Physalin H, physalin B, and isophysalin B suppress the quorum-sensing function of Staphylococcus aureus by binding to AgrA.

The virulence of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), depends on the expression of toxins and virulence factors controlled by the quorum-sensing (QS) system, encoded on the virulence accessory gene regulator (agr) locus. The aim of this study was to identify a phytochemical that inhibits Agr-QS function and to elucidate its mechanism. We screened 577 compounds and identified physalin H, physalin B, and isophysalin B--phytochemicals belonging to physalins found in plants of the Solanaceae family--as novel Agr-QS modulators. Biological analyses and in vitro protein-DNA binding assays suggested that these physalins suppress gene expression related to the Agr-QS system by inhibiting binding of the key response regulator AgrA to the agr promoters, reducing the function of hemolytic toxins downstream of these genes in MRSA. Furthermore, although physalin F suppressed gene expression in the Agr-QS system, its anti-hemolytic activity was lower than that of physalins H, B, and isophysalin B. Conversely, five physalins isolated from the same plant with the ability to suppress Agr-QS did not reduce bacterial Agr-QS activity but inhibited AgrA binding to DNA in vitro. A docking simulation revealed that physalin interacts with the DNA-binding site of AgrA in three docking states. The carbonyl oxygens at C-1 and C-18 of physalins, which can suppress Agr-QS, were directed to residues N201 and R198 of AgrA, respectively, whereas these carbonyl oxygens of physalins, without Agr-QS suppression activity, were oriented in different directions. Next, 100-ns molecular dynamics simulations revealed that the hydrogen bond formed between the carbonyl oxygen at C-15 of physalins and L186 of AgrA functions as an anchor, sustaining the interaction between the carbonyl oxygen at C-1 of physalins and N201 of AgrA. Thus, these results suggest that physalin H, physalin B, and isophysalin B inhibit the interaction of AgrA with the agr promoters by binding to the DNA-binding site of AgrA, suppressing the Agr-QS function of S. aureus. Physalins that suppress the Agr-QS function are proposed as potential lead compounds in the anti-virulence strategy for MRSA infections.

The role of Staphylococcus aureus quorum sensing in cutaneous and systemic infections.

BACKGROUND: Staphylococcus aureus is a leading cause of human bacterial infections worldwide. It is the most common causative agent of skin and soft tissue infections, and can also cause various other infections, including pneumonia, osteomyelitis, as well as life-threatening infections, such as sepsis and infective endocarditis. The pathogen can also asymptomatically colonize human skin, nasal cavity, and the intestine. S. aureus colonizes approximately 20-30% of human nostrils, being an opportunistic pathogen for subsequent infection. Its strong ability to silently spread via human contact makes it difficult to eradicate S. aureus. A major concern with S. aureus is its capacity to develop antibiotic resistance and adapt to diverse environmental conditions. The variability in the accessory gene regulator (Agr) region of the genome contributes to a spectrum of phenotypes within the bacterial population, enhancing the likelihood of survival in different environments. Agr functions as a central quorum sensing (QS) system in S. aureus, allowing bacteria to adjust gene expression in response to population density. Depending on Agr expression, S. aureus secretes various toxins, contributing to virulence in infectious diseases. Paradoxically, expressing Agr may be disadvantageous in certain situations, such as in hospitals, causing S. aureus to generate Agr mutants responsible for infections in healthcare settings. MAIN BODY: This review aims to demonstrate the molecular mechanisms governing the diverse phenotypes of S. aureus, ranging from a harmless colonizer to an organism capable of infecting various human organs. Emphasis will be placed on QS and its role in orchestrating S. aureus behavior across different contexts. SHORT CONCLUSION: The pathophysiology of S. aureus infection is substantially influenced by phenotypic changes resulting from factors beyond Agr. Future studies are expected to give the comprehensive understanding of S. aureus overall profile in various settings.

Weight loss improves inflammation by T helper 17 cells in an obese patient with psoriasis at high risk for cardiovascular events.

Psoriasis is a chronic inflammatory skin disease that is associated with obesity and myocardial infarction. Obesity-induced changes in lipid metabolism promote T helper 17 (Th17) cell differentiation, which in turn promotes chronic inflammation. Th17 cells have central roles in many inflammatory diseases, including psoriasis and atherosclerosis; however, whether treatment of obesity attenuates Th17 cells and chronic inflammatory diseases has been unknown. In this study, we found an increase in Th17 cells in a patient with obesity, type 2 diabetes and psoriasis. Furthermore, weight loss with diet and exercise resulted in a decrease in Th17 cells and improvement of psoriasis. This case supports the hypothesis that obesity leads to an increase in Th17 cells and chronic inflammation of the skin and blood vessel walls, thereby promoting psoriasis and atherosclerosis.

Pathogenic role of the staphylococcal accessory gene regulator quorum sensing system in atopic dermatitis.

The skin is home to various bacteria, archaea, fungi, and viruses, collectively referred to as the skin microbiota. Patients with certain skin diseases reportedly have unique skin "dysbiosis," a condition involving imbalanced microbiota, suggesting that dysbiosis in the skin may be either causal or a consequence of specific skin diseases. Atopic dermatitis (AD) is the most common allergic skin disease that affects 15-20% of children and 2-10% of adults worldwide. Both intrinsic genetic factors, such as susceptibility to type 2 inflammation or skin barrier dysfunction, and extrinsic environmental factors, such as air pollen and skin microbiota, contribute to AD. Staphylococcus aureus, which does not often colonize the skin of healthy individuals, is commonly identified in the lesional skin of patients with AD and is correlated with the disease flare. However, the role of S. aureus in the pathogenesis of AD has not been elucidated. Here, we discuss the pathological behavior of S. aureus, focusing on accessory gene regulator (Agr) quorum sensing, which is a fundamental bacterial cell-to-cell interaction mechanism that affects the behavior of S. aureus and other members of the microbial community. Importantly, beyond bacteria-bacteria interactions, the Agr quorum sensing system also regulates various virulence factors, which induce type 2 and IL-17-dependent skin inflammation in the host. Furthermore, the colonization of Agr-positive S. aureus in early life accelerates the development of pediatric AD. Finally, we aim to highlight the current efforts to establish novel therapeutic methods to ameliorate or prevent AD through Agr-targeted intervention.

High-Efficiency Single-Cell Containment Microdevices Based on Fluid Control.

In this study, we developed a comb-shaped microfluidic device that can efficiently trap and culture a single cell (bacterium). Conventional culture devices have difficulty in trapping a single bacterium and often use a centrifuge to push the bacterium into the channel. The device developed in this study can store bacteria in almost all growth channels using the flowing fluid. In addition, chemical replacement can be performed in a few seconds, making this device suitable for culture experiments with resistant bacteria. The storage efficiency of microbeads that mimic bacteria was significantly improved from 0.2% to 84%. We used simulations to investigate the pressure loss in the growth channel. The pressure in the growth channel of the conventional device was more than 1400 PaG, whereas that of the new device was less than 400 PaG. Our microfluidic device was easily fabricated by a soft microelectromechanical systems method. The device was highly versatile and can be applied to various bacteria, such as Salmonella enterica serovar Typhimurium and Staphylococcus aureus.

Diversities of allergic pathologies and their modifiers: Report from the second DGAKI-JSA meeting.

In October 2021, researchers from the German Society of Allergy and Clinical Immunology (DGAKI) and from the Japanese Society of Allergology (JSA) focused their attention on the pathological conditions and modifiers of various allergic diseases. Topics included 1) the pathophysiology of IgE/mast cell-mediated allergic diseases; 2) the diagnosis and prevention of IgE/mast cell-mediated diseases; 3) the pathophysiology, diagnosis, and treatment of eosinophilic airway diseases; and 4) host-pathogen interaction and allergic diseases. This report summarizes the panel discussions, which highlighted the importance of recognizing the diversity of genetics, immunological mechanisms, and modifying factors underlying allergic diseases.

Epidermal clearance of Candida albicans is mediated by IL-17 but independent of fungal innate immune receptors.

IL-17 plays important roles in host defense against Candida albicans at barrier surfaces and during invasive infection. However, the role of IL-17 in host defense after colonization of the epidermis, a main site of C. albicans infection, remains poorly understood. Using a murine model of epicutaneous candidiasis without skin abrasion, we found that skin inflammation triggered by epidermal C. albicans colonization was self-limiting with fungal clearance completed by day 7 after inoculation in wild-type mice or animals deficient in IL-17A or IL-17F. In contrast, marked neutrophilic inflammation in the epidermis and impaired fungal clearance were observed in mice lacking both IL-17A and IL-17F. Clearance of C. albicans was independent of Dectin-1, Dectin-2, CARD9 (caspase-recruitment domain family, member 9), TLR2 (Toll-like receptor 2) and MyD88 in the epidermal colonization model. We found that group 3 innate lymphoid cells (ILC3s) and γδT cells were the major IL-17 producers in the epicutaneous candidiasis model. Analyses of Rag2-/- mice and Rag2-/-Il2rg-/- mice revealed that production of IL-17A and IL-17F by ILC3s was sufficient for C. albicans clearance. Finally, we found that depletion of neutrophils impaired C. albicans clearance in the epidermal colonization model. Taken together, these findings indicate a critical and redundant function of IL-17A and IL-17F produced by ILC3s in host defense against C. albicans in the epidermis. The results also suggest that epidermal C. albicans clearance is independent of innate immune receptors or that these receptors act redundantly in fungal recognition and clearance.

The Effects of Dietary Supplementation of Lactococcus lactis Strain Plasma on Skin Microbiome and Skin Conditions in Healthy Subjects-A Randomized, Double-Blind, Placebo-Controlled Trial.

(1) Background: Lactococcus lactis strain Plasma (LC-Plasma) is a unique strain which directly activates plasmacytoid dendritic cells, resulting in the prevention against broad spectrum of viral infection. Additionally, we found that LC-Plasma intake stimulated skin immunity and prevents Staphylococcus aureus epicutaneous infection. The aim of this study was to investigate the effect of LC-Plasma dietary supplementation on skin microbiome, gene expression in the skin, and skin conditions in healthy subjects. (2) Method: A randomized, double-blind, placebo-controlled, parallel-group trial was conducted. Seventy healthy volunteers were enrolled and assigned into two groups receiving either placebo or LC-Plasma capsules (approximately 1 × 1011 cells/day) for 8 weeks. The skin microbiome was analyzed by NGS and qPCR. Gene expression was analyzed by qPCR and skin conditions were diagnosed by dermatologists before and after intervention. (3) Result: LC-Plasma supplementation prevented the decrease of Staphylococcus epidermidis and Staphylococcus pasteuri and overgrowth of Propionibacterium acnes. In addition, LC-Plasma supplementation suggested to increase the expression of antimicrobial peptide genes but not tight junction genes. Furthermore, the clinical scores of skin conditions were ameliorated by LC-Plasma supplementation. (4) Conclusions: Our findings provided the insights that the dietary supplementation of LC-Plasma might have stabilizing effects on seasonal change of skin microbiome and skin conditions in healthy subjects.

Keratinocyte IL-36 Receptor/MyD88 Signaling Mediates Malassezia-Induced IL-17-Dependent Skin Inflammation.

BACKGROUND: Among skin commensal fungi, lipophilic Malassezia species exist on nearly all human skin surfaces. The pathophysiology of Malassezia-associated skin diseases remains poorly understood due in part to the lack of appropriate animal models. Our objective was to investigate the mechanisms underlying Malassezia-induced skin inflammation using a novel murine model that physiologically recapitulates Malassezia skin infection. METHODS: Mice were inoculated epicutaneously with Malassezia yeasts without barrier disruption and in the absence of external lipid supplementation. Skin inflammation, lesional fungal loads, and expression of cytokines and antimicrobial peptides were evaluated in wild-type and mutant mouse strains. RESULTS: Malassezia-induced skin inflammation and epidermal thickening were observed on day 4 after inoculation in wild-type mice. High fungal burdens were detected in the cornified layer on day 2 and decreased thereafter with near complete clearance by day 7 after inoculation. Malassezia-induced skin inflammation and fungal clearance by the host were interleukin-17 (IL-17) dependent with contribution of group 3 innate lymphoid cells. Moreover, IL-17-dependent skin inflammation was mediated through IL-36 receptor and keratinocyte MyD88 signaling. CONCLUSION: Using a new skin infection model, it is shown that Malassezia-induced IL-17- dependent skin inflammation and control of fungal infection are mediated via keratinocyte IL-36 receptor/MyD88 signaling.

Interaction between Staphylococcus Agr virulence and neutrophils regulates pathogen expansion in the skin.

Staphylococcus aureus commonly infects the skin, but the host-pathogen interactions controlling bacterial growth remain unclear. S. aureus virulence is regulated by the Agr quorum-sensing system that controls factors including phenol-soluble modulins (PSMs), a group of cytotoxic peptides. We found a differential requirement for Agr and PSMα for pathogen growth in the skin. In neutrophil-deficient mice, S. aureus growth on the epidermis was unaffected, but the pathogen penetrated the dermis through mechanisms that require PSMα. In the dermis, pathogen expansion required Agr in wild-type mice, but not in neutrophil-deficient mice. Agr limited oxidative and non-oxidative killing in neutrophils by inhibiting pathogen late endosome localization and promoting phagosome escape. Unlike Agr, the SaeR/S virulence program was dispensable for growth in the epidermis and promoted dermal pathogen expansion independently of neutrophils. Thus, S. aureus growth and invasion are differentially regulated with Agr limiting intracellular killing within neutrophils to promote pathogen expansion in the dermis and subcutaneous tissue.

Staphylococcus Agr virulence is critical for epidermal colonization and associates with atopic dermatitis development.

Atopic dermatitis (AD) is commonly associated with colonization by Staphylococcus aureus in the affected skin. To understand the role of S. aureus in the development of AD, we performed whole-genome sequencing of S. aureus strains isolated from the cheek skin of 268 Japanese infants 1 and 6 months after birth. About 45% of infants were colonized with S. aureus at 1 month regardless of AD outcome. In contrast, skin colonization by S. aureus at 6 months of age increased the risk of developing AD. Acquisition of dysfunctional mutations in the S. aureus Agr quorum-sensing (QS) system was primarily observed in strains from 6-month-old infants who did not develop AD. Expression of a functional Agr system in S. aureus was required for epidermal colonization and the induction of AD-like inflammation in mice. Thus, retention of functional S. aureus agr virulence during infancy is associated with pathogen skin colonization and the development of AD.

Three-Dimensional Epidermal Model from Human Hair Follicle-Derived Keratinocytes.

Three-dimensional (3D) epidermal models reconstructed from human skin-derived keratinocytes have been utilized as an alternative to animal testing and models, not only in toxicology, but also in skin biology. Although there are currently several reconstructed human epidermis (RHE) models commercially available, the donors of the keratinocytes are not identified in these models. A tailor-made system is needed to investigate the individual differences in RHE derived from each donor.It is possible to make an individual RHE using each donor's keratinocytes, which are usually obtained by invasive procedures such as skin excision or biopsy. To overcome this drawback, we established an RHE model using keratinocytes derived from plucked hair follicles as a less invasive procedure under conditions without feeder cells, serum, or matrix proteins. In this chapter, we provide a method of isolation and two-dimensional (2D) culture of keratinocytes derived from adult human plucked hair follicles including the outer root sheath (ORS). We also provide a detailed protocol for establishing an RHE model by culturing the keratinocytes under a 3D culture condition. We believe that our less invasive technique will provide a useful tool for investigating individual RHE in both normal and disease settings.

Staphylococcus aureus Epicutaneous Infection Is Suppressed by Lactococcus lactis Strain Plasma via Interleukin 17A Elicitation.

BACKGROUND: Lactococcus lactis strain Plasma (LC-Plasma) was revealed to stimulate plasmacytoid dendritic cells and induce antiviral immunity in vitro and in vivo. In this study, we assessed the effects of LC-Plasma on skin immunity. METHODS: To evaluate the effect of LC-Plasma on skin immunity and Staphylococcus aureus epicutaneous infection, lymphocyte activities in skin-draining lymph nodes (SLNs) and gene expression in skin were analyzed after 2 weeks of oral administration of LC-Plasma. To evaluate the mechanisms of interleukin 17A production, SLN lymphocytes were cultured with or without LC-Plasma, and the interleukin 17A concentrations in supernatants were measured. RESULTS: Oral administration of LC-Plasma activated plasma dendritic cells in SLNs, augmented skin homeostasis, and elicited suppression of Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes proliferation. In addition, significant suppression of the S. aureus burden and reduced skin inflammation were observed following oral administration of LC-Plasma. Furthermore, a subsequent in vitro study revealed that LC-Plasma could elicit interleukin 17A production from CD8+ T cells and that its induction mechanism depended on the Toll-like receptor 9 signaling pathway, with type I interferon partially involved. CONCLUSIONS: Our results suggest that LC-Plasma oral administration enhances skin homeostasis via plasma dendritic cell activation in SLNs, resulting in suppression of S. aureus epicutaneous infection and skin inflammation.

Application of an agr-Specific Antivirulence Compound as Therapy for Staphylococcus aureus-Induced Inflammatory Skin Disease.

Atopic dermatitis (AD) is a chronic inflammatory skin disease where more than 90% of patients affected are colonized with Staphylococcus aureus. In AD, S. aureus δ-toxin is a major virulence factor causing cutaneous inflammation via mast cell degranulation. δ-toxin is controlled by the S. aureus agr quorum sensing system, and thus we addressed whether interference with agr signaling would limit skin inflammation. Indeed, treatment of S. aureus with the agr-inhibitor solonamide B (SolB) abolished δ-toxin production and reduced skin inflammation in a mouse model of inflammatory skin disease, demonstrating the potential of antivirulence therapy in treating S. aureus-induced skin disorders.

IL-36α from Skin-Resident Cells Plays an Important Role in the Pathogenesis of Imiquimod-Induced Psoriasiform Dermatitis by Forming a Local Autoamplification Loop.

IL-36α (gene symbol Il1f6), a member of the IL-36 family, is closely associated with inflammatory diseases, including colitis and psoriasis. In this study, we found that Il1f6-/- mice developed milder psoriasiform dermatitis upon treatment with imiquimod, a ligand for TLR ligand 7 (TLR7) and TLR8, whereas Il1f6-/- mice showed similar susceptibility to dextran sodium sulfate-induced colitis to wild-type mice. These effects were observed in both cohoused and separately housed conditions, and antibiotic treatment did not cancel the resistance of Il1f6-/- mice to imiquimod-induced dermatitis. Bone marrow (BM) cell transfer revealed that IL-36α expression in skin-resident cells is important for the pathogenesis of dermatitis in these mice. Following stimulation with IL-36α, the expression of Il1f6 and Il1f9 (IL-36γ), but not Il1f8 (IL-36ß), was enhanced in murine BM-derived Langerhans cells (BMLCs) and murine primary keratinocytes but not in fibroblasts from mice. Upon stimulation with agonistic ligands of TLRs and C-type lectin receptors (CLRs), Il1f6 expression was induced in BMLCs and BM-derived dendritic cells. Furthermore, IL-36α stimulation resulted in significantly increased gene expression of psoriasis-associated Th17-related cytokines and chemokines such as IL-1α, IL-1ß, IL-23, CXCL1, and CXCL2 in BMLCs and fibroblasts, and IL-1α, IL-1ß, IL-17C, and CXCL2 in keratinocytes. Collectively, these results suggest that TLR/CLR signaling-induced IL-36α plays an important role for the development of psoriasiform dermatitis by enhancing Th17-related cytokine/chemokine production in skin-resident cells via a local autoamplification loop.

Streptococcal Cysteine Protease-Mediated Cleavage of Desmogleins Is Involved in the Pathogenesis of Cutaneous Infection.

Streptococcus pyogenes is responsible for a wide variety of cutaneous infections ranging from superficial impetigo to fulminant invasive necrotizing fasciitis. Dysfunction of desmosomes is associated with the pathogenesis of cutaneous diseases. We identified streptococcal pyrogenic exotoxin B (SpeB) as a proteolytic factor that cleaves the extracellular domains of desmoglein 1 and 3. In an epicutaneous infection model, lesional skin infected with an speB deletion mutant were significantly smaller as compared to those caused by the wild-type strain. Furthermore, immunohistological analysis indicated cleavage of desmogleins that developed around the invasion site of the wild-type strain. In contrast, the speB mutant was preferentially found on the epidermis surface layer. Taken together, our findings provide evidence that SpeB-mediated degradation of desmosomes has a pathogenic role in development of S. pyogenes cutaneous infection.

Staphylococcus aureus Virulent PSMα Peptides Induce Keratinocyte Alarmin Release to Orchestrate IL-17-Dependent Skin Inflammation.

Staphylococcus aureus commonly colonizes the epidermis, but the mechanisms by which the host senses virulent, but not commensal, S. aureus to trigger inflammation remain unclear. Using a murine epicutaneous infection model, we found that S. aureus-expressed phenol-soluble modulin (PSM)α, a group of secreted virulence peptides, is required to trigger cutaneous inflammation. PSMα induces the release of keratinocyte IL-1α and IL-36α, and signaling via IL-1R and IL-36R was required for induction of the pro-inflammatory cytokine IL-17. The levels of released IL-1α and IL-36α, as well as IL-17 production by γδ T cells and ILC3 and neutrophil infiltration to the site of infection, were greatly reduced in mice with total or keratinocyte-specific deletion of the IL-1R and IL-36R signaling adaptor Myd88. Further, Il17a-/-f-/- mice showed blunted S. aureus-induced inflammation. Thus, keratinocyte Myd88 signaling in response to S. aureus PSMα drives an IL-17-mediated skin inflammatory response to epicutaneous S. aureus infection.

Role of the microbiota in skin immunity and atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects 15-20% of children and 2-5% of adults in industrialized countries. The pathogen Staphylococcus aureus selectively colonizes the lesional skin of AD patients while this bacterium is absent in the skin of the majority of healthy individuals. However, the role of S. aureus in the pathogenesis of AD remains poorly understood. In addition to S. aureus, recent studies show a contribution of the skin microbiota to the regulation of immune responses in the skin as well as to the development of inflammatory skin disease. This review summarizes current knowledge about the role of the microbiota in skin immune responses and the role of S. aureus virulent factors in the pathogenesis of AD.