Effects of linear amphiphilicity on membrane interactions of C-terminal thrombin peptides

Effects of linear amphiphilicity on membrane interactions of antimicrobial peptides were investigated by ellipsometry, dual polarization interferometry, fluorescence spectroscopy, light scattering, and circular dichroism. In doing so, the thrombin-derived GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) was compared to WFF25 (WFFFYYLIIGGGVVTHQQRKKKKDE) of identical composition, but with amino acids sorted according to hydrophobicity, the latter peptide thus displaying pronounced linear amphiphilicity. In addition, GKY25d (GKYG(f) YTH(v) FRL(k) KWI(q) KVI(d) QFGE; with an identical sequence but with selected D-amino acid substitutions) was included as a control peptide, for which conformationally induced (helix-related) amphiphilicity was suppressed. Through its pronounced linear amphiphilicity, WFF25, but not the less amphiphilic GKY25 and GKY25d, forms aggregates in solution. Through its terminal W/F stretch, WFF25 also displays pronounced selectivity, with higher membrane binding and liposome rupture than GKY25 and GKY25d for anionic membranes, but suppressed peptide insertion and lytic effects for zwitterionic ones. In addition, WFF25 binds extensively to anionic polyelectrolyte components in bacterial membranes, i.e., lipopolysaccharide and lipoteichoic acid, resulting in reduced antimicrobial effects through peptide scavenging, not seen for the less amphiphilic GKY25 and GKY25d peptides. Taken together, the results thus demonstrate a series of striking effects for highly amphiphilic peptides, which need to be recognized in the development of such compounds as potential peptide therapeutics

Vertebrate TFPI-2 C-terminal peptides exert therapeutic applications against Gram-negative infections

Background: Tissue factor pathway inhibitor-2 (TFPI-2) is a serine protease inhibitor that exerts multiple physiological and patho-physiological activities involving the modulation of coagulation, angiogenesis, tumor invasion, and apoptosis. In previous studies we reported a novel role of human TFPI-2 in innate immunity by serving as a precursor for host defense peptides. Here we employed a number of TFPI-2 derived peptides from different vertebrate species and found that their antibacterial activity is evolutionary conserved although the amino acid sequence is not well conserved. We further studied the theraputic potential of one selected TFPI-2 derived peptide (mouse) in a murine sepsis model. Results: Hydrophobicity and net charge of many peptides play a important role in their host defence to invading bacterial pathogens. In vertebrates, the C-terminal portion of TFPI-2 consists of a highly conserved cluster of positively charged amino acids which may point to an antimicrobial activity. Thus a number of selected C-terminal TFPI-2 derived peptides from different species were synthesized and it was found that all of them exert antimicrobial activity against E. coli and P. aeruginosa. The peptide-mediated killing of E. coli was enhanced in human plasma, suggesting an involvement of the classical pathway of the complement. Under in vitro conditions the peptides displayed anti-coagulant activity by modulating the intrinsic pathway of coagulation and in vivo treatment with the mouse derived VKG24 peptide protects mice from an otherwise lethal LPS shock model. Conclusions: Our results suggest that the evolutionary conserved C-terminal part of TFPI-2 is an interesting agent for the development of novel antimicrobial therapies

An antimicrobial helix A-derived peptide of heparin cofactor II blocks endotoxin responses in vivo.

Host defense peptides are key components of the innate immune system, providing multi-facetted responses to invading pathogens. Here, we describe that the peptide GKS26 (GKSRIQRLNILNAKFAFNLYRVLKDQ), corresponding to the A domain of heparin cofactor II (HCII), ameliorates experimental septic shock. The peptide displays antimicrobial effects through direct membrane disruption, also at physiological salt concentration and in the presence of plasma and serum. Biophysical investigations of model lipid membranes showed the antimicrobial action of GKS26 to be mirrored by peptide incorporation into, and disordering of, bacterial lipid membranes. GKS26 furthermore binds extensively to bacterial lipopolysaccharide (LPS), as well as its endotoxic lipid A moiety, and displays potent anti-inflammatory effects, both in vitro and in vivo. Thus, for mice challenged with ip injection of LPS, GKS26 suppresses pro-inflammatory cytokines, reduces vascular leakage and infiltration in lung tissue, and normalizes coagulation. Together, these findings suggest that GKS26 may be of interest for further investigations as therapeutic against severe infections and septic shock

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

Therapeutic S100A8/A9 blockade inhibits myocardial and systemic inflammation and mitigates sepsis-induced myocardial dysfunction

Endotoxemia; Inflammation; Mitochondrial functionEndotoxemia; Inflamación; Función mitocondrialEndotoxèmia; Inflamació; Funció mitocondrialBackground and Aims The triggering factors of sepsis-induced myocardial dysfunction (SIMD) are poorly understood and are not addressed by current treatments. S100A8/A9 is a pro-inflammatory alarmin abundantly secreted by activated neutrophils during infection and inflammation. We investigated the efficacy of S100A8/A9 blockade as a potential new treatment in SIMD. Methods The relationship between plasma S100A8/A9 and cardiac dysfunction was assessed in a cohort of 62 patients with severe sepsis admitted to the intensive care unit of Linköping University Hospital, Sweden. We used S100A8/A9 blockade with the small-molecule inhibitor ABR-238901 and S100A9−/− mice for therapeutic and mechanistic studies on endotoxemia-induced cardiac dysfunction in mice. Results In sepsis patients, elevated plasma S100A8/A9 was associated with left-ventricular (LV) systolic dysfunction and increased SOFA score. In wild-type mice, 5 mg/kg of bacterial lipopolysaccharide (LPS) induced rapid plasma S100A8/A9 increase and acute LV dysfunction. Two ABR-238901 doses (30 mg/kg) administered intraperitoneally with a 6 h interval, starting directly after LPS or at a later time-point when LV dysfunction is fully established, efficiently prevented and reversed the phenotype, respectively. In contrast, dexamethasone did not improve cardiac function compared to PBS-treated endotoxemic controls. S100A8/A9 inhibition potently reduced systemic levels of inflammatory mediators, prevented upregulation of inflammatory genes and restored mitochondrial function in the myocardium. The S100A9−/− mice were protected against LPS-induced LV dysfunction to an extent comparable with pharmacologic S100A8/A9 blockade. The ABR-238901 treatment did not induce an additional improvement of LV function in the S100A9−/− mice, confirming target specificity. Conclusion Elevated S100A8/A9 is associated with the development of LV dysfunction in severe sepsis patients and in a mouse model of endotoxemia. Pharmacological blockade of S100A8/A9 with ABR-238901 has potent anti-inflammatory effects, mitigates myocardial dysfunction and might represent a novel therapeutic strategy for patients with severe sepsis.Open access funding provided by Lund University. This study was supported by grants from the Marianne and Marcus Wallenberg Foundation the Swedish Heart and Lung Foundation, the Swedish Research Council, the Bundy Academy foundation at Lund University, Skåne Region Research Funds, Malmö University Hospital Funds, the Crafoord Foundation, the Royal Physiographic Society in Lund, Swedish Research Council, Östergotland Region Research Funds, and the Ministry of Research and Education of Romania (PNRR-C9/I8-CF148)

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

Novel Aspects of the Coagulation System in Host Defence

Bacterial infections are a major global threat to public health and sepsis is a major cause of morbidity and mortality. Sepsis incidence is rising, with supportive treatments and antibiotics being the main treatment possibilities. Sepsis is characterised by an excessive and uncontrolled immune and coagulation response, leading to tissue damage, capillary leakage, and multiple organ failure. Although sepsis treatment has improved over the last decades, it still remains a challenging disease because of the multiplicity of symptoms caused by bacteria and bacterial products and in parallel, increased bacterial resistance. Therefore, there is clearly a demand for novel therapeutic strategies for sepsis, and correspondingly, also a need for increased knowledge on host defence molecules and their actions. The major goal of this thesis was to identify and characterise novel endogenous host defence peptides (HDPs) and proteins, to define their antimicrobial and immunomodulatory effects, and to explore their potential as novel anti-infectives. In response to injury and wounding, activation of the coagulation system generates a fibrin clot that prevents excessive blood loss and provides a first protective physical barrier. During this process, various HDPs are formed which play a role in the prevention of infections. In this work, we have defined new host defence activities of proteolytically generated HDPs derived from several well-known plasma proteins involved in initiation or control of blood clotting, including the main coagulation factor thrombin, and the protease inhibitors tissue factor pathway inhibitor -1 and -2, as well as heparin cofactor II. The discovered HDPs were antibacterial against Gram-positive and Gram-negative bacteria as well as fungi. Furthermore, the HDPs also displayed other bioactive roles, including down-regulation of inflammatory responses (thrombin-derived peptide, truncated heparin cofactor II), modulation of coagulation (TFPI-2 peptide), and boosting of complement activation (TFPI-1 and -2 peptides). Moreover, in animal models of septic shock and bacterial sepsis, several of these multifunctional HDPs significantly decreased mortality. In conclusion, host defence peptides with multiple properties could be potentially interesting in the development of the next generation of therapeutics for the treatment of bacterial infections and sepsis

Roflumilast increases bacterial load and dissemination in a model of Pseudomononas aeruginosa airway infection

Exacerbations present a major clinical problem in many patients suffering from COPD. Roflumilast, an inhibitor of phosphodiesterase 4, has beneficial effects in several clinical trials and is currently widely used to prevent exacerbations in severe COPD. Roflumilast has anti-inflammatory properties that may interfere with potentially important host defense functions, including cytotoxic properties of neutrophils at sites of inflammation. Since chronic bacterial infection are prevalent in severe COPD, Pseudomonas aeruginosa being a major pathogen, we hypothesized that this drug could impair host defense against P. aeruginosa. In this study, mice were pretreated with vehicle alone or roflumilast at doses of 5 mg/kg or 10 mg/kg followed by instillation of P. aeruginosa in the airways. Bacterial load and dissemination as well as inflammatory markers and immune cells present in the airways were followed. Roflumilast increased mortality, bacterial load and dissemination in mice infected with P. aeruginosa. In addition, roflumilast-treated mice had significantly lower number of neutrophils in the bronchi but not in the lung tissue airways compared with untreated mice. Several proinflammatory cytokines decreased in roflumilast-treated mice but neither the neutrophil-recruiting chemokine KC nor IL-6. The findings show that roflumilast-treatment impair host defense against P. aeruginosa in the airways. This may imply that patients suffering from chronic bacterial infection of the airways could benefit from being withheld treatment with roflumilast

Cold Atmospheric Plasma Disarms M1 Protein, an Important Streptococcal Virulence Factor

Cold atmospheric plasma (CAP) has been demonstrated to be a successful antiseptic for chronic and infected wounds. Although experimental work has focused on elucidation of the curative power of CAP for wound healing, the molecular mechanisms behind this ability are less understood. To date, the direct effect of CAP on the activity of microbial virulence factors has not been investigated. In the present study, we therefore examined whether CAP can modulate the detrimental activity of M1 protein, one of the most studied Streptococcus pyogenes virulence determinant. Our results show that CAP abolishes the ability of M1 protein to trigger inflammatory host responses. Subsequent mass spectrometric analysis revealed that this effect was caused by oxidation of Met81 and Trp128 located at the sub-N-terminal region of M1 protein provoking a conformational change. Notably, our results also show that CAP has an insignificant effect on the host immune system, supporting the benefits of using CAP to combat infections. Considering the growing number of antibiotic-resistant bacteria, novel antimicrobial therapeutic approaches are urgently needed that do not bear the risk of inducing additional resistance. Our study therefore may open new research avenues for the development of novel approaches for the treatment of skin and wound infections caused by S. pyogenes

Lipopolysaccharide Interactions of C-Terminal Peptides from Human Thrombin

Interactions with bacterial lipopolysaccharide (LPS), both in aqueous solution and in lipid membranes, were investigated for a series of amphiphilic peptides derived from the C-terminal region of human thrombin, using ellipsometry, dual polarization interferometry, fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering, and z-potential measurements. The ability of these peptides to block endotoxic effects caused by LPS, monitored through NO production in macrophages, was compared to peptide binding to LPS and its endotoxic component lipid A, and to size, charge, and secondary structure of peptide/LPS complexes. While the antiendotoxic peptide GKY25 (GKYGFYTHVFRL-KKWIQKVIDQFGE) displayed significant binding to both LPS and lipid A, so did two control peptides with either selected D-amino acid substitutions or with maintained composition but scrambled sequence, both displaying strongly attenuated antiendotoxic effects. Hence, the extent of LPS or lipid A binding is not the sole discriminant for the antiendotoxic effect of these peptides. In contrast, helix formation in peptide/LPS complexes correlates to the antiendotoxic effect of these peptides and is potentially linked to this functionality. Preferential binding to LPS over lipid membrane was furthermore demonstrated for these peptides and preferential binding to the lipid A moiety within LPS inferred