Inhibition of the ATP synthase sensitizes Staphylococcus aureus towards human antimicrobial peptides.

Antimicrobial peptides (AMPs) are an important part of the human innate immune system for protection against bacterial infections, however the AMPs display varying degrees of activity against Staphylococcus aureus. Previously, we showed that inactivation of the ATP synthase sensitizes S. aureus towards the AMP antibiotic class of polymyxins. Here we wondered if the ATP synthase similarly is needed for tolerance towards various human AMPs, including human ß-defensins (hBD1-4), LL-37 and histatin 5. Importantly, we find that the ATP synthase mutant (atpA) is more susceptible to killing by hBD4, hBD2, LL-37 and histatin 5 than wild type cells, while no changes in susceptibility was detected for hBD3 and hBD1. Administration of the ATP synthase inhibitor, resveratrol, sensitizes S. aureus towards hBD4-mediated killing. Neutrophils rely on AMPs and reactive oxygen molecules to eliminate bacteria and the atpA mutant is more susceptible to killing by neutrophils than the WT, even when the oxidative burst is inhibited.These results show that the staphylococcal ATP synthase enhance tolerance of S. aureus towards some human AMPs and this indicates that inhibition of the ATP synthase may be explored as a new therapeutic strategy that sensitizes S. aureus to naturally occurring AMPs of the innate immune system.

Staphylococcal Enterotoxins Dose-Dependently Modulate the Generation of Myeloid-Derived Suppressor Cells.

Staphylococcus aureus is one of the major human bacterial pathogens causing a broad spectrum of serious infections. Myeloid-derived suppressor cells (MDSC) represent an innate immune cell subset capable of regulating host-pathogen interactions, yet their role in the pathogenesis of S. aureus infections remains incompletely defined. The aim of this study was to determine the influence of different S. aureus strains and associated virulence factors on human MDSC generation. Using an in vitro MDSC generation assay we demonstrate that low concentrations of supernatants of different S. aureus strains led to an induction of functional MDSC, whereas increased concentrations, conversely, reduced MDSC numbers. The concentration-dependent reduction of MDSC correlated with T cell proliferation and cytotoxicity. Several findings supported a role for staphylococcal enterotoxins in modulating MDSC generation. Staphylococcal enterotoxins recapitulated concentration-dependent MDSC induction and inhibition, T cell proliferation and cytotoxicity, while an enterotoxin-deficient S. aureus strain largely failed to alter MDSC. Taken together, we identified staphylococcal enterotoxins as main modulators of MDSC generation. The inhibition of MDSC generation by staphylococcal enterotoxins might represent a novel therapeutic target in S. aureus infections and beyond in non-infectious conditions, such as cancer.

Formyl-Peptide Receptors in Infection, Inflammation, and Cancer.

Formyl-peptide receptors (FPRs) recognize bacterial and mitochondrial formylated peptides as well as endogenous non-formylated peptides and even lipids. FPRs are expressed on various host cell types but most strongly on neutrophils and macrophages. After the discovery of FPRs on leukocytes, it was assumed that these receptors predominantly govern a proinflammatory response resulting in chemotaxis, degranulation, and oxidative burst during infection. However, it is clear that the activation of FPRs has more complex consequences and can also promote the resolution of inflammation. Recent studies have highlighted associations between FPR function and inflammatory conditions, including inflammatory disorders, cancer, and infection. In this review we discuss these recent findings.

Phenol-Soluble Modulin Peptides Contribute to Influenza A Virus-Associated Staphylococcus aureus Pneumonia.

Influenza A virus (IAV) infection is often followed by secondary bacterial lung infection, which is a major reason for severe, often fatal pneumonia. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains such as USA300 cause particularly severe and difficult-to-treat cases of IAV-associated pneumonia. CA-MRSA strains are known to produce extraordinarily large amounts of phenol-soluble modulin (PSM) peptides, which are important cytotoxins and proinflammatory molecules that contribute to several types of S. aureus infection. However, their potential role in pneumonia has remained elusive. We determined the impact of PSMs on human lung epithelial cells and found that PSMs are cytotoxic and induce the secretion of the proinflammatory cytokine interleukin-8 (IL-8) in these cells. Both effects were boosted by previous infection with the 2009 swine flu pandemic IAV H1N1 strain, suggesting that PSMs may contribute to lung inflammation and damage in IAV-associated S. aureus pneumonia. Notably, the PSM-producing USA300 strain caused a higher mortality rate than did an isogenic PSM-deficient mutant in a mouse IAV-S. aureus pneumonia coinfection model, indicating that PSMs are major virulence factors in IAV-associated S. aureus pneumonia and may represent important targets for future anti-infective therapies.

Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes.

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.

Toll-like receptor 2 activation depends on lipopeptide shedding by bacterial surfactants.

Sepsis caused by Gram-positive bacterial pathogens is a major fatal disease but its molecular basis remains elusive. Toll-like receptor 2 (TLR2) has been implicated in the orchestration of inflammation and sepsis but its role appears to vary for different pathogen species and clones. Accordingly, Staphylococcus aureus clinical isolates differ substantially in their capacity to activate TLR2. Here we show that strong TLR2 stimulation depends on high-level production of phenol-soluble modulin (PSM) peptides in response to the global virulence activator Agr. PSMs are required for mobilizing lipoproteins, the TLR2 agonists, from the staphylococcal cytoplasmic membrane. Notably, the course of sepsis caused by PSM-deficient S. aureus is similar in wild-type and TLR2-deficient mice, but TLR2 is required for protection of mice against PSM-producing S. aureus. Thus, a crucial role of TLR2 depends on agonist release by bacterial surfactants. Modulation of this process may lead to new therapeutic strategies against Gram-positive infections.

PSM Peptides of Staphylococcus aureus Activate the p38-CREB Pathway in Dendritic Cells, Thereby Modulating Cytokine Production and T Cell Priming.

The challenging human pathogen Staphylococcus aureus has highly efficient immune evasion strategies for causing a wide range of diseases, from skin and soft tissue to life-threatening infections. Phenol-soluble modulin (PSM) peptides are major pathogenicity factors of community-associated methicillin-resistant S. aureus strains. In previous work, we demonstrated that PSMs in combination with TLR2 ligand from S. aureus induce tolerogenic dendritic cells (DCs) characterized by the production of high amounts of IL-10, but no proinflammatory cytokines. This in turn promotes the activation of regulatory T cells while impairing Th1 response; however, the signaling pathways modulated by PSMs remain elusive. In this study, we analyzed the effects of PSMs on signaling pathway modulation downstream of TLR2. TLR2 stimulation in combination with PSMα3 led to increased and prolonged phosphorylation of NF-κB, ERK, p38, and CREB in mouse bone marrow-derived DCs compared with single TLR2 activation. Furthermore, inhibition of p38 and downstream MSK1 prevented IL-10 production, which in turn reduced the capacity of DCs to activate regulatory T cells. Interestingly, the modulation of the signaling pathways by PSMs was independent of the known receptor for PSMs, as shown by experiments with DCs lacking the formyl peptide receptor 2. Instead, PSMs penetrate the cell membrane most likely by transient pore formation. Moreover, colocalization of PSMs and p38 was observed near the plasma membrane in the cytosol, indicating a direct interaction. Thus, PSMs from S. aureus directly modulate the signaling pathway p38-CREB in DCs, thereby impairing cytokine production and in consequence T cell priming to increase the tolerance toward the pathogen.

Insight into structure-function relationship in phenol-soluble modulins using an alanine screen of the phenol-soluble modulin (PSM) α3 peptide.

Phenol-soluble modulins (PSMs) are a family of peptides with multiple functions in staphylococcal pathogenesis. To gain insight into the structural features affecting PSM functions, we analyzed an alanine substitution library of PSMα3, a strongly cytolytic and proinflammatory PSM of Staphylococcus aureus with a significant contribution to S. aureus virulence. Lysine residues were essential for both receptor-dependent proinflammatory and receptor-independent cytolytic activities. Both phenotypes also required additional structural features, with the C terminus being crucial for receptor activation. Biofilm formation was affected mostly by hydrophobic amino acid positions, suggesting that the capacity to disrupt hydrophobic interactions is responsible for the effect of PSMs on biofilm structure. Antimicrobial activity, absent from natural PSMα3, could be created by the exchange of large hydrophobic side chains, indicating that PSMα3 has evolved to exhibit cytolytic rather than antimicrobial activity. In addition to gaining insight into the structure-function relationship in PSMs, our study identifies nontoxic PSMα3 derivatives for active vaccination strategies and lays the foundation for future efforts aimed to understand the biological role of PSM recognition by innate host defense.

Hypoxia-inducible factor 1-regulated lysyl oxidase is involved in Staphylococcus aureus abscess formation.

Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor involved in the adaptation of mammals to hypoxia and plays a crucial role in cancer angiogenesis. Recent evidence suggests a leading role for HIF-1 in various inflammatory and infectious diseases. Here we describe the role of HIF-1 in Staphylococcus aureus infections by investigating the HIF-1-dependent host cell response. For this purpose, transcriptional profiling of HIF-1α-deficient HepG2 and control cells, both infected with Staphylococcus aureus, was performed. Four hours after infection, the expression of 190 genes, 24 of which were regulated via HIF-1, was influenced. LOX (encoding lysyl oxidase) was one of the upregulated genes with a potential impact on the course of S. aureus infection. LOX is an amine oxidase required for biosynthetic cross-linking of extracellular matrix components. LOX was upregulated in vitro in different cell cultures infected with S. aureus and also in vivo, in kidney abscesses of mice intravenously infected with S. aureus and in clinical skin samples from patients with S. aureus infections. Inhibition of LOX by ß-aminopropionitrile (BAPN) did not affect the bacterial load in kidneys or blood but significantly influenced abscess morphology and collagenization. Our data provide evidence for a crucial role of HIF-1-regulated LOX in abscess formation.

Staphylococcus aureus phenol-soluble modulin peptides modulate dendritic cell functions and increase in vitro priming of regulatory T cells.

The major human pathogen Staphylococcus aureus has very efficient strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant S. aureus depends on phenol-soluble modulin (PSM) peptide toxins, which are known to attract and lyse neutrophils. However, their influences on other immune cells remain elusive. In this study, we analyzed the impact of PSMs on dendritic cells (DCs) playing an essential role in linking innate and adaptive immunity. In human neutrophils, PSMs exert their function by binding to the formyl peptide receptor (FPR) 2. We show that mouse DCs express the FPR2 homolog mFPR2 as well as its paralog mFPR1 and that PSMs are chemoattractants for DCs at noncytotoxic concentrations. PSMs reduced clathrin-mediated endocytosis and inhibited TLR2 ligand-induced secretion of the proinflammatory cytokines TNF, IL-12, and IL-6, while inducing IL-10 secretion by DCs. As a consequence, treatment with PSMs impaired the capacity of DCs to induce activation and proliferation of CD4(+) T cells, characterized by reduced Th1 but increased frequency of FOXP3(+) regulatory T cells. These regulatory T cells secreted high amounts of IL-10, and their suppression capacity was dependent on IL-10 and TGF-ß. Interestingly, the induction of tolerogenic DCs by PSMs appeared to be independent of mFPRs, as shown by experiments with mice lacking mFPR2 (mFPR2(-/-)) and the cognate G protein (p110γ(-/-)). Thus, PSMs from highly virulent pathogens affect DC functions, thereby modulating the adaptive immune response and probably increasing the tolerance toward the pathogen.

Enterococcus faecium stimulates human neutrophils via the formyl-peptide receptor 2.

The human formyl-peptide receptor 2 (FPR2/ALX) senses phenol-soluble modulin (PSM) peptide toxins produced by pathogenic staphylococcal species and plays a crucial role in directing neutrophil influx during staphylococcal infection. However, it has remained unclear if FPR2 responds also to molecules from other bacterial pathogens. Here we analyzed a variety of gram-positive and gram-negative pathogens and found that apart from staphylococci only certain enterococcal strains have the capacity to stimulate FPR2/ALX. Most of the analyzed Enterococcus faecium but only sporadic Enterococcus faecalis strains released FPR2/ALX-stimulating molecules leading to neutrophil calcium ion fluxes, chemotaxis, and complement receptor upregulation. Among ten test strains vancomycin-resistant E. faecium had a significantly higher capacity to stimulate FPR2/ALX than vancomycin-susceptible strains, suggesting an association of strong FPR2/ALX activation with health-care associated strains. The enterococcal FPR2/ALX agonists were found to be peptides or proteins, which appear, however, to be unrelated to staphylococcal PSMs in sequence and physicochemical properties. Enterococci are among the most frequent invasive bacterial pathogens but the basis of enterococcal virulence and immune activation has remained incompletely understood. Our study indicates that previously unrecognized proteinaceous agonists contribute to Enterococcus-host interaction and underscores the importance of FPR2/ALX in host defense against major endogenous bacterial pathogens.

Mobile genetic element-encoded cytolysin connects virulence to methicillin resistance in MRSA.

Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs.

Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major human pathogen. Traditionally, MRSA infections occurred exclusively in hospitals and were limited to immunocompromised patients or individuals with predisposing risk factors. However, recently there has been an alarming epidemic caused by community-associated (CA)-MRSA strains, which can cause severe infections that can result in necrotizing fasciitis or even death in otherwise healthy adults outside of healthcare settings. In the US, CA-MRSA is now the cause of the majority of infections that result in trips to the emergency room. It is unclear what makes CA-MRSA strains more successful in causing human disease compared with their hospital-associated counterparts. Here we describe a class of secreted staphylococcal peptides that have a remarkable ability to recruit, activate and subsequently lyse human neutrophils, thus eliminating the main cellular defense against S. aureus infection. These peptides are produced at high concentrations by standard CA-MRSA strains and contribute significantly to the strains' ability to cause disease in animal models of infection. Our study reveals a previously uncharacterized set of S. aureus virulence factors that account at least in part for the enhanced virulence of CA-MRSA.