Supramolecular assembly of micellar aggregates is the basis of low endotoxin recovery (LER) in a drug formulation that can be resolved by a whole blood assay.

Lipopolysaccharide (LPS, endotoxin) is ubiquitous and represents a harmful contaminant of pharmaceutical compounds, recombinant biologicals and drug products. The pyrogen can induce severe immune responses and pathology in vitro and in vivo. Health authorities require strict control of endotoxin in parenteral drugs. However, for research and pre-clinical compound analysis, endotoxin testing is not a required quality control, which may cause potential drawbacks in the translational pipeline. Endotoxin testing is usually performed by the Limulus amebocyte lysate (LAL) assay, which is hampered by the so-called low endotoxin recovery (LER) effect when certain drug formulations are tested. A comprehensive study including structural, biophysical, and biological analyses was conducted to identify LER root cause for phosphate- and polysorbate-containing parenteral drug products. LPS in water showed extended ribbon-like aggregate structures. In placebo (formulation buffer without drug) and in drug product (drug in formulation buffer), a reaggregation of LPS into a network of interlinked micelles with hidden head group charges, and a strong reduction of the negative surface potential was observed. The non-accessibility of the LPS backbone has a direct impact leading (i) to a loss of activation of the LAL-cascade, (ii) reduced activation of the TLR4/MD-2 receptor system, and (iii) increased survival in a mouse model of endotoxemia. These data provide a structure-based explanation of the LER-underlying mechanisms. A human whole blood assay is shown to resolve LER and detect the pyrogenic activity of endotoxin with high sensitivity. This may open new test options to improve quality control in drug development and drug safety.

A Comparison between SARS-CoV-2 and Gram-Negative Bacteria-Induced Hyperinflammation and Sepsis.

Sepsis is a life-threatening condition caused by the body's overwhelming response to an infection, such as pneumonia or urinary tract infection. It occurs when the immune system releases cytokines into the bloodstream, triggering widespread inflammation. If not treated, it can lead to organ failure and death. Unfortunately, sepsis has a high mortality rate, with studies reporting rates ranging from 20% to over 50%, depending on the severity and promptness of treatment. According to the World Health Organization (WHO), the annual death toll in the world is about 11 million. One of the main toxins responsible for inflammation induction are lipopolysaccharides (LPS, endotoxin) from Gram-negative bacteria, which rank among the most potent immunostimulants found in nature. Antibiotics are consistently prescribed as a part of anti-sepsis-therapy. However, antibiotic therapy (i) is increasingly ineffective due to resistance development and (ii) most antibiotics are unable to bind and neutralize LPS, a prerequisite to inhibit the interaction of endotoxin with its cellular receptor complex, namely Toll-like receptor 4 (TLR4)/MD-2, responsible for the intracellular cascade leading to pro-inflammatory cytokine secretion. The pandemic virus SARS-CoV-2 has infected hundreds of millions of humans worldwide since its emergence in 2019. The COVID-19 (Coronavirus disease-19) caused by this virus is associated with high lethality, particularly for elderly and immunocompromised people. As of August 2023, nearly 7 million deaths were reported worldwide due to this disease. According to some reported studies, upregulation of TLR4 and the subsequent inflammatory signaling detected in COVID-19 patients "mimics bacterial sepsis". Furthermore, the immune response to SARS-CoV-2 was described by others as "mirror image of sepsis". Similarly, the cytokine profile in sera from severe COVID-19 patients was very similar to those suffering from the acute respiratory distress syndrome (ARDS) and sepsis. Finally, the severe COVID-19 infection is frequently accompanied by bacterial co-infections, as well as by the presence of significant LPS concentrations. In the present review, we will analyze similarities and differences between COVID-19 and sepsis at the pathophysiological, epidemiological, and molecular levels.

Repurposing the Antibacterial Agents Peptide 19-4LF and Peptide 19-2.5 for Treatment of Cutaneous Leishmaniasis.

The lack of safe and cost-effective treatments against leishmaniasis highlights the urgent need to develop improved leishmanicidal agents. Antimicrobial peptides (AMPs) are an emerging category of therapeutics exerting a wide range of biological activities such as anti-bacterial, anti-fungal, anti-parasitic and anti-tumoral. In the present study, the approach of repurposing AMPs as antileishmanial drugs was applied. The leishmanicidal activity of two synthetic anti-lipopolysaccharide peptides (SALPs), so-called 19-2.5 and 19-4LF was characterized in Leishmania major. In vitro, both peptides were highly active against intracellular Leishmania major in mouse macrophages without exerting toxicity in host cells. Then, q-PCR-based gene profiling, revealed that this activity was related to the downregulation of several genes involved in drug resistance (yip1), virulence (gp63) and parasite proliferation (Cyclin 1 and Cyclin 6). Importantly, the treatment of BALB/c mice with any of the two AMPs caused a significant reduction in L. major infective burden. This effect was associated with an increase in Th1 cytokine levels (IL-12p35, TNF-α, and iNOS) in the skin lesion and spleen of the L. major infected mice while the Th2-associated genes were downregulated (IL-4 and IL-6). Lastly, we investigated the effect of both peptides in the gene expression profile of the P2X7 purinergic receptor, which has been reported as a therapeutic target in several diseases. The results showed significant repression of P2X7R by both peptides in the skin lesion of L. major infected mice to an extent comparable to that of a common anti-leishmanial drug, Paromomycin. Our in vitro and in vivo studies suggest that the synthetic AMPs 19-2.5 and 19-4LF are promising candidates for leishmaniasis treatment and present P2X7R as a potential therapeutic target in cutaneous leishmaniasis (CL).

Antimicrobial Peptides in the Battle against Orthopedic Implant-Related Infections: A Review.

Prevention of orthopedic implant-related infections is a major medical challenge, particularly due to the involvement of biofilm-encased and multidrug-resistant bacteria. Current therapies, based on antibiotic administration, have proven to be insufficient, and infection prevalence may rise due to the dissemination of antibiotic resistance. Antimicrobial peptides (AMPs) have attracted attention as promising substitutes of conventional antibiotics, owing to their broad-spectrum of activity, high efficacy at very low concentrations, and, importantly, low propensity for inducing resistance. The aim of this review is to offer an updated perspective of the development of AMPs-based preventive strategies for orthopedic and dental implant-related infections. In this regard, two major research strategies are herein addressed, namely (i) AMP-releasing systems from titanium-modified surfaces and from bone cements or beads; and (ii) AMP immobilization strategies used to graft AMPs onto titanium or other model surfaces with potential translation as coatings. In overview, releasing strategies have evolved to guarantee higher loadings, prolonged and targeted delivery periods upon infection. In addition, avant-garde self-assembling strategies or polymer brushes allowed higher immobilized peptide surface densities, overcoming bioavailability issues. Future research efforts should focus on the regulatory demands for pre-clinical and clinical validation towards clinical translation.

Activity of Anti-Microbial Peptides (AMPs) against Leishmania and Other Parasites: An Overview.

Anti-microbial peptides (AMPs), small biologically active molecules, produced by different organisms through their innate immune system, have become a considerable subject of interest in the request of novel therapeutics. Most of these peptides are cationic-amphipathic, exhibiting two main mechanisms of action, direct lysis and by modulating the immunity. The most commonly reported activity of AMPs is their anti-bacterial effects, although other effects, such as anti-fungal, anti-viral, and anti-parasitic, as well as anti-tumor mechanisms of action have also been described. Their anti-parasitic effect against leishmaniasis has been studied. Leishmaniasis is a neglected tropical disease. Currently among parasitic diseases, it is the second most threating illness after malaria. Clinical treatments, mainly antimonial derivatives, are related to drug resistance and some undesirable effects. Therefore, the development of new therapeutic agents has become a priority, and AMPs constitute a promising alternative. In this work, we describe the principal families of AMPs (melittin, cecropin, cathelicidin, defensin, magainin, temporin, dermaseptin, eumenitin, and histatin) exhibiting a potential anti-leishmanial activity, as well as their effectiveness against other microorganisms.

Cathelicidin and PMB neutralize endotoxins by multifactorial mechanisms including LPS interaction and targeting of host cell membranes.

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell-directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.

An antibiotic potentiator retains its activity after being immobilized on silicone and prevents growth of multidrug-resistant Pseudomonas aeruginosa biofilms.

Device-Associated Healthcare-Associated Infections (DA-HAI) are a major threat to public health worldwide since they are associated with increased hospital stays, morbidity, mortality, financial burden, and hospital overload. A strategy to combat DA-HAI involves the use of medical devices endowed with surfaces that can kill or repel pathogens and prevent biofilm formation. We aimed to develop low-toxic protease-resistant anti-biofilm surfaces that can sensitize drug-resistant bacteria to sub-inhibitory concentrations of antibiotics. To this end, we hypothesized that polymyxin B nonapeptide (PMBN) could retain its antibiotic-enhancing potential upon immobilization on a biocompatible polymer, such as silicone. The ability of PMBN-coated silicone to sensitize a multidrug-resistant clinical isolate of Pseudomonas aeruginosa (strain Ps4) to antibiotics and block biofilm formation was assessed by viable counting, confocal microscopy and safranin uptake. These assays demonstrated that covalently immobilized PMBN enhances not only antibiotics added exogenously but also those incorporated into the functionalized coating. As a result, the functionalized surface exerted a potent bactericidal activity that precluded biofilm formation. PMBN-coated silicone displayed a high level of stability and very low cytotoxicity and hemolytic activity in the presence of antibiotics. We demonstrated for the first time that an antibiotic enhancer can retain its activity when covalently attached to a solid surface. These findings may be applied to the development of medical devices resistant to biofilm formation.

Anti-Infective and Anti-Inflammatory Mode of Action of Peptide 19-2.5.

The polypeptide Pep19-2.5 (Aspidasept®) has been described to act efficiently against infection-inducing bacteria by binding and neutralizing their most potent toxins, i.e., lipopolysaccharides (LPS) and lipoproteins/peptides (LP), independent of the resistance status of the bacteria. The mode of action was described to consist of a primary Coulomb/polar interaction of the N-terminal region of Pep19-2.5 with the polar region of the toxins followed by a hydrophobic interaction of the C-terminal region of the peptide with the apolar moiety of the toxins. However, clinical development of Aspidasept as an anti-sepsis drug requires an in-depth characterization of the interaction of the peptide with the constituents of the human immune system and with other therapeutically relevant compounds such as antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs). In this contribution, relevant details of primary and secondary pharmacodynamics, off-site targets, and immunogenicity are presented, proving that Pep19-2.5 may be readily applied therapeutically against the deleterious effects of a severe bacterial infection.

An update on endotoxin neutralization strategies in Gram-negative bacterial infections.

INTRODUCTION: Gram-negative bacterial infections represent still a severe problem of human health care, regarding the increase in multi-resistance against classical antibiotics and the lack of newly developed antimicrobials. For the fight against these germs, anti-infective agents must overcome and/or bind to the Gram-negative outer membrane consisting of a lipopolysaccharide (LPS, endotoxin) outer leaflet and an inner leaflet from phospholipids, with additional peripheral or integral membrane proteins (OMP's). AREAS COVERED: The current article reviews data of existing therapeutic options and summarizes newer approaches for targeting and neutralizing endotoxins, ranging from in vitro over in vivo animal data to clinical applications by using databases such as Medline. EXPERT OPINION: Conventional antibiotic treatment of the bacteria leads to their killing, but not necessary LPS neutralization, which may be a severe problem in particular for the systemic pathway. This is the reason why there is an increasing number of therapeutic approaches, which - besides combating whole bacteria - at the same time try to neutralize endotoxin within or outside the bacterial cells mainly responsible for the high inflammation induction in Gram-negative species.

A synthetic peptide sensitizes multi-drug resistant Pseudomonas aeruginosa to antibiotics for more than two hours and permeabilizes its envelope for twenty hours.

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative pathogen that frequently causes life-threatening infections in immunocompromised patients. We previously showed that subinhibitory concentrations of short synthetic peptides permeabilize P. aeruginosa and enhance the lethal action of co-administered antibiotics. METHODS: Long-term permeabilization caused by exposure of multidrug-resistant P. aeruginosa strains to peptide P4-9 was investigated by measuring the uptake of several antibiotics and fluorescent probes and by using confocal imaging and atomic force microscopy. RESULTS: We demonstrated that P4-9, a 13-amino acid peptide, induces a growth delay (i.e. post-antibiotic effect) of 1.3 h on a multidrug-resistant P. aeruginosa clinical isolate. Remarkably, when an independently P4-9-treated culture was allowed to grow in the absence of the peptide, cells remained sensitive to subinhibitory concentrations of antibiotics such as ceftazidime, fosfomycin and erythromycin for at least 2 h. We designated this persistent sensitization to antibiotics occurring in the absence of the sensitizing agent as Post-Antibiotic Effect associated Permeabilization (PAEP). Using atomic force microscopy, we showed that exposure to P4-9 induces profound alterations on the bacterial surface and that treated cells need at least 2 h of growth to repair those lesions. During PAEP, P. aeruginosa mutants overexpressing either the efflux pump MexAB-OprM system or the AmpC ß-lactamase were rendered sensitive to antibiotics that are known substrates of those mechanisms of resistance. Finally, we showed for the first time that the descendants of bacteria surviving exposure to a membrane disturbing peptide retain a significant level of permeability to hydrophobic compounds, including propidium iodide, even after 20 h of growth in the absence of the peptide. CONCLUSIONS: The phenomenon of long-term sensitization to antibiotics shown here may have important therapeutic implications for a combined peptide-antibiotic treatment because the peptide would not need to be present to exert its antibiotic enhancing activity as long as the target organism retains sensitization to the antibiotic.

Permeability enhancers sensitize ß-lactamase-expressing Enterobacteriaceae and Pseudomonas aeruginosa to ß-lactamase inhibitors, thereby restoring their ß-lactam susceptibility.

OBJECTIVES: ß-lactamases are the major resistance determinant for ß-lactam antibiotics in Gram-negative bacteria. Although there are ß-lactamase inhibitors (BLIs) available, ß-lactam-BLI combinations are increasingly being neutralised by diverse mechanisms of bacterial resistance. This study hypothesised that permeability-increasing antimicrobial peptides (AMPs) could lower the amount of BLIs necessary to sensitise bacteria to antibiotics that are ß-lactamase substrates. METHODS: To test this hypothesis, checkerboard assays were performed to measure the ability of several AMPs to synergise with piperacillin, ticarcillin, amoxicillin, ampicillin, and ceftazidime in the presence of either tazobactam, clavulanic acid, sulbactam, aztreonam, phenylboronic acid (PBA), or oxacillin. Assays were performed using planktonic and biofilm-forming cells of Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae overexpressing ß-lactamases. RESULTS: Synergy between polymyxin B nonapeptide (PMBN) and tazobactam boosted piperacillin activity by a factor of 128 in Escherichia coli (from 256 to 2 mg/L, fractional inhibitory concentration index (FICI) ≤ 0.02) and by a factor of at least 64 in Klebsiella pneumoniae (from 1024 mg/L to 16 mg/L, FICI ≤ 0.05). Synergy between PMBN and PBA enhanced ceftazidime activity 133 times in Pseudomonas aeruginosa (from 16 mg/L to 0.12 mg/L, FICI ≤ 0.03). As a consequence, MICs of all the tested antibiotics were brought down to therapeutic range. In addition, the combinations also reduced several orders of magnitude the amount of inhibitor needed for antibiotic sensitisation. Ceftazidime/PBA/PMBN at 50 times the planktonic MIC caused a 10 million-fold reduction in the viability of mature biofilms. CONCLUSION: This study proved that AMPs can synergise with BLIs and that this phenomenon can be exploited to sensitise bacteria to antibiotics.

Inactivation of Bacteria by γ-Irradiation to Investigate the Interaction with Antimicrobial Peptides.

The activity of antimicrobial peptides (AMPs) has been investigated extensively using model membranes composed of phospholipids or lipopolysaccharides in aqueous environments. However, from a biophysical perspective, there is a large scientific interest regarding the direct interaction of membrane-active peptides with whole bacteria. Working with living bacteria limits the usability of experimental setups and the interpretation of the resulting data because of safety risks and the overlap of active and passive effects induced by AMPs. We killed or inactivated metabolic-active bacteria using γ-irradiation or sodium azide, respectively. Microscopy, flow cytometry, and SYTOX green assays showed that the cell envelope remained intact to a high degree at the minimal bactericidal dose. Furthermore, the tumor-necrosis-factor-α-inducing activity of the lipopolysaccharides and the chemical lipid composition was unchanged. Determining the binding capacity of AMPs to the bacterial cell envelope by calorimetry is difficult because of an overlapping of the binding heat and metabolic activities of the bacteria-induced by the AMPs. The inactivation of all active processes helps to decipher the complex thermodynamic information. From the isothermal titration calorimetry (ITC) results, we propose that the bacterial membrane potential (Δψ) is possibly an underestimated modulator of the AMP activity. The negative surface charge of the outer leaflet of the outer membrane of Gram-negative bacteria is already neutralized by peptide concentrations below the minimal inhibitory concentration. This proves that peptide aggregation on the bacterial membrane surface plays a decisive role in the degree of antimicrobial activity. This will not only enable many biophysical approaches for the investigation between bacteria and membrane-active peptides in the future but will also make it possible to compare biophysical parameters of active and inactive bacteria. This opens up new possibilities to better understand the active and passive interaction processes between AMPs and bacteria.

LPS-neutralizing peptides reduce outer membrane vesicle-induced inflammatory responses.

Outer membrane vesicles (OMVs) are secreted by Gram-negative bacteria and induce a stronger inflammatory response than pure LPS. After endocytosis of OMVs by macrophages, lipopolysaccharide (LPS) is released from early endosomes to activate its intracellular receptors followed by non-canonical inflammasome activation and pyroptosis, which are critically involved in sepsis development. Previously, we could show that the synthetic anti-endotoxin peptide Pep19-2.5 neutralizes inflammatory responses induced by intracellular LPS. Here, we aimed to investigate whether Pep19-2.5 is able to suppress cytoplasmic LPS-induced inflammation under more physiological conditions by using OMVs which naturally transfer LPS to the cytosol. Isothermal titration calorimetry revealed an exothermic reaction between Pep19-2.5 and Escherichia coli OMVs and the Limulus Amebocyte Lysate assay indicated a strong endotoxin blocking activity. In THP-1 macrophages and primary human macrophages Pep19-2.5 and polymyxin B reduced interleukin (IL)-1ß and tumor necrosis factor (TNF) release as well as pyroptosis induced by OMVs, while the Toll-like receptor 4 signaling inhibitor TAK-242 suppressed OMV-induced TNF and IL-1ß secretion, but not pyroptosis. Internalization of Pep19-2.5 was at least partially mediated by the P2X7 receptor in macrophages but not in monocytes. Additionally, a cell-dependent difference in the neutralization efficiency of Pep19-2.5 became evident in macrophages and monocytes, indicating a critical role for peptide-mediated IL-1ß secretion via the P2X7 receptor. In conclusion, we provide evidence that LPS-neutralizing peptides inhibit OMV-induced activation of the inflammasome/IL-1 axis and give new insights into the mechanism of peptide-mediated neutralization of cytoplasmic LPS suggesting an essential and cell-type specific role for the P2X7 receptor.

Synthetic Anti-lipopolysaccharide Peptides (SALPs) as Effective Inhibitors of Pathogen-Associated Molecular Patterns (PAMPs).

Antimicrobial peptides (AMPs) are in the focus of scientific research since the 1990s. In most cases, the main aim was laid on the design of AMP to kill bacteria effectively, with particular emphasis on broadband action and independency on antibiotic resistance. However, so far no approved drug on the basis of AMP has entered the market.Our approach of constructing AMP, called synthetic anti-lipopolysaccharide peptides (SALPs), on the basis of inhibiting the inflammatory action of lipopolysaccharide (LPS, endotoxin) from Gram-negative bacteria was focused on the neutralization of the decisive toxins. These are, beside LPS from Gram-negative bacteria, the lipoproteins (LP) from Gram-positive origin. Although some of the SALPs have an antibacterial action, the most important property is the high-affinity binding to LPS and LP, whether as constituent of the bacteria or in free form which prevents the damaging inflammation, that could otherwise lead to life-threatening septic shock. Most importantly, the SALP may inhibit inflammation independently of the resistance status of the bacteria, and so far the repeated use of the peptides apparently does not cause resistance of the attacking pathogens.In this chapter, an overview is given over the variety of possible applications in the field of fighting against severe bacterial infections, from the use in systemic infection/inflammation up to various topical applications such as anti-biofilm action and severe skin and soft tissue infections.

AMPs as Anti-biofilm Agents for Human Therapy and Prophylaxis.

Microbial cells show a strong natural tendency to adhere to surfaces and to colonize them by forming complex communities called biofilms. In this growth mode, biofilm-forming cells encase themselves inside a dense matrix which efficiently protects them against antimicrobial agents and effectors of the immune system. Moreover, at the physiological level, biofilms contain a very heterogeneous cell population including metabolically inactive organisms and persisters, which are highly tolerant to antibiotics. The majority of human infectious diseases are caused by biofilm-forming microorganisms which are responsible for pathologies such as cystic fibrosis, infective endocarditis, pneumonia, wound infections, dental caries, infections of indwelling devices, etc. AMPs are well suited to combat biofilms because of their potent bactericidal activity of broad spectrum (including resting cells and persisters) and their ability to first penetrate and then to disorganize these structures. In addition, AMPs frequently synergize with antimicrobial compounds and were recently reported to repress the molecular pathways leading to biofilm formation. Finally, there is a very active research to develop AMP-containing coatings that can prevent biofilm formation by killing microbial cells on contact or by locally releasing their active principle. In this chapter we will describe these strategies and discuss the perspectives of the use of AMPs as anti-biofilm agents for human therapy and prophylaxis.

A permeability-increasing drug synergizes with bacterial efflux pump inhibitors and restores susceptibility to antibiotics in multi-drug resistant Pseudomonas aeruginosa strains.

Resistance to antibiotics poses a major global threat according to the World Health Organization. Restoring the activity of existing drugs is an attractive alternative to address this challenge. One of the most efficient mechanisms of bacterial resistance involves the expression of efflux pump systems capable of expelling antibiotics from the cell. Although there are efflux pump inhibitors (EPIs) available, these molecules are toxic for humans. We hypothesized that permeability-increasing antimicrobial peptides (AMPs) could lower the amount of EPI necessary to sensitize bacteria to antibiotics that are efflux substrates. To test this hypothesis, we measured the ability of polymyxin B nonapeptide (PMBN), to synergize with antibiotics in the presence of EPIs. Assays were performed using planktonic and biofilm-forming cells of Pseudomonas aeruginosa strains overexpressing the MexAB-OprM efflux system. Synergy between PMBN and EPIs boosted azithromycin activity by a factor of 2,133 and sensitized P. aeruginosa to all tested antibiotics. This reduced several orders of magnitude the amount of inhibitor needed for antibiotic sensitization. The selected antibiotic-EPI-PMBN combination caused a 10 million-fold reduction in the viability of biofilm forming cells. We proved that AMPs can synergize with EPIs and that this phenomenon can be exploited to sensitize bacteria to antibiotics.

Antibacterial action of synthetic antilipopolysaccharide peptides (SALP) involves neutralization of both membrane-bound and free toxins.

Increasing failure of conventional antibiotics to combat bacterial infections requires the urgent development of new antibacterial drugs; a promising class of new drugs based on antimicrobial peptides. Here, we studied the molecular interaction of polycationic synthetic antilipopolysaccharide peptides (SALPs) with various gram-negative and gram-positive bacteria, including resistant strains. The analysis of antimicrobial activity by conventional techniques and atomic force microscopy showed a strict dependence on amino acid (aa) sequences, with the type of amino acid, its position within the primary structure, and the sequence length being critical parameters. By monitoring lipopolysaccharide (LPS)- or bacteria-induced cytokine production in human mononuclear cells and whole blood, we found a direct link between the binding of the lead compound Pep19-2.5 to Salmonella enterica and the anti-inflammatory activity of the peptide. Thermodynamic analysis of Pep19-2.5 binding to the bacterial cell envelope showed an exothermic reaction with saturation characteristics, whereas small-angle X-ray scattering data indicated a direct attachment of Pep19-2.5 to the bacterial cell envelope. This binding preferentially takes place to the LPS outer monolayer, as evidenced by the change in the LPS acyl chain and phosphate vibrational bands seen by Fourier-transform infrared spectroscopy. We report here that the anti-inflammatory activity of Pep19-2.5 is not only connected with neutralization of cell-free bacterial toxins but also with a direct binding of the peptide to the outer leaflet of the bacterial outer membrane.

Inhibition of Lipopolysaccharide- and Lipoprotein-Induced Inflammation by Antitoxin Peptide Pep19-2.5.

The most potent cell wall-derived inflammatory toxins ("pathogenicity factors") of Gram-negative and -positive bacteria are lipopolysaccharides (LPS) (endotoxins) and lipoproteins (LP), respectively. Despite the fact that the former signals via toll-like receptor 4 (TLR4) and the latter via TLR2, the physico-chemistry of these compounds exhibits considerable similarity, an amphiphilic molecule with a polar and charged backbone and a lipid moiety. While the exterior portion of the LPS (i.e., the O-chain) represents the serologically relevant structure, the inner part, the lipid A, is responsible for one of the strongest inflammatory activities known. In the last years, we have demonstrated that antimicrobial peptides from the Pep19-2.5 family, which were designed to bind to LPS and LP, act as anti-inflammatory agents against sepsis and endotoxic shock caused by severe bacterial infections. We also showed that this anti-inflammatory activity requires specific interactions of the peptides with LPS and LP leading to exothermic reactions with saturation characteristics in calorimetry assays. Parallel to this, peptide-mediated neutralization of LPS and LP involves changes in various physical parameters, including both the gel to liquid crystalline phase transition of the acyl chains and the three-dimensional aggregate structures of the toxins. Furthermore, the effectivity of neutralization of pathogenicity factors by peptides was demonstrated in several in vivo models together with the finding that a peptide-based therapy sensitizes bacteria (also antimicrobial resistant) to antibiotics. Finally, a significant step in the understanding of the broad anti-inflammatory function of Pep19-2.5 was the demonstration that this compound is able to block the intracellular endotoxin signaling cascade.

Antimicrobial endotoxin-neutralizing peptides promote keratinocyte migration via P2X7 receptor activation and accelerate wound healing in vivo.

BACKGROUND AND PURPOSE: Wound healing is a complex process that is essential to provide skin homeostasis. Infection with pathogenic bacteria such as Staphylococcus aureus can lead to chronic wounds, which are challenging to heal. Previously, we demonstrated that the antimicrobial endotoxin-neutralizing peptide Pep19-2.5 promotes artificial wound closure in keratinocytes. Here, we investigated the mechanism of peptide-induced cell migration and if Pep19-2.5 accelerates wound closure in vivo. EXPERIMENTAL APPROACH: Cell migration was examined in HaCaT keratinocytes and P2X7 receptor-overexpressing HEK293 cells using the wound healing scratch assay. The protein expression of phosphorylated ERK1/2, ATP release, calcium influx and mitochondrial ROS were analysed to characterize Pep19-2.5-mediated signalling. For in vivo studies, female BALB/c mice were wounded and infected with methicillin-resistant S. aureus (MRSA) or left non-infected and treated topically with Pep19-2.5 twice daily for 6 days. KEY RESULTS: Specific P2X7 receptor antagonists inhibited Pep19-2.5-induced cell migration and ERK1/2 phosphorylation in keratinocytes and P2X7 receptor-transfected HEK293 cells. ATP release was not increased by Pep19-2.5; however, ATP was required for cell migration. Pep19-2.5 increased cytosolic calcium and mitochondrial ROS, which were involved in peptide-induced migration and ERK1/2 phosphorylation. In both non-infected and MRSA-infected wounds, the wound diameter was reduced already at day 2 post-wounding in the Pep19-2.5-treated groups compared to vehicle, and remained decreased until day 6. CONCLUSIONS AND IMPLICATIONS: Our data suggest the potential application of Pep19-2.5 in the treatment of non-infected and S. aureus-infected wounds and provide insights into the mechanism involved in Pep19-2.5-induced wound healing.

Coupling killing to neutralization: combined therapy with ceftriaxone/Pep19-2.5 counteracts sepsis in rabbits.

Sepsis, which is induced by severe bacterial infections, is a major cause of death worldwide, and therapies combating the disease are urgently needed. Because many drugs have failed in clinical trials despite their efficacy in mouse models, the development of reliable animal models of sepsis is in great demand. Several studies have suggested that rabbits reflect sepsis-related symptoms more accurately than mice. In this study, we evaluated a rabbit model of acute sepsis caused by the intravenous inoculation of Salmonella enterica. The model reproduces numerous symptoms characteristic of human sepsis including hyperlactatemia, hyperglycemia, leukopenia, hypothermia and the hyperproduction of several pro-inflammatory cytokines. Hence, it was chosen to investigate the proposed ability of Pep19-2.5-an anti-endotoxic peptide with high affinity to lipopolysaccharide and lipoprotein-to attenuate sepsis-associated pathologies in combination with an antibiotic (ceftriaxone). We demonstrate that a combination of Pep19-2.5 and ceftriaxone administered intravenously to the rabbits (1) kills bacteria and eliminates bacteremia 30 min post challenge; (2) inhibits Toll-like receptor 4 agonists in serum 90 min post challenge; (3) reduces serum levels of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor α); and (4) reverts to hypothermia and gives rise to temperature values indistinguishable from basal levels 330 min post challenge. The two components of the combination displayed synergism in some of these activities, and Pep19-2.5 notably counteracted the endotoxin-inducing potential of ceftriaxone. Thus, the combination therapy of Pep19-2.5 and ceftriaxone holds promise as a candidate for human sepsis therapy.