Skin-Derived SPINK9 Kills Escherichia coli.

Antimicrobial peptides play a critical role in the barrier function of human skin. They offer a fast response to invading microorganisms and protect from external microbial infection. Here we show the isolation of the kallikrein-related peptidase inhibitor SPINK9 as a major antibacterial factor from healthy stratum corneum. In total, six N-terminal SPINK9 variants were identified in the stratum corneum. Whereas all variants exhibited similar inhibition activities against kallikrein-related peptidase, only three variants with either lysine or glutamine as their first N-terminal residues were able to kill various Escherichia coli strains, but not other bacteria or fungi. The killing activity also depended on the sequence essential for kallikrein-related peptidase inhibition. Ultrastructural electron microscopy analyses suggested that SPINK9 entered the cell and killed growing bacteria. A bacterial chaperone, SKP, was identified as the major SPINK9 interacting partner in E. coli cells. The Skp-deleted mutant was more sensitive to SPINK9 than the wild-type control, suggesting that the bactericidal activity of SPINK9 should first overcome the resistance from the bacterial chaperone SKP. Thus, SPINK9 is a member of epidermal antimicrobial peptides for selective killing of E. coli, which might contribute to the innate barrier function of human skin.

The serine protease inhibitor of Kazal-type 7 (SPINK7) is expressed in human skin.

Proteases and their inhibitors play an important role in epidermal homeostasis. Their imbalance contributes to severe skin diseases. SPINK7 is a member of the SPINK protease inhibitor family and has been described so far as a cancer-related gene in the esophagus. Herein, we describe for the first time its expression in healthy human skin. Moreover, SPINK7 is up-regulated in inflammatory skin diseases like psoriasis and eczema as demonstrated by immunohistochemistry, though real-time PCR analyses revealed no significant up-regulation. In cultured keratinocytes, SPINK7 mRNA expression was up-regulated by IL-17A together with IFNγ. Our observation points to a role of SPINK7 in skin homeostasis and its involvement in inflammatory skin diseases.

Gingipains of Porphyromonas gingivalis Affect the Stability and Function of Serine Protease Inhibitor of Kazal-type 6 (SPINK6), a Tissue Inhibitor of Human Kallikreins.

Periodontitis, a chronic inflammation driven by dysbiotic subgingival bacterial flora, is linked on clinical levels to the development of a number of systemic diseases and to the development of oral and gastric tract tumors. A key pathogen, Porphyromonas gingivalis, secretes gingipains, cysteine proteases implicated as the main factors in the development of periodontitis. Here we hypothesize that gingipains may be linked to systemic pathologies through the deregulation of kallikrein-like proteinase (KLK) family members. KLKs are implicated in cancer development and are clinically utilized as tumor progression markers. In tissues, KLK activity is strictly controlled by a limited number of tissue-specific inhibitors, including SPINK6, an inhibitor of these proteases in skin and oral epithelium. Here we identify gingipains as the only P. gingivalis proteases responsible for SPINK6 degradation. We further show that gingipains, even at low nanomolar concentrations, cleaved SPINK6 in concentration- and time-dependent manner. The proteolysis was accompanied by loss of inhibition against KLK13. We also mapped the cleavage by Arg-specific gingipains to the reactive site loop of the SPINK6 inhibitor. Moreover, we identified a significant fraction of SPINK6-sensitive proteases in healthy saliva and confirmed the ability of gingipains to inactivate SPINK6 under ex vivo conditions. Finally, we demonstrate the double-edge action of gingipains, which, in addition, can activate KLKs because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13. Altogether, the results indicate the potential of P. gingivalis to disrupt the control system of KLKs, providing a possible mechanistic link between periodontal disease and tumor development.

The solution structure of the kallikrein-related peptidases inhibitor SPINK6.

Kallikrein-related peptidases (KLKs) are crucial for epidermal barrier function and are involved in the proteolytic regulation of the desquamation process. Elevated KLK levels were reported in atopic dermatitis. In skin, the proteolytic activity of KLKs is regulated by specific inhibitors of the serine protease inhibitor of Kazal-type (SPINK) family. SPINK6 was shown to be expressed in human stratum corneum and is able to inhibit several KLKs such as KLK4, -5, -12, -13 and -14. In order to understand the structural traits of the specific inhibition we solved the structure of SPINK6 in solution by NMR-spectroscopy and studied its interaction with KLKs. Thereby, beside the conserved binding mode, we identified an alternate binding mode which has so far not been observed for SPINK inhibitors.

The serine protease inhibitor of Kazal-type 9 (SPINK9) is expressed in lichen simplex chronicus, actinic keratosis and squamous cell carcinoma.

The serine protease inhibitor of Kazal-type (SPINK) 9 was reported to be exclusively expressed in palmoplantar skin. SPINK9 is a specific inhibitor of the serine protease kallikrein-related peptidase 5 (KLK5), which contributes to the desquamation process of the stratum corneum. Herein, we demonstrated that SPINK9 is also expressed in lichen simplex chronicus. Moreover, we observed expression of SPINK9 in 51 % of lesions of actinic keratosis and squamous cell carcinoma. In conclusion, we demonstrate that SPINK9 is not only expressed in healthy palmoplantar skin, but also in dermatologic disorders like lichen simplex chronicus, actinic keratosis and squamous cell carcinoma.

Kallikreins - The melting pot of activity and function.

The human tissue kallikrein and kallikrein-related peptidases (KLKs), encoded by the largest contiguous cluster of protease genes in the human genome, are secreted serine proteases with diverse expression patterns and physiological roles. Because of the broad spectrum of processes that are modulated by kallikreins, these proteases are the subject of extensive investigations. This review brings together basic information about the biochemical properties affecting enzymatic activity, with highlights on post-translational modifications, especially glycosylation. Additionally, we present the current state of knowledge regarding the physiological functions of KLKs in major human organs and outline recent discoveries pertinent to the involvement of kallikreins in cell signaling and in viral infections. Despite the current depth of knowledge of these enzymes, many questions regarding the roles of kallikreins in health and disease remain unanswered.

Decreased susceptibility of Staphylococcus aureus small-colony variants toward human antimicrobial peptides.

Staphylococcus aureus is a frequent resident of human nose and skin in many individuals, but it is also able to cause a variety of serious infections including those of the skin and soft tissue. There is increasing evidence that particularly persistent, relapsing, and difficult-to-treat infections caused by S. aureus are associated with the formation of the small-colony variant (SCV) phenotype. The aim of this study was to investigate the hypothesis that (i) skin-derived antimicrobial peptides (AMPs) exhibit a reduced activity against SCVs and (ii) that switching into the SCV phenotype may endow S. aureus with a decreased susceptibility toward the killing activity of human stratum corneum. Here, we show that clinically derived S. aureus SCVs are less susceptible to the bactericidal activity of different human skin-derived AMPs as compared with their isogenic corresponding wild-type strains. Similarly, a S. aureus hemB mutant displaying the SCV phenotype was less susceptible to the antimicrobial activity of AMPs than its hemB-complemented mutant. These findings were accompanied by a higher resistance of SCVs to the killing activity of human stratum corneum. Switching into the SCV phenotype may help S. aureus to subvert cutaneous innate defense, thus contributing to the establishment and persistence of infection.

SPINK9 stimulates metalloprotease/EGFR-dependent keratinocyte migration via purinergic receptor activation.

Serine protease inhibitors of the Kazal-type 9 (SPINK9) is a keratinocyte-derived cationic peptide that is found most abundantly in the upper layers of the palmar-plantar epidermis. In vitro, the peptide displays the capacity to inhibit specifically kallikrein-related peptidase 5 (KLK5). Here, we report that cells expressing SPINK9 secrete the peptide constitutively. Recombinant SPINK9 (rSPINK9) provoked transactivation of the EGFR in human keratinocytes, resulting in efficient downstream triggering of cell migration. Transactivation occurred via functional upregulation of a disintegrin and metalloproteases (ADAMs), as evidenced by suppression with a metalloproteinase inhibitor and an EGFR-blocking antibody. SPINK9 preparations isolated from human skin also displayed EGFR-transactivating capacity. The classical purinergic receptor antagonists oxidized ATP and pyridoxalphosphate-6-azophenyl-2',4',-disulfonic acid effectively suppressed EGFR transactivation by rSPINK9, indicating that in analogy to what has recently been reported for the cationic antimicrobial peptides cathelicidin LL-37 and bee venom melittin, purinergic receptors have an essential bridging role in promoting the upregulation of ADAM function by the cationic peptide. SPINK9 could represent an example of how a cationic peptide may subserve multiple and interrelated functions that contribute to the maintenance of the physical and immunological barrier of the skin.

Characterization of Spink6 in mouse skin: the conserved inhibitor of kallikrein-related peptidases is reduced by barrier injury.

The proteolytic regulation of the desquamation process by kallikrein-related peptidases (KLKs) is crucial for epidermal barrier function, and elevated KLK levels have been reported in atopic dermatitis. KLKs are controlled by specific inhibitors of the serine protease inhibitor of Kazal-type (Spink) family. Recently, SPINK6 was shown to be present in human stratum corneum. In order to investigate its role in epidermal barrier function, we studied mouse Spink6. Sequence alignment revealed that the Kazal domain of Spink6 is highly conserved in animals. Recombinant Spink6 efficiently inhibited mouse Klk5 and human KLK2, KLK4, KLK5, KLK6, KLK7, KLK12, KLK13, and KLK14, whereas human KLK1 and KLK8 were not inhibited. Spink6 was expressed in mouse epidermis mainly in the stratum granulosum, and the inner root sheath of hair follicles. Stimulation with flagellin, EGF, and IL-1ß did not alter Spink6 expression, whereas stimulation with tumor necrosis factor-α (TNFα)/IFNγ and all-trans retinoic acid resulted in a significant downregulation of Spink6 expression in cultured primary mouse keratinocytes. Mechanically and metabolically induced skin barrier dysfunction resulted both in a downregulation of Spink6 expression. Our study indicates that Spink6 is a potent inhibitor of KLKs and involved in skin barrier function.

Focal targeting by human ß-defensin 2 disrupts localized virulence factor assembly sites in Enterococcus faecalis.

Virulence factor secretion and assembly occurs at spatially restricted foci in some Gram-positive bacteria. Given the essentiality of the general secretion pathway in bacteria and the contribution of virulence factors to disease progression, the foci that coordinate these processes are attractive antimicrobial targets. In this study, we show in Enterococcus faecalis that SecA and Sortase A, required for the attachment of virulence factors to the cell wall, localize to discrete domains near the septum or nascent septal site as the bacteria proceed through the cell cycle. We also demonstrate that cationic human ß-defensins interact with E. faecalis at discrete septal foci, and this exposure disrupts sites of localized secretion and sorting. Modification of anionic lipids by multiple peptide resistance factor, a protein that confers antimicrobial peptide resistance by electrostatic repulsion, renders E. faecalis more resistant to killing by defensins and less susceptible to focal targeting by the cationic antimicrobial peptides. These data suggest a paradigm in which focal targeting by antimicrobial peptides is linked to their killing efficiency and to disruption of virulence factor assembly.

Immunomodulatory effects of vitamin D on innate and adaptive immune responses to Streptococcus pneumoniae.

BACKGROUND: Streptococcus pneumoniae forms part of the normal nasopharyngeal flora but can also cause a broad spectrum of inflammatory diseases. Vitamin D has potent effects on human immunity, including induction of antimicrobial peptides and suppression of T-cell proliferation, but its ability to modulate the immune response to pneumococci is unknown. METHODS: Monocyte-derived dendritic cells (DCs) were stimulated with pneumococcal peptidoglycan (PGN) in the presence or absence of vitamin D. Expression of maturation markers, cytokines, pattern recognition receptors, and antimicrobial peptides were measured with flow cytometry, enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Stimulated DCs were cocultured with autologous T-helper cells, and concentrations of T-helper (Th) 1-, Th17-, and regulatory T-cell-related cytokines were measured with enzyme-linked immunosorbent assay. RESULTS: Vitamin D enhanced DC maturation and expression of the migration marker C-C chemokine receptor type 7 (CCR7) in PGN-stimulated cells. It also enhanced expression of key pattern recognition receptors (Toll-like receptor 2, Nucleotide-binding oligomerization domain-containing protein 2 [Nod2]) and induced a synergistic up-regulation of the inflammatory mediator IL-1ß and the ß-defensin Human Beta Defensin 3 (hBD-3). Furthermore, vitamin D skewed the DC-mediated T-helper response to PGN from an inflammatory Th1/Th17 phenotype toward a regulatory T-cell phenotype. CONCLUSION: Vitamin D modulates key elements of innate immunity while dampening adaptive immune responses in DCs after pneumococcal challenge, which may have implications for prevention and treatment of pneumococcus-induced inflammation.

High-density genotyping study identifies four new susceptibility loci for atopic dermatitis.

Atopic dermatitis is a common inflammatory skin disease with a strong heritable component. Pathogenetic models consider keratinocyte differentiation defects and immune alterations as scaffolds, and recent data indicate a role for autoreactivity in at least a subgroup of patients. FLG (encoding filaggrin) has been identified as a major locus causing skin barrier deficiency. To better define risk variants and identify additional susceptibility loci, we densely genotyped 2,425 German individuals with atopic dermatitis (cases) and 5,449 controls using the Immunochip array followed by replication in 7,196 cases and 15,480 controls from Germany, Ireland, Japan and China. We identified four new susceptibility loci for atopic dermatitis and replicated previous associations. This brings the number of atopic dermatitis risk loci reported in individuals of European ancestry to 11. We estimate that these susceptibility loci together account for 14.4% of the heritability for atopic dermatitis.

Differential expression and in vivo secretion of the antimicrobial peptides psoriasin (S100A7), RNase 7, human beta-defensin-2 and -3 in healthy human skin.

Antimicrobial peptides (AMP) are key players in the skin's defense system. Previous observations suggest a site- and age-dependent expression of individual AMP. We investigated the expression and secretion patterns of four important AMP in a representative collective of healthy human skin samples. Levels of psoriasin, RNase 7 and hBD-3 expression - assessed by immunohistochemistry - varied between different body localisations. Older individuals expressed hBD-2 more frequently. No gender-related expression was observed. The in vivo secretion of psoriasin, measured in skin washing fluids using ELISA, was related to body localisation and age, whereas RNase 7 secretion showed no significant differences regarding these variables. HBD-2 and -3 secretion could not be detected. Our findings suggest the usage of control samples matching localisation and approximate age (in the case of hBD-2) for comparative immunohistochemical analysis. To avoid bias through great interindividual differences, sufficient large collectives should be used for in vivo secretion analyses.

Staphylococcus aureus subverts cutaneous defense by D-alanylation of teichoic acids.

The Gram-positive bacterium Staphylococcus aureus is a frequent skin colonizer that often causes severe skin infections. It has been reported that neutralizing the negatively charged bacterial surface through the incorporation of d-alanine in its teichoic acids confers reduced susceptibility of S. aureus towards cationic antimicrobial peptides (AMPs). Using a S. aureus strain deficient in d-alanylated teichoic acids (dltA mutant), we demonstrate that d-alanylation of its surface reduces the susceptibility of S. aureus to skin-derived AMPs such as RNase 7 and human beta-defensins. This is accompanied by a higher killing activity of skin extracts towards the S. aureus dltA mutant as well as towards clinical isolates expressing lower levels of dltA. We conclude that modulation of cell envelope d-alanylation may help S. aureus to persist on human skin through evasion of cutaneous innate defense provided by cationic skin-derived AMPs.

Cross-linking of SPINK6 by transglutaminases protects from epidermal proteases.

Extracellular kallikrein-related peptidases (KLKs) are involved in the desquamation process and the initiation of epidermal inflammation by different mechanisms. Their action is tightly controlled by specific protease inhibitors. Recently, we have identified the serine protease inhibitor of Kazal-type (SPINK) 6 as a selective inhibitor of KLKs in human stratum corneum extracts. As SPINK6 is expressed in the same localization as transglutaminases (TGM) and contains TGM substrate motifs, SPINK6 was tested to be cross-linked in the epidermis. Recombinant SPINK6 was shown to be cross-linked to fibronectin (FN) by TGM1 by western blot analyses. Moreover, SPINK6 was cross-linked in epidermal extracts and cultured keratinocytes by immunoblotting analyses. The use of TGM1 and TGM3 resulted in different immunoreactivities in western blot analyses of SPINK6 and epidermal extracts, suggesting substrate specifities of different TGMs for SPINK6 cross-linking in the epidermis. Conjugated SPINK6 exhibited protease inhibitory activity in keratinocytes and stratum corneum extracts; cross-linked SPINK6 protected FN from KLK5-mediated cleavage, whereas a lower KLK-inhibiting SPINK6-GM mutation did not. In conclusion, we demonstrated that SPINK6 is cross-linked in keratinocytes and human epidermis and remains inhibitory active. Thus, cross-linked SPINK6 might protect specific substrates such as FN from KLK cleavage and contributes to the regulation of proteases in the epidermis.

Extracellular cathepsin K exerts antimicrobial activity and is protective against chronic intestinal inflammation in mice.

OBJECTIVE: Cathepsin K is a lysosomal cysteine protease that has pleiotropic roles in bone resorption, arthritis, atherosclerosis, blood pressure regulation, obesity and cancer. Recently, it was demonstrated that cathepsin K-deficient (Ctsk(-/-) ) mice are less susceptible to experimental autoimmune arthritis and encephalomyelitis, which implies a functional role for cathepsin K in chronic inflammatory responses. Here, the authors address the relevance of cathepsin K in the intestinal immune response during chronic intestinal inflammation. DESIGN: Chronic colitis was induced by administration of 2% dextran sodium sulphate (DSS) in distilled water. Mice were assessed for disease severity, histopathology and endoscopic appearance. Furthermore, DSS-exposed Ctsk(-/-) mice were treated by rectal administration of recombinant cathepsin K. Intestinal microflora was assessed by real-time PCR and 16srDNA molecular fingerprinting of ileal and colonic mucosal and faecal samples. RESULTS: Using Ctsk(-/-) mice, the authors demonstrate a protective role of cathepsin K against chronic DSS colitis. Dissecting the underlying mechanisms the authors found cathepsin K to be present in intestinal goblet cells and the mucin layer. Furthermore, a direct cathepsin K-mediated bactericidal activity against intestinal bacteria was demonstrated, which potentially explains the alteration of intestinal microbiota observed in Ctsk(-/-) mice. Rectal administration of recombinant cathepsin K in DSS-treated Ctsk(-/-) mice ameliorates the severity of intestinal inflammation. CONCLUSION: These data identify extracellular cathepsin K as an intestinal antibacterial factor with anti-inflammatory potential and suggest that topical administration of cathepsin K might provide a therapeutic option for patients with inflammatory bowel disease.

Susceptibility of Staphylococcus aureus bacteremia strains to different skin-derived antimicrobial proteins.

Staphylococcus aureus is a major human pathogen causing cutaneous infections to life-threatening bacteremia. These infections are often caused by strains derived from the own microflora suggesting that a disturbed epidermal barrier may promote invasion of S. aureus. Antimicrobial peptides and proteins (AMP) such as human beta-defensin-3 and RNase 7 contribute to control the colonization of S. aureus on the skin surface. This leads to the hypothesis that strains with a decreased susceptibility toward skin-derived AMP may better overcome the innate cutaneous defence barrier increasing the possibility of invading into the blood stream. To address this hypothesis we determined whether S. aureus strains from bacteremia patients are less susceptible to various skin-derived AMP than strains from healthy carriers. No differences in the AMP-killing activity against bacteremia-derived S. aureus and control strains were detected suggesting that the onset of S. aureus bacteremia is not based on the varying susceptibilities against skin-derived AMP.