G protein-coupled estrogen receptor agonist G-1 decreases ADAM10 levels and NLRP3-inflammasome component activation in response to Staphylococcus aureus alpha-hemolysin.

The G protein-coupled estrogen receptor, also known as GPER1 or originally GPR30, is found in various tissues, indicating its diverse functions. It is typically present in immune cells, suggesting its role in regulating immune responses to infectious diseases. Our previous studies have shown that G-1, a selective GPER agonist, can limit the pathogenesis mediated by Staphylococcus aureus alpha-hemolysin (Hla). It aids in clearing bacteria in a mouse skin infection model and restricts the surface display of the Hla receptor, ADAM10 (a disintegrin and metalloprotease 10) in HaCaT keratinocytes. In this report, we delve into the modulation of GPER in human immune cells in relation to the NLRP3 inflammasome. We used macrophage-like differentiated THP-1 cells for our study. We found that treating these cells with G-1 reduces ATP release, decreases the activity of the caspase-1 enzyme, and lessens cell death following Hla intoxication. This is likely due to the reduced levels of ADAM10 and NLRP3 proteins, as well as the decreased display of the ADAM10 receptor in the G-1-treated THP-1 cells. Our studies, along with our previous work, suggest the potential therapeutic use of G-1 in reducing Hla susceptibility in humans. This highlights the importance of GPER in immune regulation and its potential as a therapeutic target.

Genomic characterization of Staphylococcus aureus isolates causing osteoarticular infections in otherwise healthy children.

BACKGROUND: Pediatric osteoarticular infections are commonly caused by Staphylococcus aureus. The contribution of S. aureus genomic variability to pathogenesis of these infections is poorly described. METHODS: We prospectively enrolled 47 children over 3 1/2 years from whom S. aureus was isolated on culture-12 uninfected with skin colonization, 16 with skin abscesses, 19 with osteoarticular infections (four with septic arthritis, three with acute osteomyelitis, six with acute osteomyelitis and septic arthritis and six with chronic osteomyelitis). Isolates underwent whole genome sequencing, with assessment for 254 virulence genes and any mutations as well as creation of a phylogenetic tree. Finally, isolates were compared for their ability to form static biofilms and compared to the genetic analysis. RESULTS: No sequence types predominated amongst osteoarticular infections. Only genes involved in evasion of host immune defenses were more frequently carried by isolates from osteoarticular infections than from skin colonization (p = .02). Virulence gene mutations were only noted in 14 genes (three regulating biofilm formation) when comparing isolates from subjects with osteoarticular infections and those with skin colonization. Biofilm results demonstrated large heterogeneity in the isolates' capacity to form static biofilms, with healthy control isolates producing more robust biofilm formation. CONCLUSIONS: S. aureus causing osteoarticular infections are genetically heterogeneous, and more frequently harbor genes involved in immune evasion than less invasive isolates. However, virulence gene carriage overall is similar with infrequent mutations, suggesting that pathogenesis of S. aureus osteoarticular infections may be primarily regulated at transcriptional and/or translational levels.

Complement Receptor 3 Contributes to the Sexual Dimorphism in Neutrophil Killing of Staphylococcus aureus.

We previously reported sex differences in innate susceptibility to Staphylococcus aureus skin infection and that bone marrow neutrophils (BMN) from female mice have an enhanced ability to kill S. aureus ex vivo compared with those of male mice. However, the mechanism(s) driving this sex bias in neutrophil killing have not been reported. Given the role of opsonins such as complement, as well as their receptors, in S. aureus recognition and clearance, we investigated their contribution to the enhanced bactericidal capacity of female BMN. We found that levels of C3 in the serum and CR3 (CD11b/CD18) on the surface of BMN were higher in female compared with male mice. Consistent with increased CR3 expression following TNF-α priming, production of reactive oxygen species (ROS), an important bactericidal effector, was also increased in female versus male BMN in response to serum-opsonized S. aureus Furthermore, blocking CD11b reduced both ROS levels and S. aureus killing by murine BMN from both sexes. However, at the same concentration of CD11b blocking Ab, S. aureus killing by female BMN was greatly reduced compared with those from male mice, suggesting CR3-dependent differences in bacterial killing between sexes. Overall, this work highlights the contributions of CR3, C3, and ROS to innate sex bias in the neutrophil response to S. aureus Given that neutrophils are crucial for S. aureus clearance, understanding the mechanism(s) driving the innate sex bias in neutrophil bactericidal capacity could identify novel host factors important for host defense against S. aureus.

Vaccination with VLPs Presenting a Linear Neutralizing Domain of S. aureus Hla Elicits Protective Immunity.

The pore-forming cytotoxin α-hemolysin, or Hla, is a critical Staphylococcus aureus virulence factor that promotes infection by causing tissue damage, excessive inflammation, and lysis of both innate and adaptive immune cells, among other cellular targets. In this study, we asked whether a virus-like particle (VLP)-based vaccine targeting Hla could attenuate S. aureus Hla-mediated pathogenesis. VLPs are versatile vaccine platforms that can be used to display target antigens in a multivalent array, typically resulting in the induction of high titer, long-lasting antibody responses. In the present study, we describe the first VLP-based vaccines that target Hla. Vaccination with either of two VLPs displaying a 21 amino-acid linear neutralizing domain (LND) of Hla protected both male and female mice from subcutaneous Hla challenge, evident by reduction in lesion size and neutrophil influx to the site of intoxication. Antibodies elicited by VLP-LND vaccination bound both the LND peptide and the native toxin, effectively neutralizing Hla and preventing toxin-mediated lysis of target cells. We anticipate these novel and promising vaccines being part of a multi-component S. aureus vaccine to reduce severity of S. aureus infection.

Staphylococcus aureus Fatty Acid Kinase FakA Modulates Pathogenesis during Skin Infection via Proteases.

Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro, and that the ΔfakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla ΔfakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the ΔfakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the ΔfakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the ΔfakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in ΔfakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δaur and ΔsspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the ΔfakA mutant during skin infection.

GPER activation protects against epithelial barrier disruption by Staphylococcus aureus α-toxin.

Sex bias in innate defense against Staphylococcus aureus skin and soft tissue infection (SSTI) is dependent on both estrogen production by the host and S. aureus secretion of the virulence factor, α-hemolysin (Hla). The impact of estrogen signaling on the immune system is most often studied in terms of the nuclear estrogen receptors ERα and ERß. However, the potential contribution of the G protein-coupled estrogen receptor (GPER) to innate defense against infectious disease, particularly with respect to skin infection, has not been addressed. Using a murine model of SSTI, we found that GPER activation with the highly selective agonist G-1 limits S. aureus SSTI and Hla-mediated pathogenesis, effects that were absent in GPER knockout mice. Specifically, G-1 reduced Hla-mediated skin lesion formation and pro-inflammatory cytokine production, while increasing bacterial clearance. In vitro, G-1 reduced surface expression of the Hla receptor, ADAM10, in a human keratinocyte cell line and increased resistance to Hla-mediated permeability barrier disruption. This novel role for GPER activation in skin innate defense against infectious disease suggests that G-1 may have clinical utility in patients with epithelial permeability barrier dysfunction or who are otherwise at increased risk of S. aureus infection, including those with atopic dermatitis or cancer.

Prenylated Diresorcinols Inhibit Bacterial Quorum Sensing.

Current treatment options for bacterial infections are dependent on antibiotics that inhibit microbial growth and viability. These approaches result in the evolution of drug-resistant strains of bacteria. An anti-infective strategy that is less likely to lead to the development of resistance is the disruption of quorum sensing mechanisms, which are involved in promoting virulence. The goal of this study was to identify fungal metabolites effective as quorum sensing inhibitors. Three new prenylated diresorcinols (1-3), along with two known compounds, (4 R) -regiolone and decarboxycitrinone, were isolated from a freshwater fungus (Helotiales sp.) from North Carolina. Their structures were assigned on the basis of HRESIMS and NMR experiments. The structure of compound 1 was confirmed via X-ray diffraction analysis, and its absolute configuration was established by TDDFT-ECD and optical rotation calculations. Compounds 1-3 suppressed quorum sensing in a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), with IC50 values ranging from 0.3 to 12.5 µM. These compounds represent potential leads in the development of antivirulence therapeutics.

Innate Sex Bias of Staphylococcus aureus Skin Infection Is Driven by α-Hemolysin.

Numerous studies have reported sex bias in infectious diseases, with bias direction dependent on pathogen and site of infection. Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs), yet sex bias in susceptibility to S. aureus SSTI has not been described. A search of electronic health records revealed an odds ratio of 2.4 for S. aureus SSTI in males versus females. To investigate the physiological basis of this bias, we compared outcomes between male and female mice in a model of S. aureus dermonecrosis. Consistent with the epidemiological data, female mice were better protected against SSTI, with reduced dermonecrosis followed later by increased bacterial clearance. Protection in females was disrupted by ovariectomy and restored by short-term estrogen administration. Importantly, this sex bias was mediated by a sex-specific response to the S. aureus-secreted virulence factor α-hemolysin (Hla). Infection with wild-type S. aureus suppressed inflammatory cytokine production in the skin of female, but not male, mice when compared with infection with an isogenic hla deletion mutant. This differential response was conserved following injection with Hla alone, demonstrating a direct response to Hla independent of bacterial burden. Additionally, neutrophils, essential for clearing S. aureus, demonstrated sex-specific S. aureus bactericidal capacity ex vivo. This work suggests that sex-specific skin innate responsiveness to Hla and neutrophil bactericidal capacity play important roles in limiting S. aureus SSTI in females. Understanding the molecular mechanisms controlling this sex bias may reveal novel targets to promote host innate defense against S. aureus skin infection.

VLP-based vaccine induces immune control of Staphylococcus aureus virulence regulation.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) and mounting antibiotic resistance requires innovative treatment strategies. S. aureus uses secreted cyclic autoinducing peptides (AIPs) and the accessory gene regulator (agr) operon to coordinate expression of virulence factors required for invasive infection. Of the four agr alleles (agr types I-IV and corresponding AIPs1-4), agr type I isolates are most frequently associated with invasive infection. Cyclization via a thiolactone bond is essential for AIP function; therefore, recognition of the cyclic form of AIP1 may be necessary for antibody-mediated neutralization. However, the small sizes of AIPs and labile thiolactone bond have hindered vaccine development. To overcome this, we used a virus-like particle (VLP) vaccine platform (PP7) for conformationally-restricted presentation of a modified AIP1 amino acid sequence (AIP1S). Vaccination with PP7-AIP1S elicited AIP1-specific antibodies and limited agr-activation in vivo. Importantly, in a murine SSTI challenge model with a highly virulent agr type I S. aureus isolate, PP7-AIP1S vaccination reduced pathogenesis and increased bacterial clearance compared to controls, demonstrating vaccine efficacy. Given the contribution of MRSA agr type I isolates to human disease, vaccine targeting of AIP1-regulated virulence could have a major clinical impact in the fight against antibiotic resistance.

Serum Lipoproteins Are Critical for Pulmonary Innate Defense against Staphylococcus aureus Quorum Sensing.

Hyperlipidemia has been extensively studied in the context of atherosclerosis, whereas the potential health consequences of the opposite extreme, hypolipidemia, remain largely uninvestigated. Circulating lipoproteins are essential carriers of insoluble lipid molecules and are increasingly recognized as innate immune effectors. Importantly, severe hypolipidemia, which may occur with trauma or critical illness, is clinically associated with bacterial pneumonia. To test the hypothesis that circulating lipoproteins are essential for optimal host innate defense in the lung, we used lipoprotein-deficient mice and a mouse model of Staphylococcus aureus pneumonia in which invasive infection requires virulence factor expression controlled by the accessory gene regulator (agr) operon. Activation of agr and subsequent virulence factor expression is inhibited by apolipoprotein B, the structural protein of low-density lipoprotein, which binds and sequesters the secreted agr-signaling peptide (AIP). In this article, we report that lipoprotein deficiency impairs early pulmonary innate defense against S. aureus quorum-sensing-dependent pathogenesis. Specifically, apolipoprotein B levels in the lung early postinfection are significantly reduced with lipoprotein deficiency, coinciding with impaired host control of S. aureus agr-signaling and increased agr-dependent morbidity (weight loss) and inflammation. Given that lipoproteins also inhibit LTA- and LPS-mediated inflammation, these results suggest that hypolipidemia may broadly impact posttrauma pneumonia susceptibility to both Gram-positive and -negative pathogens. Together with previous reports demonstrating that hyperlipidemia also impairs lung innate defense, these results suggest that maintenance of normal serum lipoprotein levels is necessary for optimal host innate defense in the lung.

Apolipoprotein B48, the Structural Component of Chylomicrons, Is Sufficient to Antagonize Staphylococcus aureus Quorum-Sensing.

Serum lipoproteins (LP) are increasingly being recognized as dual purpose molecules that contribute to both cholesterol homeostasis and host innate defense. In fact, very low LP levels are associated with increased risk of bacterial infection in critically ill patients. In this respect, we reported that apolipoprotein B100 (apoB100), the 4536 amino acid structural protein of very low density lipoprotein (VLDL) produced by the liver, limits Staphylococcus aureus pathogenesis. S. aureus uses quorum-sensing (QS) via the accessory gene regulator (agr) operon and an autoinducing peptide (AIP) to coordinate expression of over 200 virulence genes. ApoB100 prevents agr activation by binding and sequestering secreted AIP. Importantly, human serum LP are produced not only by the liver, but are also produced by enterocytes, in the form of chylomicrons, during uptake of dietary lipids. In contrast to apoB100 in VLDL, human enterocytes use apoB48, the N-terminal 2152 amino acids (48%) of apoB100, as the structural component of chylomicrons. Interestingly, enteral feeding of critically ill patients has been associated with decreased risk of infectious complications, suggesting chylomicrons could contribute to host innate defense in critically ill patients when serum LP production by the liver is limited during the acute phase response. Therefore, we hypothesized that apoB48 would be sufficient to antagonize S. aureus QS. As expected, isolated apoB48-LP bound immobilized AIP and antagonized agr-signaling. ApoB48- and apoB100-LP inhibited agr activation with IC50s of 3.5 and 2.3 nM, respectively, demonstrating a conserved AIP binding site. Importantly, apoB48-LP antagonized QS, limited morbidity and promoted bacterial clearance in a mouse model of S. aureus infection. This work demonstrates that both naturally occurring forms of apolipoprotein B can antagonize S. aureus QS, and may suggest a previously unrecognized role for chylomicrons and enterocytes in host innate defense against S. aureus QS-mediated pathogenesis.

ω-Hydroxyemodin limits staphylococcus aureus quorum sensing-mediated pathogenesis and inflammation.

Antibiotic-resistant pathogens are a global health threat. Small molecules that inhibit bacterial virulence have been suggested as alternatives or adjuncts to conventional antibiotics, as they may limit pathogenesis and increase bacterial susceptibility to host killing. Staphylococcus aureus is a major cause of invasive skin and soft tissue infections (SSTIs) in both the hospital and community settings, and it is also becoming increasingly antibiotic resistant. Quorum sensing (QS) mediated by the accessory gene regulator (agr) controls virulence factor production essential for causing SSTIs. We recently identified ω-hydroxyemodin (OHM), a polyhydroxyanthraquinone isolated from solid-phase cultures of Penicillium restrictum, as a suppressor of QS and a compound sought for the further characterization of the mechanism of action. At concentrations that are nontoxic to eukaryotic cells and subinhibitory to bacterial growth, OHM prevented agr signaling by all four S. aureus agr alleles. OHM inhibited QS by direct binding to AgrA, the response regulator encoded by the agr operon, preventing the interaction of AgrA with the agr P2 promoter. Importantly, OHM was efficacious in a mouse model of S. aureus SSTI. Decreased dermonecrosis with OHM treatment was associated with enhanced bacterial clearance and reductions in inflammatory cytokine transcription and expression at the site of infection. Furthermore, OHM treatment enhanced the immune cell killing of S. aureus in vitro in an agr-dependent manner. These data suggest that bacterial disarmament through the suppression of S. aureus QS may bolster the host innate immune response and limit inflammation.

Development of a mimotope vaccine targeting the Staphylococcus aureus quorum sensing pathway.

A major hurdle in vaccine development is the difficulty in identifying relevant target epitopes and then presenting them to the immune system in a context that mimics their native conformation. We have engineered novel virus-like-particle (VLP) technology that is able to display complex libraries of random peptide sequences on a surface-exposed loop in the coat protein without disruption of protein folding or VLP assembly. This technology allows us to use the same VLP particle for both affinity selection and immunization, integrating the power of epitope discovery and epitope mimicry of traditional phage display with the high immunogenicity of VLPs. Previously, we showed that using affinity selection with our VLP platform identifies linear epitopes of monoclonal antibodies and subsequent immunization generates the proper antibody response. To test if our technology could identify immunologic mimotopes, we used affinity selection on a monoclonal antibody (AP4-24H11) that recognizes the Staphylococcus aureus autoinducing peptide 4 (AIP4). AIP4 is a secreted eight amino acid, cyclized peptide produced from the S. aureus accessory gene regulator (agrIV) quorum-sensing operon. The agr system coordinates density dependent changes in gene expression, leading to the upregulation of a host of virulence factors, and passive transfer of AP4-24H11 protects against S. aureus agrIV-dependent pathogenicity. In this report, we identified a set of peptides displayed on VLPs that bound with high specificity to AP4-24H11. Importantly, similar to passive transfer with AP4-24H11, immunization with a subset of these VLPs protected against pathogenicity in a mouse model of S. aureus dermonecrosis. These data are proof of principle that by performing affinity selection on neutralizing antibodies, our VLP technology can identify peptide mimics of non-linear epitopes and that these mimotope based VLP vaccines provide protection against pathogens in relevant animal models.