Skin Immune Landscape: Inside and Outside the Organism.

The skin is an essential organ to the human body protecting it from external aggressions and pathogens. Over the years, the skin was proven to have a crucial immunological role, not only being a passive protective barrier but a network of effector cells and molecular mediators that constitute a highly sophisticated compound known as the "skin immune system" (SIS). Studies of skin immune sentinels provided essential insights of a complex and dynamic immunity, which was achieved through interaction between the external and internal cutaneous compartments. In fact, the skin surface is cohabited by microorganisms recognized as skin microbiota that live in complete harmony with the immune sentinels and contribute to the epithelial barrier reinforcement. However, under stress, the symbiotic relationship changes into a dysbiotic one resulting in skin disorders. Hence, the skin microbiota may have either positive or negative influence on the immune system. This review aims at providing basic background information on the cutaneous immune system from major cellular and molecular players and the impact of its microbiota on the well-coordinated immune responses in host defense.

Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion.

The Salmonella Rck outer membrane protein binds to the cell surface, which leads to bacterial internalization via a Zipper mechanism. This invasion process requires induction of cellular signals, including phosphorylation of tyrosine proteins, and activation of c-Src and PI3K, which arises as a result of an interaction with a host cell surface receptor. In this study, epidermal growth factor receptor (EGFR) was identified as the cell signaling receptor required for Rck-mediated adhesion and internalization. First, Rck-mediated adhesion and internalization were shown to be altered when EGFR expression and activity were modulated. Then, immunoprecipitations were performed to demonstrate the Rck-EGFR interaction. Furthermore, surface plasmon resonance biosensor and homogeneous time-resolved fluorescence technologies were used to demonstrate the direct interaction of Rck with the extracellular domain of human EGFR. Finally, our study strongly suggests a noncompetitive binding of Rck and EGF to EGFR. Overall, these results demonstrate that Rck is able to bind to EGFR and thereby establish a tight adherence to provide a signaling cascade, which leads to internalization of Rck-expressing bacteria.-Wiedemann, A., Mijouin, L., Ayoub, M. A., Barilleau, E., Canepa, S., Teixeira-Gomes, A. P., Le Vern, Y., Rosselin, M., Reiter, E., Velge, P. Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion.

Biological Activity of Polynesian Calophyllum inophyllum Oil Extract on Human Skin Cells.

Oil from the nuts of Calophyllum inophyllum, locally called "Tamanu oil" in French Polynesia, was traditionally used for wound healing and to cure various skin problems and ailments. The skin-active effect of "Tamanu oil emulsion" was investigated on human skin cells (keratinocytes and dermal fibroblasts) and showed cell proliferation, glycosaminoglycan and collagen production, and wound healing activity. Transcriptomic analysis of the treated cells revealed gene expression modulation including genes involved in the metabolic process implied in O-glycan biosynthesis, cell adhesion, and cell proliferation. The presence of neoflavonoids as bioactive constituents in Tamanu oil emulsion may contribute to these biological activities. Altogether, consistent data related to targeted histological and cellular functions brought new highlights on the mechanisms involved in these biological processes induced by Tamanu oil effects in skin cells.

Effect of Substance P in Staphylococcus aureus and Staphylococcus epidermidis Virulence: Implication for Skin Homeostasis.

Staphylococcus aureus and Staphylococcus epidermidis are two major skin associated bacteria, and Substance P (SP) is a major skin neuropeptide. Since bacteria are known to sense and response to many human hormones, we investigated the effects of SP on Staphylococci virulence in reconstructed human epidermis model and HaCaT keratinocytes. We show that SP is stimulating the virulence of S. aureus and S. epidermidis in a reconstructed human epidermis model. qRT-PCR array analysis of 64 genes expressed by keratinocytes in the response to bacterial infection revealed a potential link between the action of SP on Staphylococci and skin physiopathology. qRT-PCR and direct assay of cathelicidin and human ß-defensin 2 secretion also provided that demonstration that the action of SP on bacteria is independent of antimicrobial peptide expression by keratinocytes. Considering an effect of SP on S. aureus and S. epidermidis, we observed that SP increases the adhesion potential of both bacteria on keratinocytes. However, SP modulates the virulence of S. aureus and S. epidermidis through different mechanisms. The response of S. aureus is associated with an increase in Staphylococcal Enterotoxin C2 (SEC2) production and a reduction of exolipase processing whereas in S. epidermidis the effect of SP appears mediated by a rise in biofilm formation activity. The Thermo unstable ribosomal Elongation factor Ef-Tu was identified as the SP-interacting protein in S. aureus and S. epidermidis. SP appears as an inter-kingdom communication factor involved in the regulation of bacterial virulence and essential for skin microflora homeostasis.

Pseudomonas aeruginosa Expresses a Functional Human Natriuretic Peptide Receptor Ortholog: Involvement in Biofilm Formation.

UNLABELLED: Considerable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa can detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF(4-23) mimics the effects of natriuretic peptides on P. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression. IMPORTANCE: The bacterium Pseudomonas aeruginosa is a highly dangerous opportunist pathogen for immunocompromised hosts, especially cystic fibrosis patients. The sites of P. aeruginosa infection are varied, with predominance in the human lung, in which bacteria are in contact with host molecular messengers such as hormones. The C-type natriuretic peptide (CNP), a hormone produced by lung cells, has been described as a bacterial virulence enhancer. In this study, we showed that the CNP hormone counteracts P. aeruginosa biofilm formation and we identified the bacterial protein AmiC as the sensor involved in the CNP effects. We showed that AmiC could bind specifically CNP. These results show for the first time that a human hormone could be sensed by bacteria through a specific protein, which is an ortholog of the human receptor NPR-C. The bacterium would be able to modify its lifestyle by favoring virulence factor production while reducing biofilm formation.

Effects of a skin neuropeptide (substance p) on cutaneous microflora.

BACKGROUND: Skin is the largest human neuroendocrine organ and hosts the second most numerous microbial population but the interaction of skin neuropeptides with the microflora has never been investigated. We studied the effect of Substance P (SP), a peptide released by nerve endings in the skin on bacterial virulence. METHODOLOGY/PRINCIPAL FINDINGS: Bacillus cereus, a member of the skin transient microflora, was used as a model. Exposure to SP strongly stimulated the cytotoxicity of B. cereus (+553±3% with SP 10(-6) M) and this effect was rapid (<5 min). Infection of keratinocytes with SP treated B. cereus led to a rise in caspase1 and morphological alterations of the actin cytoskeleton. Secretome analysis revealed that SP stimulated the release of collagenase and superoxide dismutase. Moreover, we also noted a shift in the surface polarity of the bacteria linked to a peel-off of the S-layer and the release of S-layer proteins. Meanwhile, the biofilm formation activity of B. cereus was increased. The Thermo unstable ribosomal Elongation factor (Ef-Tu) was identified as the SP binding site in B. cereus. Other Gram positive skin bacteria, namely Staphylococcus aureus and Staphylococcus epidermidis also reacted to SP by an increase of virulence. Thermal water from Uriage-les-Bains and an artificial polysaccharide (Teflose®) were capable to antagonize the effect of SP on bacterial virulence. CONCLUSIONS/SIGNIFICANCE: SP is released in sweat during stress and is known to be involved in the pathogenesis of numerous skin diseases through neurogenic inflammation. Our study suggests that a direct effect of SP on the skin microbiote should be another mechanism.

Antimicrobial peptides and pro-inflammatory cytokines are differentially regulated across epidermal layers following bacterial stimuli.

The skin is a natural barrier between the body and the environment and is colonised by a large number of microorganisms. Here, we report a complete analysis of the response of human skin explants to microbial stimuli. Using this ex vivo model, we analysed at both the gene and protein level the response of epidermal cells to Staphylococcus epidermidis (S. epidermidis) and Pseudomonas fluorescens (P. fluorescens), which are present in the cutaneous microbiota. We showed that both bacterial species affect the structure of skin explants without penetrating the living epidermis. We showed by real-time quantitative polymerase chain reaction (qPCR) that S. epidermidis and P. fluorescens increased the levels of transcripts that encode antimicrobial peptides (AMPs), including human ß defensin (hBD)2 and hBD3, and the pro-inflammatory cytokines interleukin (IL)-1α and (IL)-1-ß, as well as IL-6. In addition, we analysed the effects of bacterial stimuli on the expression profiles of genes related to innate immunity and the inflammatory response across the epidermal layers, using laser capture microdissection (LCM) coupled to qPCR. We showed that AMP transcripts were principally upregulated in suprabasal keratinocytes. Conversely, the expression of pro-inflammatory cytokines was upregulated in the lower epidermis. These findings were confirmed by protein localisation using specific antibodies coupled to optical or electron microscopy. This work underscores the potential value of further studies that use LCM on human skin explants model to study the roles and effects of the epidermal microbiota on human skin physiology.

Comparative study of normal and sensitive skin aerobic bacterial populations.

The purpose of this study was to investigate if the sensitive skin syndrome, a frequent skin disorder characterized by abnormal painful reactions to environmental factors in the absence of visible inflammatory response, could be linked to a modification in the skin bacterial population. A total of 1706 bacterial isolates was collected at the levels of the forehead, cheekbone, inner elbow, and lower area of the scapula on the skin of normal and sensitive skin syndrome-suffering volunteers of both sexes and of different ages. Among these isolates, 21 strains were randomly selected to validate in a first step the Matrix-Assisted Laser Desorption/Ionization (MALDI)-Biotyper process as an efficient identification tool at the group and genus levels, by comparison to API(®) strips and 16S ribosomal RNA gene sequencing identification techniques. In a second step, identification of the skin microbiota isolates by the MALDI-Biotyper tool allowed to pinpoint some differences in terms of bacterial diversity with regard to the collection area, and the volunteer's age and gender. Finally, comparison of the skin microbiota from normal and sensitive skin syndrome-suffering volunteers pointed out gender-related variations but no detectable correlation between a phylum, a genus or a dominant bacterial species and the sensitive skin phenotype. This study reveals that there is no dysbiosis of aerobic cultivable bacteria associated with the sensitive skin syndrome and further demonstrates that the MALDI-Biotyper is a powerful technique that can be efficiently employed to the study of cultivable human skin bacteria. To our knowledge, this is the first study focusing on bacteria in the sensitive skin syndrome. These results are of potential importance for pharmaceutical and cosmetic industries, which are looking for new strategies to treat this multiparametric disorder.

Effect of GABA, a bacterial metabolite, on Pseudomonas fluorescens surface properties and cytotoxicity.

Different bacterial species and, particularly Pseudomonas fluorescens, can produce gamma-aminobutyric acid (GABA) and express GABA-binding proteins. In this study, we investigated the effect of GABA on the virulence and biofilm formation activity of different strains of P. fluorescens. Exposure of a psychotropic strain of P. fluorescens (MF37) to GABA (10-5 M) increased its necrotic-like activity on eukaryotic (glial) cells, but reduced its apoptotic effect. Conversely, muscimol and bicuculline, the selective agonist and antagonist of eukaryote GABAA receptors, respectively, were ineffective. P. fluorescens MF37 did not produce biosurfactants, and its caseinase, esterase, amylase, hemolytic activity or pyoverdine productions were unchanged. In contrast, the effect of GABA was associated to rearrangements of the lipopolysaccharide (LPS) structure, particularly in the lipid A region. The surface hydrophobicity of MF37 was marginally modified, and GABA reduced its biofilm formation activity on PVC, but not on glass, although the initial adhesion was increased. Five other P. fluorescens strains were studied, and only one, MFP05, a strain isolated from human skin, showed structural differences of biofilm maturation after exposure to GABA. These results reveal that GABA can regulate the LPS structure and cytotoxicity of P. fluorescens, but that this property is specific to some strains.

In Planta Biocontrol of Pectobacterium atrosepticum by Rhodococcus erythropolis Involves Silencing of Pathogen Communication by the Rhodococcal Gamma-Lactone Catabolic Pathway.

The virulence of numerous Gram-negative bacteria is under the control of a quorum sensing process based on synthesis and perception of N-acyl homoserine lactones. Rhodococcus erythropolis, a Gram-positive bacterium, has recently been proposed as a biocontrol agent for plant protection against soft-rot bacteria, including Pectobacterium. Here, we show that the γ-lactone catabolic pathway of R. erythropolis disrupts Pectobacterium communication and prevents plant soft-rot. We report the first characterization and demonstration of N-acyl homoserine lactone quenching in planta. In particular, we describe the transcription of the R. erythropolis lactonase gene, encoding the key enzyme of this pathway, and the subsequent lactone breakdown. The role of this catabolic pathway in biocontrol activity was confirmed by deletion of the lactonase gene from R. erythropolis and also its heterologous expression in Escherichia coli. The γ-lactone catabolic pathway is induced by pathogen communication rather than by pathogen invasion. This is thus a novel and unusual biocontrol pathway, differing from those previously described as protecting plants from phytopathogens. These findings also suggest the existence of an additional pathway contributing to plant protection.

Pseudomonas fluorescens can induce and divert the human ß-defensin-2 secretion in intestinal epithelial cells to enhance its virulence.

The effect of intestinal molecules produced by the host on the virulence of Pseudomonas fluorescens is poorly documented. In the present work, we evaluated the secretion of human ß-defensin-2 (hBD-2) by enterocytes after infection with P. fluorescens (a species previously suggested to be involved in inflammatory bowel disease) and investigated the effect of this host-defense peptide on the bacterial virulence. The results showed that P. fluorescens can induce hBD-2 production in Caco-2/TC7 cells via P38 and ERK MAPK-dependent pathways. Surprisingly, the exposure of P. fluorescens to low doses of the antimicrobial peptide was found to enhance its cytotoxic and proinflammatory effects suggesting a potential feedback mechanism in the dialog between bacteria and the host.

Virulence of the Pseudomonas fluorescens clinical strain MFN1032 towards Dictyostelium discoideum and macrophages in relation with type III secretion system.

BACKGROUND: Pseudomonas fluorescens biovar I MFN1032 is a clinical isolate able to grow at 37°C. This strain displays secretion-mediated hemolytic activity involving phospholipase C and cyclolipopeptides, and a cell-associated hemolytic activity distinct from the secreted hemolytic activity. Cell-associated hemolysis is independent of biosurfactant production and remains in a gacA mutant. Disruption of the hrpU-like operon (the basal part of type III secretion system from rhizospheric strains) suppresses this activity. We hypothesized that this phenotype could reflect evolution of an ancestral mechanism involved in the survival of this species in its natural niche. In this study, we evaluated the hrpU-like operon's contribution to other virulence mechanisms using a panel of Pseudomonas strains from various sources. RESULTS: We found that MFN1032 inhibited the growth of the amoebae Dictyostelium discoideum and that this inhibition involved the hrpU-like operon and was absent in a gacA mutant. MFN1032 was capable of causing macrophage lysis, if the hrpU-like operon was intact, and this cytotoxicity remained in a gacA mutant. Cell-associated hemolytic activity and macrophage necrosis were found in other P. fluorescens clinical isolates, but not in biocontrol P. fluorescens strains harbouring hrpU-like operon. The growth of Dictyostelium discoideum was inhibited to a different extent by P. fluorescens strains without correlation between this inhibition and hrpU-like operon sequences. CONCLUSIONS: In P. fluorescens MFN1032, the basal part of type III secretion system plays a role in D. discoideum growth inhibition and macrophage necrosis. The inhibition of D. discoideum growth is dependent on the GacS/GacA system, while cell-associated hemolytic activity and macrophage lysis are not. Virulence against eukaryotic cells based on the hrpU-like operon may be more than just a stochastic evolution of a conserved system dedicated to survival in competition with natural predators such as amoebae. It may also mean that there are some important modifications of other type III secretion system components, which remain unknown. Cell-associated hemolysis might be a good indicator of the virulence of Pseudomonas fluorescens strain.

Involvement of c-Src tyrosine kinase upstream of class I phosphatidylinositol (PI) 3-kinases in Salmonella Enteritidis Rck protein-mediated invasion.

The Salmonella outer membrane protein Rck mediates a Zipper entry mechanism controlled by tyrosine phosphorylation and class I phosphatidylinositol 3-kinase (PI 3-kinase). However, the underlying mechanism leading to this signaling cascade remains unclear. The present study showed that using Rck-coated beads or Rck-overexpressing Escherichia coli, Rck-mediated actin polymerization and invasion were blocked by PP2, a Src family tyrosine kinase inhibitor. In addition, phosphorylation of Src family kinases significantly increased after stimulation with Rck. The specific contribution of c-Src, one member of the Src family kinases, was demonstrated using c-Src-deficient fibroblasts or c-Src siRNA transfected epithelial cells. We also observed that Rck-mediated internalization led to the formation of a complex between c-Src and at least one tyrosine-phosphorylated protein. Furthermore, our results revealed that the c-Src signal molecule was upstream of PI 3-kinase during the Rck-mediated signaling pathway as Rck-mediated PI 3-kinase activation was blocked by PP2, and PI 3-kinase inhibitor had no effect on the Src phosphorylation. These results demonstrate the involvement of c-Src upstream of the PI 3-kinase in the Zipper entry process mediated by Rck.

Salmonella enteritidis Rck-mediated invasion requires activation of Rac1, which is dependent on the class I PI 3-kinases-Akt signaling pathway.

The Salmonella outer membrane protein Rck mediates a Zipper-like entry mechanism controlled by Rac, the Arp2/3 complex, and actin polymerization. However, little is known about the early steps leading to Rac activation and Rck-mediated internalization. The use of pharmacological inhibitors or PI 3-kinase dominant-negative mutant induced more than 80% less invasion without affecting attachment. Moreover, Rck-mediated internalization caused an increase in the association of p85 with at least one tyrosine-phosphorylated protein, indicating that class I PI 3-kinase activity was stimulated. We also report that this PI 3-kinase activity is essential for Rac1 activation. However, Rac recruitment at the Rck-mediated entry site was independent of its activation. Using a pharmacological approach or Akt-knockout cells, we also demonstrated that Akt was phosphorylated in response to Rck-mediated internalization as demonstrated by immunoblotting analysis and that all three Akt isoforms were required during this process. Overall, our results describe a signaling pathway involving tyrosine phosphorylation, class I PI 3-kinase, Akt activation, and Rac activation, leading to Rck-dependent Zipper entry. The specificity of this signaling pathway with regard to that of the type 3 secretion system, which is the other invasion process of Salmonella, is discussed.

Rck of Salmonella enterica, subspecies enterica serovar enteritidis, mediates zipper-like internalization.

Salmonella can invade non-phagocytic cells through its type III secretion system (T3SS-1), which induces a Trigger entry process. This study showed that Salmonella enterica, subspecies enterica serovar Enteritidis can also invade cells via the Rck outer membrane protein. Rck was necessary and sufficient to enable non-invasive E. coli and Rck-coated beads to adhere to and invade different cells. Internalization analysis of latex beads coated with different Rck peptides showed that the peptide containing amino acids 140-150 promoted adhesion, whereas amino acids between 150 and 159 modulated invasion. Expression of dominant-negative derivatives and use of specific inhibitors demonstrated the crucial role of small GTPases Rac1 and Cdc42 in activating the Arp2/3 complex to trigger formation of actin-rich accumulation, leading to Rck-dependent internalization. Finally, scanning and transmission electron microscopy with Rck-coated beads and E. coli expressing Rck revealed microvillus-like extensions that formed a Zipper-like structure, engulfing the adherent beads and bacteria. Overall, our results provide new insights into the Salmonella T3SS-independent invasion mechanisms and strongly suggest that Rck induces a Zipper-like entry mechanism. Consequently, Salmonella seems to be the first bacterium found to be able to induce both Zipper and Trigger mechanisms to invade host cells.