Discovery of natural bispecific antibodies: Is psoriasis induced by a toxigenic Corynebacterium simulans and maintained by CIDAMPs as autoantigens?

The high abundance of Corynebacterium simulans in psoriasis skin suggests a contribution to the psoriasis aetiology. This hypothesis was tested in an exploratory study, where western blot (WB) analyses with extracts of heat-treated C. simulans and psoriasis serum-derived IgG exhibited a single 16 kDa-WB-band. Proteomic analyses revealed ribosomal proteins as candidate C. s.-antigens. A peptidomic analysis unexpectedly showed that psoriasis serum-derived IgG already contained 31 immunopeptides of Corynebacteria ssp., suggesting the presence of natural bispecific antibodies (BsAbs). Moreover, peptidomic analyses gave 372 DECOY-peptides with similarity to virus- and phage proteins, including Corynebacterium diphtheriae phage, and similarity to diphtheria toxin. Strikingly, a peptidomic analysis for human peptides revealed 64 epitopes of major psoriasis autoantigens such as the spacer region of filaggrin, hornerin repeats and others. Most identified immunopeptides represent potential cationic intrinsically disordered antimicrobial peptides (CIDAMPs), which are generated within the epidermis. These may form complexes with bacterial disordered protein regions, representing chimeric antigens containing discontinuous epitopes. In addition, among 128 low-abundance immunopeptides, 48 are putatively psoriasis-relevant such as epitope peptides of PGE2-, vitamin D3- and IL-10-receptors. Further, 47 immunopeptides originated from tumour antigens, and the endogenous retrovirus HERV-K. I propose that persistent infection with a toxigenic C. simulans initiates psoriasis, which is exacerbated as an autoimmune disease by CIDAMPs as autoantigens. The discovery of natural BsAbs allows the identification of antigen epitopes from microbes, viruses, autoantigens and tumour-antigens, and may help to develop epitope-specific peptide-vaccines and therapeutic approaches with antigen-specific regulatory T cells to improve immune tolerance in an autoimmune disease-specific-manner.

Evolution of innate defense in human skin.

More often as compared to other barrier systems (gastrointestinal, urogenital, and respiratory linings) human skin over millions of years has been subject to fundamental changes in structure and function. When life on land started, the first changes consisted in the formation of a coherent impermeable stratum corneum. Two-legged locomotion was followed by loss of body hair and formation of sweat glands. Major changes took place after the agricultural revolution, investigating settlements with domestication of animals and plants. Living together after giving up nomadic life, hairless skin became a battlefield for pathogens, members of the skin microbiome, and arthropod visits. Human skin became exceptional in showing a boosted, highly developed immune system which is much more complex as compared to the "skins" of other species. A recently found skin disinfection system ("Cationic Intrinsically Disordered Antimicrobial Peptides, CIDAMPs") dates back to the origins of life and still is active in present-day integuments. As a skin-restricted and effective principle, keratinocyte- myeloid synergy (KMS) is recognized. As a consequence of such highly developed immune defense, the basic contributions of KMS - cells (keratinocytes, neutrophils, macrophages) in regulating innate immunity is emphasized. Antimicrobial peptides and chemokines became major keratinocyte products. The formation of impermeable str. corneum membrane has enabled KMS - cells to accumulate within upper skin levels and cause a special group of human skin diseases, pustular dermatoses.

Cationic Intrinsically Disordered Antimicrobial Peptides (CIDAMPs) Represent a New Paradigm of Innate Defense with a Potential for Novel Anti-Infectives.

In the search for potential mechanisms underlying the remarkable resistance of healthy skin against infection by soil bacteria like Pseudomonas (P.) aeruginosa we identified fragments of the intrinsically disordered protein hornerin as potent microbicidal agents in the stratum corneum. We found that, independent of the amino acid (AA)-sequence, any tested linear cationic peptide containing a high percentage of disorder-promoting AA and a low percentage of order-promoting AA is a potent microbicidal antimicrobial. We further show that the antimicrobial activity of these cationic intrinsically disordered antimicrobial peptides (CIDAMPs) depends on the peptide chain length, its net charge, lipidation and environmental conditions. The ubiquitous presence of latent CIDAMP sources in nature suggests a common and yet overlooked adapted innate disinfection system of body surfaces. The simple structure and virtually any imaginable sequence or composition of disorder-promoting AA allow the generation of a plethora of CIDAMPs. These are potential novel microbicidal anti-infectives for various bacterial pathogens, including P. aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA) and fungal pathogens like Candida albicans and Cryptococcus neoformans.

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.

Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics.

Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) belong to a novel class of epithelial peptide antibiotics with microbicidal activity against various pathogens, including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Here we show that treatment of distinct bacteria with different hornerin (HRNR)-derived CIDAMPs cause formation of unique cytoplasmic protein aggregates, suggesting a common intracellular mode of action. We further found that, unlike most amphipathic antimicrobial peptides, HRNR traverses bacterial membranes energy-dependently and accumulates within the cytoplasm. Strikingly, certain structurally different, HRNR-based CIDAMPs were found to bind to an identical panel of distinct bacterial ribosomal proteins, thereby manifesting features of several known classes of antibiotics. This may cause the formation of aberrant proteins and toxic protein aggregates in HRNR-treated pathogens which eventually may induce its death. Our study reveals evidence that structurally distinct CIDAMPs of an abundant body surface protein simultaneously target multiple sites of the bacterial protein synthesis machinery.

Expression and Regulation of S100 Fused-Type Protein Hornerin at the Ocular Surface and Lacrimal Apparatus.

Purpose: The S100 fused-type proteins hornerin (HRNR) and filaggrin-2 (FLG2) are members of the epidermal differentiation complex, which is involved in terminal differentiation of keratinocytes via cornification as well as maintenance of the epidermal antimicrobial barrier. We investigated the expression and possible regulation of HRNR and FLG2 at the ocular surface and in the lacrimal apparatus. Methods: Tissues of the lacrimal apparatus and ocular surface were analyzed systematically by means of RT-PCR, immunohistochemistry, and immuntransmission electron microscopy (iTEM) for their ability to express and produce HRNR and FLG2. In addition, inducibility and regulation of HRNR were studied in cultivated human corneal (HCE), conjunctival (HCjE), as well as meibomian gland (HMGEC) epithelial cell line by real-time RT-PCR. Results: RT-PCR, immunohistochemistry, and iTEM revealed constitutive expression of HRNR in the epithelium of cornea, conjunctiva, nasolacrimal ducts, and acinus cells of lacrimal and meibomian glands. HRNR also was detected in tears of healthy volunteers. No expression of FLG2 could be detected in tissue samples of the ocular surface and lacrimal apparatus. Real-time RT-PCR revealed a decreased HRNR gene expression after challenge with proinflammatory cytokines and supernatants of Escherichia coli and Pseudomonas aeroginosa in HCE cells, whereas HCjE cells revealed no changes. In HMGECs serum-induced differentiation and application of all-trans retinoic acid significantly increased HRNR gene expression. Conclusions: The data suggest that HRNR, but not FLG2, is a component of the ocular surface and lacrimal apparatus, including meibomian glands. HRNR seems to contribute to the maintenance of the epithelial barrier at the ocular surface and, thus, also may be involved in ocular surface diseases.

Molecular Basis of the Receptor Interactions of Polysialic Acid (polySia), polySia Mimetics, and Sulfated Polysaccharides.

Polysialic acid (polySia) and polySia glycomimetic molecules support nerve cell regeneration, differentiation, and neuronal plasticity. With a combination of biophysical and biochemical methods, as well as data mining and molecular modeling techniques, it is possible to correlate specific ligand-receptor interactions with biochemical processes and in vivo studies that focus on the potential therapeutic impact of polySia, polySia glycomimetics, and sulfated polysaccharides in neuronal diseases. With this strategy, the receptor interactions of polySia and polySia mimetics can be understood on a submolecular level. As the HNK-1 glycan also enhances neuronal functions, we tested whether similar sulfated oligo- and polysaccharides from seaweed could be suitable, in addition to polySia, for finding potential new routes into patient care focusing on an improved cure for various neuronal diseases. The knowledge obtained here on the structural interplay between polySia or sulfated polysaccharides and their receptors can be exploited to develop new drugs and application routes for the treatment of neurological diseases and dysfunctions.

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.

Revealing the Achilles heel of bacterial toxins.

In addition to having antimicrobial properties, defensins inactivate various structurally unrelated bacterial toxins by a yet unknown manner. In this issue of Immunity, Kudryashova et al. (2014b) provide insights into mechanisms by which human ?-defensins destabilize and inactivate bacterial toxins.