Proteolytic Degradation of reduced Human Beta Defensin 1 generates a Novel Antibiotic Octapeptide.

Microbial resistance against clinical used antibiotics is on the rise. Accordingly, there is a high demand for new innovative antimicrobial strategies. The host-defense peptide human beta-defensin 1 (hBD-1) is produced continuously by epithelial cells and exhibits compelling antimicrobial activity after reduction of its disulphide bridges. Here we report that proteolysis of reduced hBD-1 by gastrointestinal proteases as well as human duodenal secretions produces an eight-amino acid carboxy-terminal fragment. The generated octapeptide retains antibiotic activity, yet with distinct characteristics differing from the full-length peptide. We modified the octapeptide by stabilizing its termini and by using non-natural D-amino acids. The native and modified peptide variants showed antibiotic activity against pathogenic as well as antibiotic-resistant microorganisms, including E. coli, P. aeruginosa and C. albicans. Moreover, in an in vitro C. albicans infection model the tested peptides demonstrated effective amelioration of C. albicans infection without showing cytotoxity on human cells. In summary, protease degradation of hBD-1 provides a yet unknown mechanism to broaden antimicrobial host defense, which could be used to develop defensin-derived therapeutic applications.

ß-Defensin 1 Is Prominent in the Liver and Induced During Cholestasis by Bilirubin and Bile Acids via Farnesoid X Receptor and Constitutive Androstane Receptor.

Background & aims: Knowledge about innate antimicrobial defense of the liver is limited. We investigated hepatic expression and regulation of antimicrobial peptides with focus on the human beta defensin-1 (hBD-1). Methods: Radial diffusion assay was used to analyze antimicrobial activity of liver tissue. Different defensins including hBD-1 and its activator thioredoxin-1 (TXN) were analyzed in healthy and cholestatic liver samples by qPCR and immunostaining. Regulation of hBD-1 expression was studied in vitro and in vivo using bile duct-ligated mice. Regulation of hBD-1 via bilirubin and bile acids (BAs) was studied using siRNA. Results: We found strong antimicrobial activity of liver tissue against Escherichia coli. As a potential mediator of this antimicrobial activity we detected high expression of hBD-1 and TXN in hepatocytes, whereas other defensins were minimally expressed. Using a specific antibody for the reduced, antimicrobially active form of hBD-1 we found hBD-1 in co-localization with TXN within hepatocytes. hBD-1 was upregulated in cholestasis in a graded fashion. In cholestatic mice hepatic AMP expression (Defb-1 and Hamp) was enhanced. Bilirubin and BAs were able to induce hBD-1 in hepatic cell cultures in vitro. Treatment with siRNA and/or agonists demonstrated that the farnesoid X receptor (FXR) mediates basal expression of hBD-1, whereas both constitutive androstane receptor (CAR) and FXR seem to be responsible for the induction of hBD-1 by bilirubin. Conclusion: hBD-1 is prominently expressed in hepatocytes. It is induced during cholestasis through bilirubin and BAs, mediated by CAR and especially FXR. Reduction by TXN activates hBD-1 to a potential key player in innate antimicrobial defense of the liver.

Antifungal defense of probiotic Lactobacillus rhamnosus GG is mediated by blocking adhesion and nutrient depletion.

Candida albicans is an inhabitant of mucosal surfaces in healthy individuals but also the most common cause of fungal nosocomial blood stream infections, associated with high morbidity and mortality. As such life-threatening infections often disseminate from superficial mucosal infections we aimed to study the use of probiotic Lactobacillus rhamnosus GG (LGG) in prevention of mucosal C. albicans infections. Here, we demonstrate that LGG protects oral epithelial tissue from damage caused by C. albicans in our in vitro model of oral candidiasis. Furthermore, we provide insights into the mechanisms behind this protection and dissect direct and indirect effects of LGG on C. albicans pathogenicity. C. albicans viability was not affected by LGG. Instead, transcriptional profiling using RNA-Seq indicated dramatic metabolic reprogramming of C. albicans. Additionally, LGG had a significant impact on major virulence attributes, including adhesion, invasion, and hyphal extension, whose reduction, consequently, prevented epithelial damage. This was accompanied by glucose depletion and repression of ergosterol synthesis, caused by LGG, but also due to blocked adhesion sites. Therefore, LGG protects oral epithelia against C. albicans infection by preventing fungal adhesion, invasion and damage, driven, at least in parts, by metabolic reprogramming due to nutrient limitation caused by LGG.

Ubiquitously expressed Human Beta Defensin 1 (hBD1) forms bacteria-entrapping nets in a redox dependent mode of action.

Ever since the discovery of endogenous host defense antimicrobial peptides it has been discussed how these evolutionary conserved molecules avoid to induce resistance and to remain effective. Human ß-defensin 1 (hBD1) is an ubiquitously expressed endogenous antimicrobial peptide that exhibits qualitatively distinct activities between its oxidized and reduced forms. Here, we explore these antimicrobial mechanisms. Surprisingly, using electron microscopy we detected a so far unknown net-like structure surrounding bacteria, which were treated with the reduced but not the oxidized form of hBD1. A transmigration assay demonstrated that hBD1-derived nets capture bacteria and inhibit bacterial transmigration independent of bacterial killing. The presence of nets could completely prevent migration of hBD1 resistant pathogens and are stable in the presence of human duodenal secretion with a high amount of proteases. In contrast to HD6, cysteins are necessary for net formation. This redox-dependent function serves as an additional mechanism of action for hBD1 and differs from net formation by other defensins such as Paneth cell-derived human α-defensin 6 (HD6). While hBD1red and hBD1ox have distinct antimicrobial profiles and functions, only the reduced form provides additional host protection by entrapping bacteria in extracellular net structures preventing bacterial invasion. Better understanding of the modes of action of endogenous host peptides will help to find new antimicrobial strategies.

Crohn's disease-derived monocytes fail to induce Paneth cell defensins.

Crohn's disease (CD) is associated with a multitude of genetic defects, many of which likely affect Paneth cell function. Paneth cells reside in the small intestine and produce antimicrobial peptides essential for the host barrier, principally human α-defensin 5 (HD5) and HD6. Patients with CD of the ileum are characterized by reduced constitutive expression of these peptides and, accordingly, compromised antimicrobial barrier function. Here, we present a previously unidentified regulatory mechanism of Paneth cell defensins. Using cultures of human ileal tissue, we showed that the secretome of peripheral blood mononuclear cells (PBMCs) from healthy controls restored the attenuated Paneth cell α-defensin expression characteristic of patients with ileal CD. Analysis of the Wnt pathway in both cultured biopsies and intestinal epithelial cells implicated Wnt ligands driving the PBMC effect, whereas various tested cytokines were ineffective. We further detected another defect in patients with ileal CD, because the PBMC secretomes derived from patients with CD were unable to restore the reduced HD5/HD6 expression. Accordingly, analysis of PBMC subtypes showed that monocytes of patients with CD express significantly lower levels of canonical Wnt ligands, including Wnt3, Wnt3a, Wnt1, and wntless Wnt ligand secretion mediator (Evi/Wls). These studies reveal an important cross-talk between bone marrow-derived cells and epithelial secretory Paneth cells. Defective Paneth cell-mediated innate immunity due to inadequate Wnt ligand stimulation by monocytes provides an additional mechanism in CD. Because defects of Paneth cell function stemming from various etiologies are overcome by Wnt ligands, this mechanism is a potential therapeutic target for this disease.

A peptide derived from the highly conserved protein GAPDH is involved in tissue protection by different antifungal strategies and epithelial immunomodulation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has an important role not only in glycolysis but also in nonmetabolic processes, including transcription activation and apoptosis. We report the isolation of a human GAPDH (hGAPDH) (2-32) fragment peptide from human placental tissue exhibiting antimicrobial activity. The peptide was internalized by cells of the pathogenic yeast Candida albicans and initiated a rapid apoptotic mechanism, leading to killing of the fungus. Killing was dose-dependent, with 10 µg ml (3.1 µM) and 100 µg ml hGAPDH (2-32) depolarizing 45% and 90% of the fungal cells in a population, respectively. Experimental C. albicans infection induced epithelial hGAPDH (2-32) expression. Addition of the peptide significantly reduced the tissue damage as compared with untreated experimental infection. Secreted aspartic proteinase (Sap) activity of C. albicans was inhibited by the fragment at higher concentrations, with a median effective dose of 160 mg l(-1) (50 µM) for Sap1p and 200 mg l(-1) (63 µM) for Sap2p, whereas Sap3 was not inhibited at all. Interestingly, hGAPDH (2-32) induced significant epithelial IL-8 and GM-CSF secretion and stimulated Toll-like receptor 4 expression at low concentrations independently of the presence of C. albicans, without any toxic mucosal effects. In the future, the combination of different antifungal strategies, e.g., a conventional fungicidal with immunomodulatory effects and the inhibition of fungal virulence factors, might be a promising treatment option.

Glycosylation of Candida albicans cell wall proteins is critical for induction of innate immune responses and apoptosis of epithelial cells.

C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.

Alternative approaches to antifungal therapies.

The expansive use of immunosuppressive medications in fields such as transplantational medicine and oncology, the higher frequency of invasive procedures in an ageing population and the HIV/AIDS pandemic have increased the frequency of systemic fungal infections. At the same time, increased resistance of pathogenic fungi to classical antifungal agents has led to sustained research efforts targeting alternative antifungal strategies. In this review, we focus on two promising approaches: cationic peptides and the targeting of fungal virulence factors. Cationic peptides are small, predominantly positively charged protein fragments that exert direct and indirect antifungal activities, one mechanism of action being the permeabilization of the fungal membrane. They include lysozyme, defensins and cathelicidins as well as novel synthetic peptides. Among fungal virulence factors, the targeting of candidal secreted aspartic proteinases seems to be a particularly promising approach.

Immune responses to Candida albicans in models of in vitro reconstituted human oral epithelium.

In this protocol, we describe the application of commercially available three-dimensional organotypic tissues of human oral mucosa to study the interaction between Candida albicans and epithelial cells. Infection experiments show high reproducibility and can be used to analyse directly pathogen/epithelial cell interactions. However, the system is also very flexible. Using histological, biochemical, immunological, and molecular methods, it is possible to analyse several stages of infection by C. albicans wild type or mutant strains and demonstrate the consequence of disrupting genes encoding putative virulence factors required for host cell invasion and immune defence induction. This model provides information about host and pathogen protein and gene expression during direct interactions with each other. It can additionally be supplemented with other host factors, such as immune cells, saliva, and probiotic bacteria, which are relevant for host immune defence in the oral cavity.

Potential role of probiotic bacteria in the treatment and prevention of localised candidosis.

The extensive use of immunosuppressive therapies in recent years has increased the number of patients prone to or actually suffering from localised candidosis. As Candida species gain increasing resistance towards common antifungal drugs, new strategies are needed to prevent and treat infections caused by these pathogens. Probiotic bacteria have been in vogue in the past two decades. More and more dairy products containing such organisms offer promising potential beneficial effects on human health and well-being. Because of the ability of probiotic bacteria to inhibit the growth of pathogens and to modulate human immune responses, these bacteria could provide new possibilities in antifungal therapy. We summarise the recent findings concerning the usefulness of probiotic treatment in localised candidosis, as well as discussing possible risks of probiotic treatment and highlighting the molecular mechanisms that are believed to contribute to probiotic effects.