacterial and mitochondrial N-formyl peptides-induced neutrophil activation. Therefore, FPR1 is an important therapeutic target for drugs to treat septic or sterile inflammatory diseases. Honokiol, a major bioactive compound of Magnoliaceae plants, possesses several anti-inflammatory activities. Here, we show that honokiol exhibits an inhibitory effect on FPR1 binding in human neutrophils. Honokiol inhibited superoxide anion generation, reactive oxygen species formation, and elastase release in bacterial or mitochondrial N-formyl peptides (FPR1 agonists)-activated human neutrophils. Adhesion of FPR1-induced human neutrophils to cerebral endothelial cells was also reduced by honokiol. The receptor-binding results revealed that honokiol repressed FPR1-specific ligand N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein binding to FPR1 in human neutrophils, neutrophil-like THP-1 cells, and hFPR1-transfected HEK293 cells. However, honokiol did not inhibit FPR2-specific ligand binding to FPR2 in human neutrophils. Furthermore, honokiol inhibited FPR1 agonist-induced calcium mobilization as well as phosphorylation of p38 MAPK, ERK, and JNK in human neutrophils. In conclusion, our data demonstrate that honokiol may have therapeutic potential for treating FPR1-mediated inflammatory diseases.
Project description:Staphylococcus aureus causes a wide range of human disease ranging from localized skin and soft tissue infections to potentially lethal systemic infections. S. aureus has the biosynthetic ability to generate numerous virulence factors that assist in circumventing the innate immune system during disease pathogenesis. Recent studies have uncovered a set of extracellular peptides produced by community-associated methicillin-resistant S. aureus (CA-MRSA) with homology to the phenol-soluble modulins (PSMs) from Staphylococcus epidermidis. CA-MRSA PSMs contribute to skin infection and recruit and lyse neutrophils, and truncated versions of these peptides possess antimicrobial activity. In this study, novel CA-MRSA PSM derivatives were discovered by the use of microbial imaging mass spectrometry. The novel PSM derivatives are compared with their parent full-length peptides for changes in hemolytic, cytolytic, and neutrophil-stimulating activity. A potential contribution of the major S. aureus secreted protease aureolysin in processing PSMs is demonstrated. Finally, we show that PSM processing occurs in multiple CA-MRSA strains by structural confirmation of additional novel derivatives. This work demonstrates that IMS can serve as a useful tool to go beyond genome predictions and expand our understanding of the important family of small peptide virulence factors.
Project description:Staphylococcus aureus is a Gram-positive human pathogen that is readily internalized by professional phagocytes such as macrophages and neutrophils but also by non-professional phagocytes such as epithelial or endothelial cells. Intracellular bacteria have been proposed to play a role in evasion of the innate immune system and may also lead to dissemination within migrating phagocytes. Further, S.?aureus efficiently lyses host cells with a battery of cytolytic toxins. Recently, phenol-soluble modulins (PSM) have been identified to comprise a genus-specific family of cytolytic peptides. Of these the PSM? peptides have been implicated in killing polymorphonuclear leucocytes after phagocytosis. We questioned if the peptides were active in destroying endosomal membranes to avoid lysosomal killing of the pathogen and monitored integrity of infected host cell endosomes by measuring the acidity of the intracellular bacterial microenvironment via flow cytometry and by a reporter recruitment technique. Isogenic mutants of the methicillin-resistant S.?aureus (MRSA) strains USA300 LAC, USA400 MW2 as well as the strongly cytolytic methicillin-sensitive strain 6850 were compared with their respective wild type strains. In all three genetic backgrounds, PSM? mutants were unable to escape from phagosomes in non-professional (293, HeLa, EAhy.926) and professional phagocytes (THP-1), whereas mutants in PSM? and ?-toxin as well as ?-toxin, phosphatidyl inositol-dependent phospholipase C and Panton Valentine leucotoxin escaped with efficiencies of the parental strains. S.?aureus replicated intracellularly only in presence of a functional PSM? operon thereby illustrating that bacteria grow in the host cell cytoplasm upon phagosomal escape.
Project description:Leukocytes express formyl-peptide receptors (FPRs), which sense microbe-associated molecular pattern (MAMP) molecules, leading to leukocyte chemotaxis and activation. We recently demonstrated that phenol-soluble modulin (PSM) peptides from highly pathogenic Staphylococcus aureus are efficient ligands for the human FPR2. How PSM detection by FPR2 impacts on the course of S. aureus infections has remained unknown. We characterized the specificity of mouse FPR2 (mFpr2) using a receptor-transfected cell line, homeobox b8 (Hoxb8), and primary neutrophils isolated from wild-type (WT) or mFpr2-/- mice. The influx of leukocytes into the peritoneum of WT and mFpr2-/- mice was analyzed. We demonstrate that mFpr2 is specifically activated by PSMs in mice, and they represent the first secreted pathogen-derived ligands for the mFpr2. Intraperitoneal infection with S. aureus led to lower numbers of immigrated leukocytes in mFpr2-/- compared with WT mice at 3 h after infection, and this difference was not observed when mice were infected with an S. aureus PSM mutant. Our data support the hypothesis that the mFpr2 is the functional homolog of the human FPR2 and that a mouse infection model represents a suitable model for analyzing the role of PSMs during infection. PSM recognition by mFpr2 shapes leukocyte influx in local infections, the typical infections caused by S. aureus-Weiss, E., Hanzelmann, D., Fehlhaber, B., Klos, A., von Loewenich, F. D., Liese, J., Peschel, A., Kretschmer, D. Formyl-peptide receptor 2 governs leukocyte influx in local Staphylococcus aureus infections.
Project description:Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (?34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.