Proteinexpressionsanalysen und Funktionsuntersuchungen von Ifapsoriasin in der menschlichen Haut

Ifapsoriasin ist ein Mitglied der "S100 Fused Type Protein"-Familie, deren Mitglieder wie z. B. Profilaggrin, Trichohyalin und Hornerin hauptsächlich in Haut und Hautanhangsgebilden exprimiert werden und am Prozess der terminalen Differenzierung von Keratinozyten beteiligt sind. Ifapsoriasin (auch Filaggrin-2 genannt) zeigt als Mitglied die für diese "S100 Fused Type Protein"-Familie typische Proteinstruktur. Teilfragmente des humanen Proteins wurden rekombinant exprimiert und auf Expression sowie mögliche Funktion mittels generierter Antikörper untersucht. Ifapsoriasin wird vermutlich während des Prozesses der terminalen Differenzierung verstärkt exprimiert und prozessiert. Einige Ifapsoriasin-Fragmente zeigten zudem eine hohe antimikrobielle Aktivität gegen vorwiegend gram-negative Bakterien. Ifapsoriasin könnte also nicht nur eine strukturelle Rolle während der Differenzierung von Keratinozyten einnehmen, sondern auch Teil der epidermalen antimikrobiellen Abwehr sein

Molecular Identification and Expression Analysis of Filaggrin-2, a Member of the S100 Fused-Type Protein Family

Genes of the S100 fused-type protein (SFTP) family are clustered within the epidermal differentiation complex and encode essential components that maintain epithelial homeostasis and barrier functions. Recent genetic studies have shown that mutations within the gene encoding the SFTP filaggrin cause ichthyosis vulgaris and are major predisposing factors for atopic dermatitis. As a vital component of healthy skin, filaggrin is also a precursor of natural moisturizing factors. Here we present the discovery of a member of this family, designated as filaggrin-2 (FLG2) that is expressed in human skin. The FLG2 gene encodes a histidine- and glutamine-rich protein of approximately 248 kDa, which shares common structural features with other SFTP members, in particular filaggrin. We found that FLG2 transcripts are present in skin, thymus, tonsils, stomach, testis and placenta. In cultured primary keratinocytes, FLG2 mRNA expression displayed almost the same kinetics as that of filaggrin following Ca2+ stimulation, suggesting an important role in molecular regulation of epidermal terminal differentiation. We provide evidences that like filaggrin, FLG2 is initially expressed by upper granular cells, proteolytically processed and deposited in the stratum granulosum and stratum corneum (SC) layers of normal epidermis. Thus, FLG2 and filaggrin may have overlapping and perhaps synergistic roles in the formation of the epidermal barrier, protecting the skin from environmental insults and the escape of moisture by offering precursors of natural moisturizing factors

Microbe-host interplay in atopic dermatitis and psoriasis

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.Peer reviewe

Skin-Derived C-Terminal Filaggrin-2 Fragments Are Pseudomonas aeruginosa-Directed Antimicrobials Targeting Bacterial Replication.

Soil- and waterborne bacteria such as Pseudomonas aeruginosa are constantly challenging body surfaces. Since infections of healthy skin are unexpectedly rare, we hypothesized that the outermost epidermis, the stratum corneum, and sweat glands directly control the growth of P. aeruginosa by surface-provided antimicrobials. Due to its high abundance in the upper epidermis and eccrine sweat glands, filaggrin-2 (FLG2), a water-insoluble 248 kDa S100 fused-type protein, might possess these innate effector functions. Indeed, recombinant FLG2 C-terminal protein fragments display potent antimicrobial activity against P. aeruginosa and other Pseudomonads. Moreover, upon cultivation on stratum corneum, P. aeruginosa release FLG2 C-terminus-containing FLG2 fragments from insoluble material, indicating liberation of antimicrobially active FLG2 fragments by the bacteria themselves. Analyses of the underlying antimicrobial mechanism reveal that FLG2 C-terminal fragments do not induce pore formation, as known for many other antimicrobial peptides, but membrane blebbing, suggesting an alternative mode of action. The association of the FLG2 fragment with the inner membrane of treated bacteria and its DNA-binding implicated an interference with the bacterial replication that was confirmed by in vitro and in vivo replication assays. Probably through in situ-activation by soil- and waterborne bacteria such as Pseudomonads, FLG2 interferes with the bacterial replication, terminates their growth on skin surface and thus may contributes to the skin's antimicrobial defense shield. The apparent absence of FLG2 at certain body surfaces, as in the lung or of burned skin, would explain their higher susceptibility towards Pseudomonas infections and make FLG2 C-terminal fragments and their derivatives candidates for new Pseudomonas-targeting antimicrobials

FLG2-4 does not permeabilize the bacterial membrane.

<p>A: Lysis by 50 μg/ml lysozyme (expressed as OD_595nm against time) of chloramphenicol-treated <i>P</i>. <i>aeruginosa</i> PAO1 cells (gray line) in presence of Polymyxin B (PMB, 7.7 μM; dotted line) or FLG2-4 (0.8 μM; black line). B: SYTOX Green uptake by <i>P</i>. <i>aeruginosa</i> PAO1 treated with PMB (2 μM; dotted line), FLG2-4 (0.8μM; black line) or kept untreated (gray line). Results are expressed as percentage of maximum SYTOX Green uptake in the presence of PMB. Data represent means of three experiments ± SD.</p

Effect and localization of FLG2-4 on <i>P</i>. <i>aeruginosa</i>.

<p>A-D: Electron microscopy of <i>P</i>. <i>aeruginosa</i> ATCC 33354 treated with FLG2-4 for 30 min (A-C) and untreated (D); E: Confocal laser scanning microscopy of FLG2-4-treated <i>P</i>. <i>aeruginosa</i> ATCC 33354, left panels: DRAQ5 staining, middle panels: FLG2-4 staining, left panels: merged images, upper panels: FLG2-4 treated bacteria, lower panels: untreated bacteria; F: Western Blot of fractionated, FLG2-4-treated, <i>P</i>. <i>aeruginosa</i> PAO1. Beneath, inner membrane fraction, localized by western blotting using antibodies directed against XcpY; SN: sample supernatant, CYT: cytoplasmic, IM: inner membrane, PP: periplasmic, OM: outer membrane enriched fractions, FLG2-4: FLG2-4 recombinant fragment. Approximate molecular mass in kDa is indicated on the left.</p

FLG2-4 is able to inhibit bacterial replication.

<p>A: PCR <i>in vitro</i> inhibition assay. Different concentrations of FLG2-4 were tested for PCR-inhibiting properties. A GAPDH fragment was amplified from human keratinocyte cDNA. Control: PCR reaction without FLG2-4, solvent: solvent of FLG2-4 (0.01% acetic acid/0.02% NaN₃). A representative experiment out of twelve is shown. B: Densitometric analyses of semiquantitative PCRs of <i>in vivo</i> replication assays. Amplicons separated by agarose gels were stained with ethidium bromide and subjected to densitometric analysis. The error bars represents SD, significance was calculated using a two-way ANOVA and a Bonferroni post hoc test, n = 4. C: Exemplary <i>in vivo</i> replication inhibition PCR over indicated time periods. NTC: no template control, M: molecular mass marker,*: p<0.05, pBR322: positive control using 5ng pBR322 directly as template.</p

Antimicrobial activity of FLG2 C-terminal fragments tested against various bacteria in the radial diffusion assay.

<p>The corresponding amino acids of the full length protein are indicated below each fragment. The MEC is defined as the “minimal effective concentration” by the x-axis intersect in graphical analyses of clearing zones. The MECs stated represent the x-axis intersect of a regression calculated over all measured clearing zone units, the 95% confidence interval is indicated in parentheses; n = 3–12. Shaded gray: turbid clearing zones.</p

Interaction of FLG2-4 with DNA.

<p>A: Electrophoretic shift assays of ~120 ng supercoiled (left panel) and linearized (right panel) plasmid DNA with increasing concentrations of the FLG2-4 fragment. Used concentrations are indicated below the lanes. B: <i>In vivo</i> crosslinking of FLG2-4 to DNA. <i>P</i>. <i>aeruginosa</i> (Pa) were treated with FLG2-4 or left untreated, subsequently FLG2-4 DNA interactions were crosslinked with formaldehyde (F) and isolated DNA and non-precipitated fraction (Supn) were monitored for the presence of FLG2-4 by dot-blot analyses. Crl: FLG2-4.</p

FLG2 is detected in eccrine sweat glands and sweat.

<p>A-C: Immunohistochemistry in skin sections. Especially eccrine sweat glands were stained using the antibody directed against FLG2-4 (A, B). C: control section without primary antibody. D: Western Blot analysis of sweat shows an intense staining for FLG2-4 above 70 kDa only in samples pretreated for 1h with 10 mM DTT. A representative experiment out of four is shown. S/C: complete sweat sample, S/P: sweat pellet resuspended in PBS, S/S: sweat supernatant, separated by centrifugation. FLG2: FLG2-4 recombinant fragment. Approximate molecular mass in kDa is indicated on the left.</p