Structure-Activity Studies and Therapeutic Potential of Host Defense Peptides of Human Thrombin

Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length-and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (> 12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest

Antimicrobial Activity of Human Prion Protein Is Mediated by Its N-Terminal Region

BACKGROUND: Cellular prion-related protein (PrP(c)) is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c), and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c) could exert antimicrobial activity. METHODOLOGY AND PRINCIPAL FINDINGS: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking) liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro. CONCLUSIONS: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense

Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

The coagulation system is characterized by the sequential and highly localized activation of a series of serine proteases, culminating in the conversion of fibrinogen into fibrin, and formation of a fibrin clot. Here we show that C-terminal peptides of thrombin, a key enzyme in the coagulation cascade, constitute a novel class of host defense peptides, released upon proteolysis of thrombin in vitro, and detected in human wounds in vivo. Under physiological conditions, these peptides exert antimicrobial effects against Gram-positive and Gram-negative bacteria, mediated by membrane lysis, as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, they are protective against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock. Moreover, the thrombin-derived peptides exhibit helical structures upon binding to lipopolysaccharide and can also permeabilize liposomes, features typical of “classical” helical antimicrobial peptides. These findings provide a novel link between the coagulation system and host-defense peptides, two fundamental biological systems activated in response to injury and microbial invasion

Histidine-Rich Glycoprotein Protects from Systemic Candida Infection

Fungi, such as Candida spp., are commonly found on the skin and at mucosal surfaces. Yet, they rarely cause invasive infections in immunocompetent individuals, an observation reflecting the ability of our innate immune system to control potentially invasive microbes found at biological boundaries. Antimicrobial proteins and peptides are becoming increasingly recognized as important effectors of innate immunity. This is illustrated further by the present investigation, demonstrating a novel antifungal role of histidine-rich glycoprotein (HRG), an abundant and multimodular plasma protein. HRG bound to Candida cells, and induced breaks in the cell walls of the organisms. Correspondingly, HRG preferentially lysed ergosterol-containing liposomes but not cholesterol-containing ones, indicating a specificity for fungal versus other types of eukaryotic membranes. Both antifungal and membrane-rupturing activities of HRG were enhanced at low pH, and mapped to the histidine-rich region of the protein. Ex vivo, HRG-containing plasma as well as fibrin clots exerted antifungal effects. In vivo, Hrg−/− mice were susceptible to infection by C. albicans, in contrast to wild-type mice, which were highly resistant to infection. The results demonstrate a key and previously unknown antifungal role of HRG in innate immunity

β-Microseminoprotein Endows Post Coital Seminal Plasma with Potent Candidacidal Activity by a Calcium- and pH-Dependent Mechanism

The innate immune factors controlling Candida albicans are mostly unknown. Vulvovaginal candidiasis is common in women and affects approximately 70–75% of all women at least once. Despite the propensity of Candida to colonize the vagina, transmission of Candida albicans following sexual intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as β-microseminoprotein. At neutral pH, the fungicidal activity of β-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E71 was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of β-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. A homologous fragment from porcine β-microseminoprotein demonstrated calcium-dependent fungicidal activity in a CFU assay, suggesting this may be a common feature for members of the β-microseminoprotein family. By electron microscopy, β-microseminoprotein was found to cause lysis of Candida. Liposome experiments demonstrated that β-microseminoprotein was active towards ergosterol-containing liposomes that mimic fungal membranes, offering an explanation for the selectivity against fungi. These data identify β-microseminoprotein as an important innate immune factor active against C. albicans and may help explain the low sexual transmission rate of Candida

ANTIMICROBIAL ACTIVITIES OF HISTIDINE-RICH GLYCOPROTEIN AND CATIONIC PEPTIDES

In an environment full of potential pathogens it is of importance for organisms to mount a fast and effective defence. Antimicrobial peptides are ancient and integral effector molecules of the innate immune system. They are found in all kinds of species from bacteria to plants and animals, indicating their importance during evolution. They possess a broad-spectrum antimicrobial activity and some peptides can also participate in wound healing and connect the innate and adaptive immune systems. Results presented in this thesis show that structural motifs connected with heparin-binding may confer antimicrobial activity to a given peptide. Peptides from various heparin-binding endogenous proteins exerted antimicrobial activity against Gram-positive and Gram-negative bacteria and similar results were obtained with consensus sequences for heparin-binding. Furthermore, we demonstrated that replacement of lysine and arginine by histidine in the consensus motifs abrogated the antibacterial effects of these peptides. Antibacterial effects of the histidine-rich consensus peptides were restored by the addition of Zn2+ or low pH. Similar results were obtained with histidine-rich peptides derived from domain 5 of kininogen and histidine-rich glycoprotein (HRGP). HRGP, an abundant heparin-binding plasma protein, exerted antimicrobial effects against Gram-positive and Gram-negative bacteria and fungi. The antibacterial activity of HRGP was dependent on Zn2+-ions or low pH, and the antifungal activity was increased under low pH conditions. Electron microscopy demonstrated that HRGP induced lysis of bacteria and fungi. Truncated HRGP, devoid of the heparin-binding and histidine-rich domain, was not antimicrobial. In addition, HRGP was found to have antifungal effects ex vivo when bound to fibrin clots

Histidine-rich glycoprotein exerts antibacterial activity.

Histidine-rich glycoprotein (HRGP), an abundant heparin-binding protein found in plasma and thrombocytes, exerts antibacterial effects against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Fluorescence studies and electron microscopy to assess membrane permeation showed that HRGP induces lysis of E. faecalisbacteria in the presence of Zn2+ or at low pH. Heparin blocked binding of the protein to E. faecalis and abolished antibacterial activity. Furthermore, truncated HRGP, devoid of the heparin-binding and histidine-rich domain, was not antibacterial. It has previously been shown that peptides containing consensus heparin-binding sequences (Cardin and Weintraub motifs) are antibacterial. Thus, the peptide (GHHPH)(4), derived from the histidine-rich region of HRGP and containing such a heparin-binding motif, was antibacterial for E. faecalis in the presence of Zn2+ or at low pH. The results show a previously undisclosed antibacterial activity of HRGP and suggest that the histidine-rich and heparin-binding domain of HRGP mediates the antibacterial activity of the protein

Domain 5 of high molecular weight kininogen is antibacterial.

Antimicrobial peptides are important effectors of the innate immune system. These peptides belong to a multifunctional group of molecules that apart from their antibacterial activities also interact with mammalian cells and glycosaminoglycans and control chemotaxis, apoptosis, and angiogenesis. Here we demonstrate a novel antimicrobial activity of the heparin-binding and cell-binding domain 5 of high molecular weight kininogen. Antimicrobial epitopes of domain 5 were characterized by analysis of overlapping peptides. A peptide, HKH20 (His(479) - His(498)), efficiently killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa and the Gram-positive Enterococcus faecalis. Fluorescence microscopy and electron microscopy demonstrated that HKH20 binds to and induces breaks in bacterial membranes. Furthermore, no discernible hemolysis or membrane-permeabilizing effects on eukaryotic cells were noted. Proteolytic degradation of high molecular weight kininogen by neutrophil-derived proteases as well as the metalloproteinase elastase from P. aeruginosa yielded fragments comprising HKH20 epitopes, indicating that kininogen-derived antibacterial peptides are released during proteolysis

Antimicrobial activity of histidine-rich peptides is dependent on acidic conditions.

Synthetic peptides composed of multiples of the consensus heparin-binding Cardin and Weintraub sequences AKKARA and ARKKAAKA are antimicrobial. Replacement of lysine and arginine by histidine in these peptides completely abrogates their antimicrobial and heparin-binding activities at neutral pH. However, the antibacterial activity against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) as well as the fungus Candida albicans, was restored at acidic conditions (pH 5.5). Fluorescence microscopy and FACS analysis showed that the binding of the histidine-rich peptides to E. coli and Candida was significantly enhanced at pH 5.5. Likewise, fluorescence studies for assessment of membrane permeation as well as electron microscopy analysis of peptide-treated bacteria, paired with studies of peptide effects on liposomes, demonstrated that the peptides induce membrane lysis only at acidic pH. No discernible hemolysis was noted for the histidine-rich peptides. Similar pH-dependent antimicrobial activities were demonstrated for peptides derived from histidine-rich and heparin-binding regions of human kininogen and histidine-rich glycoprotein. The results demonstrate that the presence of an acidic environment is an important regulator of the activity of histidine-rich antimicrobial peptides