d 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 -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.

Numerous studies have reported sex bias in infectious diseases, with bias direction dependent on pathogen and site of infection. is the most common cause of skin and soft tissue infections (SSTIs), yet sex bias in susceptibility to SSTI has not been described. A search of electronic health records revealed an odds ratio of 2.4 for 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 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 secreted virulence factor α-hemolysin (Hla). Infection with wild-type suppressed inflammatory cytokine production in the skin of female, but not male, mice when compared with infection with an isogenic 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, demonstrated sex-specific bactericidal capacity ex vivo. This work suggests that sex-specific skin innate responsiveness to Hla and neutrophil bactericidal capacity play important roles in limiting SSTI in females. Understanding the molecular mechanisms controlling this sex bias may reveal novel targets to promote host innate defense against skin infection.

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, (4R)-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.

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.

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.