The chemerin-CMKLR1 axis in keratinocytes impairs innate host defense against cutaneous Staphylococcus aureus infection.

The skin is the most common site of Staphylococcus aureus infection, which can lead to various diseases, including invasive and life-threatening infections, through evasion of host defense. However, little is known about the host factors that facilitate the innate immune evasion of S. aureus in the skin. Chemerin, which is abundantly expressed in the skin and can be activated by proteases derived from S. aureus, has both direct bacteria-killing activity and immunomodulatory effects via interactions with its receptor CMKLR1. Here, we demonstrate that a lack of the chemerin/CMKLR1 axis increases the neutrophil-mediated host defense against S. aureus in a mouse model of cutaneous infection, whereas chemerin overexpression, which mimics high levels of chemerin in obese individuals, exacerbates S. aureus cutaneous infection. Mechanistically, we identified keratinocytes that express CMKLR1 as the main target of chemerin to suppress S. aureus-induced IL-33 expression, leading to impaired skin neutrophilia and bacterial clearance. CMKLR1 signaling specifically inhibits IL-33 expression induced by cell wall components but not secreted proteins of S. aureus by inhibiting Akt activation in mouse keratinocytes. Thus, our study revealed that the immunomodulatory effect of the chemerin/CMKLR1 axis mediates innate immune evasion of S. aureus in vivo and likely increases susceptibility to S. aureus infection in obese individuals.

Plasmonic Nanozymes: Localized Surface Plasmonic Resonance Regulates Reaction Kinetics and Antibacterial Performance.

Among the members of the rapidly growing nanozyme family, plasmonic nanozymes stand out because of their unique localized surface plasmon resonance (LSPR) characteristics and tunable catalytic activity. We prepared a plasmonic nanozyme of Au gold nanoparticles (AuNPs) and Cu metal-organic framework nanosheets (Cu-MOFNs). The Cu-MOFNs have peroxidase-like activity, while AuNPs present unique LSPR characteristics. We found that the as-prepared AuNPs/Cu-MOFNs composite presents 1.6-fold faster reaction kinetics under LSPR excitation compared to that in the dark. Investigations of energy levels, radical capture, and dark-field scattering spectroscopy revealed that LSPR of AuNPs as well as matched energy levels can facilitate efficient hot electron transfer, which could readily cleave the chemical bond of the substrate and accelerate the reaction kinetics. On the basis of these results, we achieved enhanced antibacterial therapy and wound healing using plasmonic AuNPs/Cu-MOFNs. This study spotlights the superiority of plasmonic nanozymes in improving the enzyme-like performance of nanozymes.

Cationic amphiphilic dendrons with effective antibacterial performance.

Bacterial infections and antibiotic resistance have become a global healthcare crisis. Herein, we designed and synthesized a series of cationic amphiphilic dendrons with cationic dendrons and hydrophobic alkyl chains for potential antibacterial applications. Our results showed that the antimicrobial activities of the cationic amphiphilic dendrons were highly dependent upon the length of the hydrophobic alkyl chain, whereas the number of cationic charges was less important. Among these cationic amphiphilic dendrons, a prime candidate was identified, which possessed excellent antimicrobial activity against various pathogens (minimum inhibitory concentrations of 9, 3, and 3 µg mL-1 for Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus, respectively). Scanning electron microscopy and fluorescence microscopy analyses showed that it could disrupt the integrity of a pathogen's membrane, leading to cell lysis and death. In addition, in vitro bacteria-killing kinetics showed that it had rapid bactericidal efficiency. It also had excellent antimicrobial activities against MRSA in vivo and promoted wound healing. In general, the synthesized cationic amphiphilic dendrons, which exhibited rapid and broad-spectrum bactericidal activity, may have great potential in antimicrobial applications.

Universal Antifouling and Photothermal Antibacterial Surfaces Based on Multifunctional Metal-Phenolic Networks for Prevention of Biofilm Formation.

Biofilms formed from the pathogenic bacteria that attach to the surfaces of biomedical devices and implantable materials result in various persistent and chronic bacterial infections, posing serious threats to human health. Compared to the elimination of matured biofilms, prevention of the formation of biofilms is expected to be a more effective way for the treatment of biofilm-associated infections. Herein, we develop a facile method for endowing diverse substrates with long-term antibiofilm property by deposition of a hybrid film composed of tannic acid/Cu ion (TA/Cu) complex and poly(ethylene glycol) (PEG). In this system, the TA/Cu complex acts as a multifunctional building block with three different roles: (i) as a versatile "glue" with universal adherent property for substrate modification, (ii) as a photothermal biocidal agent for bacterial elimination under irradiation of near-infrared (NIR) laser, and (iii) as a potent linker for immobilization of PEG with inherent antifouling property to inhibit adhesion and accumulation of bacteria. The resulted hybrid film shows negligible cytotoxicity and good histocompatibility and could prevent biofilm formation for at least 15 days in vitro and suppress bacterial infection in vivo, showing great potential for practical applications to solve the biofilm-associated problems of biomedical materials and devices.

Chitosan modified ultra-thin hollow nanoparticles for photosensitizer loading and enhancing photodynamic antibacterial activities.

Antibacterial photodynamic therapy (PDT) has attracted extremely attention due to not inducing bacteria to generate resistance. However, the poor utilization and low reactive oxygen species (ROS) field of photosensitizers hinder their further application for antibacterial. Here, we designed ultra-thin hollow silica nanoparticles (UHSN), followed by pore-engineering including covalent anchoring of chitosan (UHSN@CS) for enhanced loading and photodynamic property of photosensitizer. The UHSN@CS exhibit high loading efficiency (80.6%, pH = 6.0) and controllable pH-responsive release for Ce6. Additionally, UHSN@CS can enhance the ROS yield of photosensitizers and effectively adhere to S. aureus, thus enormously enhancing antibacterial performance toward bacteria. Moreover, UHSN@CS-Ce6 can obliterate mature S. aureus biofilm and cause an 81% decrease in the biomass, showing a better therapeutic effect than Ce6 (59.2%) under laser irradiation. In vivo results confirm that UHSN@CS-Ce6 is effective to promote infectious wound regeneration. As photodynamic-based nanoplatforms, UHSN@CS-Ce6 are potential antibacterial agents for skin infection therapy.

Fabrication of Bacterial Cellulose-Based Dressings for Promoting Infected Wound Healing.

Bacterial cellulose (BC) holds several unique properties such as high water retention capability, flexibility, biocompatibility, and high absorption capacity. All these features make it a potential material for wound healing applications. However, it lacks antibacterial properties, which hampers its applications for infectious wound healings. This study reported BC-based dressings containing ε-polylysine (ε-PL), cross-linked by a biocompatible and mussel-inspired polydopamine (PDA) for promoting infectious wound healing. BC membranes were coated with PDA by a simple self-polymerization process, followed by treating with different contents of ε-PL. The resulted membranes showed strong antibacterial properties against tested bacteria by both in vitro and in vivo evaluations. The membranes also exhibited hemocompatibility and cytocompatibility by in vitro investigations. Moreover, the functionalized membranes promoted infected wound healing using Sprague-Dawley rats as a model animal. A complete wound healing was observed in the group treated with functionalized membranes, while wounds were still open for control and pure BC groups in the same duration. Histological investigations indicated that the thickness of newborn skin was greater and smoother in the groups treated with modified membranes in comparison to neat BC or control groups. These results revealed that the functionalized membranes have great potential as a dressing material for infected wounds in future clinical applications.

Small Molecule-Capped Gold Nanoclusters for Curing Skin Infections.

With the long-term and extensive abuse of antibiotics, bacteria can mutate into multidrug-resistant (MDR) strains, resist the existing antibiotics, and escape the danger of being killed. MDR bacteria-caused skin infections are intractable and chronic, becoming one of the most significant and global public-health issues. Thus, the development of novel antimicrobial materials is urgently needed. Non-antibiotic small molecule-modified gold nanoclusters (AuNCs) have great potential as a substitute for commercial antibiotics. Still, their narrow antibacterial spectrum hinders their wide clinical applications. Herein, we report that 4,6-diamino-2-pyrimidinethiol (DAPT)-modified AuNCs (DAPT-AuNCs) can fight against Gram-negative and Gram-positive bacterial strains as well as their MDR counterparts. By modifying DAPT-AuNCs on nanofibrous films, we develop an antibiotic film as innovative dressings for curing incised wounds, which exhibits excellent therapeutic effects on wounds infected by MDR bacteria. Compared to the narrow-spectral one, the broad-spectral antibacterial activity of the DAPT-AuNCs-modified film is more suitable for preventing and treating skin infections caused by various kinds of unknown bacteria. Moreover, the antibacterial films display excellent biocompatibility, implying the great potential for clinical applications.

Dialdehyde Nanocrystalline Cellulose as Antibiotic Substitutes against Multidrug-Resistant Bacteria.

Antibiotic abuse resulted in the emergence of multidrug-resistant Gram-positive pathogens, which pose a severe threat to public health. It is urgent to develop antibiotic substitutes to kill multidrug-resistant Gram-positive pathogens effectively. Herein, the antibacterial dialdehyde nanocrystalline cellulose (DNC) was prepared and characterized. The antibacterial activity and biosafety of DNC were studied. With the increasing content of aldehyde groups, DNC exhibited high antibacterial activity against Gram-positive pathogens in vitro. DNC3 significantly reduced the amounts of methicillin-resistant Staphylococcus aureus (MRSA) on the skin of infected mice models, which showed low cytotoxicity, excellent skin compatibility, and no acute oral toxicity. DNC exhibited potentials as antibiotic substitutes to fight against multidrug-resistant bacteria, such as ingredients in salves to treat skin infection and other on-skin applications.

Recombinant production of Trx-Ib-AMP4 and Trx-E50-52 antimicrobial peptides and antimicrobial synergistic assessment on the treatment of methicillin-resistant Staphylococcus aureus under in vitro and in vivo situations.

PURPOSE: The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA). METHOD: At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested. RESULTS AND CONCLUSION: The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log10 CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects.

Antifracture, Antibacterial, and Anti-inflammatory Hydrogels Consisting of Silver-Embedded Curdlan Nanofibrils.

The bacterial exopolysaccharide Curdlan has a unique collagen-like triple helical structure and immune-modulation activities. Although there have been several types of Curdlan gels reported for antibacterial or wound healing purposes, none of them exhibit favorable mechanical properties for clinically applicable wound healing materials. Herein, we present a two-step approach for preparing Ag-embedded Curdlan hydrogels that are highly soft but are very stretchable compared with common polysaccharide-based hydrogels. Ag ions were first reduced in a diluted Curdlan solution to form AgNP-decorated triple helices. Then, the aqueous solution consisting of Curdlan/Ag nanoparticles was mixed with a dimethyl sulfoxide solution consisting of a high concentration of Curdlan. This mixing triggered the conformation transformation of Curdlan random coils into triple helices, and then the helices were further packed into semicrystalline nanofibrils of ∼20 nm in diameter. Due to the presence of semicrystalline fibrils, this novel Curdlan hydrogel exhibits a fracture strain of ∼350% and fracture stress of ∼0.2 MPa at a water content of ∼97%. This nanofibril hydrogel supported the attachment, spreading, and growth of fibroblasts and effectively inhibited the growth of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Moreover, the hydrogels downregulated NO production and proinflammatory gene expression levels in lipopolysaccharide (LPS)-stimulated macrophages but did not change the anti-inflammatory gene expression levels in IL-4-stimulated macrophages. In an animal study, these hydrogels accelerated wound healing in a bacteria-infected mice skin wound model. These results validate the further development of Curdlan/AgNPs nanofibril hydrogels in clinical wound management.

A dose response model for Staphylococcus aureus.

Dose-response models (DRMs) are used to predict the probability of microbial infection when a person is exposed to a given number of pathogens. In this study, we propose a new DRM for Staphylococcus aureus (SA), which causes skin and soft-tissue infections. The current approach to SA dose-response is only partially mechanistic and assumes that individual bacteria do not interact with each other. Our proposed two-compartment (2C) model assumes that bacteria that have not adjusted to the host environment decay. After adjusting to the host, they exhibit logistic/cooperative growth, eventually causing disease. The transition between the adjusted and un-adjusted states is a stochastic process, which the 2C DRM explicitly models to predict response probabilities. By fitting the 2C model to SA pathogenesis data, we show that cooperation between individual SA bacteria is sufficient (and, within the scope of the 2C model, necessary) to characterize the dose-response. This is a departure from the classical single-hit theory of dose-response, where complete independence is assumed between individual pathogens. From a quantitative microbial risk assessment standpoint, the mechanistic basis of the 2C DRM enables transparent modeling of dose-response of antibiotic-resistant SA that has not been possible before. It also enables the modeling of scenarios having multiple/non-instantaneous exposures, with minimal assumptions.

The biological synthesis of gold/perlite nanocomposite using Urtica dioica extract and its chitosan-capped derivative for healing wounds infected with methicillin-resistant Staphylococcus aureus.

The preparation of ointments from natural compounds is essential for accelerating infected wounds. This study investigated the effects of topical uses of gold nanoparticles (Au)/perlite (Au/Perl) nanocomposites (NCs) by the help of Urtica dioica extract and its chitosan-capped derivative (Chit) on methicillin-resistant Staphylococcus aureus (MRSA)-infected wound healing in a mouse model. Furthermore, Au/Perl/Chit nanocomposite was prepared using protonated chitosan solution. The physicochemical properties of the as-synthesized nanocomposites were also investigated. The effects of Au/Perl/Chit NC were assessed by antibacterial, histopathological parameters as well as molecular evaluations. Then, they were compared with synthetic agent of mupirocin. The results revealed that Au/Perl NC was mesoporous and spherical in a range of 13-15 nm. Topical administration of Au/Perl/Chit ointment accelerated wound healing by reducing bacteria colonization and wound rate enhancing collagen biosynthesis and re-epithelialization, the expressions of IL-10, PI3K, AKT, bFGF, and COL1A genes, which is in agreement with the obtained results for mupirocin. In conclusion, the results strongly demonstrated that administration of ointments prepared from Au/Perl and Au/Perl/Chit nanocomposites stimulates MRSA-infected wound healing by decreasing the length of healing time and regulating PI3K/AKT/bFGF signaling pathway and is a promising candidate in stimulating MRSA-infected wound regeneration.

Antimicrobial and antibiofilm photodynamic therapy against vancomycin resistant Staphylococcus aureus (VRSA) induced infection in vitro and in vivo.

Biofilm mediated infection caused by multi-drug resistant bacteria are difficult to treat since it protects the microorganisms by host defense system, making them resistant to antibiotics and other antimicrobial agents. Combating such type of nosocomial infection, especially in immunocompromised patients, is an urgent need and foremost challenge faced by clinicians. Therefore, antimicrobial photodynamic therapy (aPDT) has been intensely pursued as an alternative therapy for bacterial infections. aPDT leads to the generation of reactive oxygen species (ROS) that destroy bacterial cells in the presence of a photosensitizer, visible light and oxygen. Here, we elucidated a possibility of its clinical application by reducing the treatment time and exposing curcumin to 20 J/cm2 of blue laser light, which corresponds to only 52 s to counteract vancomycin resistant Staphylococcus aureus (VRSA) both in vitro and in vivo. To understand the mechanism of action, the generation of total reactive oxygen species (ROS) was quantified by 2'-7'-dichlorofluorescein diacetate (DCFH-DA) and the type of phototoxicity was confirmed by fluorescence spectroscopic analysis. The data showed more production of singlet oxygen, indicating type-II phototoxicity. Different anti-biofilm assays (crystal violet and congo red assays) and microscopic studies were performed at sub-MIC concentration of curcumin followed by treatment with laser light against preformed biofilm of VRSA. The result showed significant reduction in the preformed biofilm formation. Finally, its therapeutic potential was validated in skin abrasion wistar rat model. The result showed significant inhibition of bacterial growth. Furthermore, immunomodulatory analysis with rat serum was performed. A significant reduction in expression of proinflammatory cytokines TNF-α and IL-6 were observed. Hence, we conclude that curcumin mediated aPDT with 20 J/cm2 of blue laser treatment (for 52 s) could be used against multi-drug resistant bacterial infections and preformed biofilm formation as a potential therapeutic approach.

Antibody-Mediated Protection against Staphylococcus aureus Dermonecrosis: Synergy of Toxin Neutralization and Neutrophil Recruitment.

The staphylococcal α-hemolysin is critical for the pathogenesis of Staphylococcus aureus skin and soft tissue infection. Vaccine and infection-elicited α-hemolysin-specific antibodies protect against S. aureus‒induced dermonecrosis, a key feature of skin and soft tissue infection. Many interactions between α-hemolysin and host cells have been identified that promote tissue damage and modulate immune responses, but the mechanisms by which protective adaptive responses cross talk with innate responses at the tissue level are not clear. Using an established mouse model of skin and soft tissue infection and a newly developed histopathologic scoring system, we observed pathologic correlates early after infection, predicting protection against dermonecrosis by anti-α-hemolysin antibody. Protection was characterized by robust neutrophilic inflammation and compartmentalization of bacteria into discrete abscesses, which led to the attenuation of dermonecrosis and enhancement of bacterial clearance later in the infection. The ultimate outcome of infection was driven by the recruitment of neutrophils within the first day after infection but not later. Antibody-mediated protection was dependent on toxin neutralization rather than on enhanced opsonophagocytic killing by neutrophils or protection against toxin-mediated neutrophil lysis. Together, these findings advance our understanding of the mechanisms by which the early synergism between antibody-mediated toxin neutralization and tissue-specific neutrophilic inflammation preserve tissue integrity during infection.

Staphylococcus epidermidis protease EcpA can be a deleterious component of the skin microbiome in atopic dermatitis.

BACKGROUND: Staphylococcus aureus and Staphylococcus epidermidis are the most abundant bacteria found on the skin of patients with atopic dermatitis (AD). S aureus is known to exacerbate AD, whereas S epidermidis has been considered a beneficial commensal organism. OBJECTIVE: In this study, we hypothesized that S epidermidis could promote skin damage in AD by the production of a protease that damages the epidermal barrier. METHODS: The protease activity of S epidermidis isolates was compared with that of other staphylococcal species. The capacity of S epidermidis to degrade the barrier and induce inflammation was examined by using human keratinocyte tissue culture and mouse models. Skin swabs from atopic and healthy adult subjects were analyzed for the presence of S epidermidis genomic DNA and mRNA. RESULTS: S epidermidis strains were observed to produce strong cysteine protease activity when grown at high density. The enzyme responsible for this activity was identified as EcpA, a cysteine protease under quorum sensing control. EcpA was shown to degrade desmoglein-1 and LL-37 in vitro, disrupt the physical barrier, and induce skin inflammation in mice. The abundance of S epidermidis and expression of ecpA mRNA were increased on the skin of some patients with AD, and this correlated with disease severity. Another commensal skin bacterial species, Staphylococcus hominis, can inhibit EcpA production by S epidermidis. CONCLUSION: S epidermidis has commonly been regarded as a beneficial skin microbe, whereas S aureus has been considered deleterious. This study suggests that the overabundance of S epidermidis found on some atopic patients can act similarly to S aureus and damage the skin by expression of a cysteine protease.

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.

Panton-Valentine leukocidin-positive Staphylococcus aureus in skin and soft tissue infections from primary care patients.

OBJECTIVES: To characterize deep skin and soft tissue infections (dSSTI) caused by Panton-Valentine leukocidin (PVL)-positive versus PVL-negative Staphylococcus aureus isolates. METHODS: We performed a retrospective analysis of patients' records including S. aureus isolates from outpatients with dSSTI. Samples had been submitted by primary care physicians, i.e. general practitioners, surgeons, dermatologists and paediatricians, located in Berlin, Germany, in 2007-2017. Bacterial isolates were identified and tested for antimicrobial susceptibility by VITEK 2; PVL was detected by PCR. RESULTS: In total, 1199 S. aureus isolates from 1074 patients with dSSTI were identified, and 613 (51.1%) of 1199 samples were PVL+. The median age of patients with PVL+S. aureus was lower than in patients with PVL- S. aureus (34 years, range 0-88 years, vs. 44 years, range 0-98 years; p < 0.0001). PVL was associated with repeated/multiple samples compared to single sample submission (69/92, 75% vs. 448/982, 45.6%, p < 0.0001; odds ratio (OR), 3.6; 95% confidence interval (CI), 2.2-5.8). Interestingly, the highest PVL positivity rate was found in isolates from gluteal (82/108, 75.9%; OR, 3.6; 95% CI, 2-5) or axillary (76/123, 61.8%; OR, 2; 95% CI, 1.1-3.3) localizations compared to isolates from the arm. The PVL positivity rate did not increase over time. Yet we noticed an increase in the trimethoprim/sulfamethoxazole (SXT) resistance rate in PVL+ isolates, mainly methicillin-sensitive S. aureus, when considering SXT resistance rates of 2007-2012 versus 2013-2017 (35/226, 15.5% vs. 74/289, 25.6%; p 0.01). CONCLUSIONS: In outpatients, gluteal and axillary dSSTI are indicative of PVL+S. aureus. Providing SXT as a complementary treatment for dSSTI should be based on susceptibility testing.

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.

A Small Membrane Stabilizing Protein Critical to the Pathogenicity of Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen, and the emergence of antibiotic-resistant strains is making all types of S. aureus infections more challenging to treat. With a pressing need to develop alternative control strategies to use alongside or in place of conventional antibiotics, one approach is the targeting of established virulence factors. However, attempts at this have had little success to date, suggesting that we need to better understand how this pathogen causes disease if effective targets are to be identified. To address this, using a functional genomics approach, we have identified a small membrane-bound protein that we have called MspA. Inactivation of this protein results in the loss of the ability of S. aureus to secrete cytolytic toxins, protect itself from several aspects of the human innate immune system, and control its iron homeostasis. These changes appear to be mediated through a change in the stability of the bacterial membrane as a consequence of iron toxicity. These pleiotropic effects on the ability of the pathogen to interact with its host result in significant impairment in the ability of S. aureus to cause infection in both a subcutaneous and sepsis model of infection. Given the scale of the effect the inactivation of MspA causes, it represents a unique and promising target for the development of a novel therapeutic approach.

Dual-wavelength photo-killing of methicillin-resistant Staphylococcus aureus.

With the effectiveness of antimicrobials declining as antimicrobial resistance continues to threaten public health, we must look to alternative strategies for the treatment of infections. In this study, we investigated an innovative, drug-free, dual-wavelength irradiation approach that combines 2 wavelengths of light, 460 nm and 405 nm, against methicillin-resistant Staphylococcus aureus (MRSA). MRSA was initially irradiated with 460-nm light (90-360 J/cm2) and subsequently irradiated with aliquots of 405-nm light (54-324 J/cm2). For in vivo studies, mouse skin was abraded and infected with approximately 107 CFUs of MRSA and incubated for 3 hours before irradiating with 460 nm (360 J/cm2) and 405 nm (342 J/cm2). Naive mouse skin was also irradiated to investigate apoptosis. We found that staphyloxanthin, the carotenoid pigment in MRSA cells, promoted resistance to the antimicrobial effects of 405-nm light. In addition, we found that the photolytic effect of 460-nm light on staphyloxanthin attenuated resistance of MRSA to 405-nm light killing. Irradiation of 460 nm alone did not elicit any antimicrobial effect on MRSA. In a proof-of-principle mouse skin abrasion infection model, we observed significant killing of MRSA using the dual-wavelength irradiation approach. However, when either wavelength of light was administered alone, no significant decrease in bacterial viability was observed. Moreover, exposure of the dual-wavelength irradiation to naive mouse skin did not result in any visible apoptosis. In conclusion, a dual-wavelength irradiation strategy may offer an innovative, effective, and safe approach for the treatment of skin infections caused by MRSA.