Journals
Magazine:
PHARMACEUTICS
ISSN:
1999-4923
Year:
2022
Vol:
14
N°:
11
Pp:
2528
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-alpha, 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).
Magazine:
MATERIALS SCIENCE AND ENGINEERING C-MATERIALS FOR BIOLOGICAL APPLICATIONS
ISSN:
0928-4931
Year:
2021
Vol:
121
Ppgs:
111876
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 haemolytic 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 so
Authors:
Heinbockel, L.; Weindl, G.; Correa, W.; et al.
Magazine:
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
ISSN:
1422-0067
Year:
2021
Vol:
22
N°:
3
Pp:
1465
The polypeptide Pep19-2.5 (Aspidasept(R)) 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.
Authors:
Schromm, A. B. (Corresponding author); Paulowski, L.; Kaconis, Y.; et al.
Magazine:
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN:
0027-8424
Year:
2021
Vol:
118
N°:
27
Pp:
e2101721118
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 peptidemediated 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.
Magazine:
JOURNAL OF BIOMEDICAL SCIENCE
ISSN:
1021-7770
Year:
2020
Vol:
27
N°:
1
Pp:
85
Background Pseudomonas aeruginosais a Gram-negative pathogen that frequently causes life-threatening infections in immunocompromised patients. We previously showed that subinhibitory concentrations of short synthetic peptides permeabilizeP. aeruginosaand enhance the lethal action of co-administered antibiotics. Methods Long-term permeabilization caused by exposure of multidrug-resistantP. aeruginosastrains 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-resistantP. aeruginosaclinical 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. aeruginosamutants overexpressing either the efflux pump MexAB-OprM system or the AmpC beta-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.
Magazine:
INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS
ISSN:
0924-8579
Year:
2020
Vol:
56
N°:
1
Pp:
105986
Objectives: beta-lactamases are the major resistance determinant for beta-lactam antibiotics in Gram-negative bacteria. Although there are beta-lactamase inhibitors (BLIs) available, beta-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 beta-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 beta-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. (c) 2020 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.
Magazine:
SCIENTIFIC REPORTS
ISSN:
2045-2322
Year:
2019
Vol:
9
N°:
1
Pp:
3452
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.
Magazine:
ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
ISSN:
0065-2598
Year:
2019
Vol:
1117
Pgs:
257 - 279
Microbial cells show a strong natural tendency to adhere to surfaces and to colonise 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.
Authors:
Correa, W.; Heinbockel, L.; Behrends, J.; et al.
Magazine:
FEBS JOURNAL
ISSN:
1742-464X
Year:
2019
Vol:
286
N°:
8
Pgs:
1576 - 1593
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.
Authors:
Correa, W. (Corresponding author); Brandenburg, J.; Behrends, J.; et al.
Magazine:
BIOPHYSICAL JOURNAL
ISSN:
0006-3495
Year:
2019
Vol:
117
N°:
10
Pp:
1805 - 1819
The activity of antimicrobial peptides (AMPs) has been extensively investigated 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 gamma-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 tumour-necrosis-factor-alpha-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 bacteriainduced 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 (Delta psi) 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.
Authors:
Pfalzgraff, A.; Correa, W.; Heinbocke,l .; et al.
Magazine:
BIOCHIMICA ET BIOPHYSICA certificate-MOLECULAR AND CELL BIOLOGY OF LIPIDS
ISSN:
1388-1981
Year:
2019
Vol:
1864
N°:
10
Pgs:
1503 - 1513
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)-1 beta 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-1 beta 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, in
Magazine:
ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
ISSN:
0065-2598
Year:
2019
Vol:
1117
Pgs.:
111 - 129
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.
Magazine:
BRITISH JOURNAL OF PHARMACOLOGY
ISSN:
0007-1188
Year:
2018
Vol:
175
N°:
17
Pgs:
3581 - 3593
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.
Magazine:
EXPERIMENTAL AND MOLECULAR MEDICINE
ISSN:
1226-3613
Year:
2017
Vol:
49
N°:
6
Pp:
e345
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 ¿); 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.
Authors:
Sanchez-Gomez; Ferrer-Espada; Stewart; et al.
Magazine:
BMC MICROBIOLOGY
ISSN:
1471-2180
Year:
2015
Vol:
15
Pgs:
137
BACKGROUND:
Infections by Pseudomonas aeruginosa constitute a serious health threat because this pathogen -particularly when it forms biofilms- can acquire resistance to the majority of conventional antibiotics. This study evaluated the antimicrobial activity of synthetic peptides based on LF11, an 11-mer peptide derived from human lactoferricin against P. aeruginosa planktonic and biofilm-forming cells. We included in this analysis selected N-acylated derivatives of the peptides to analyze the effect of acylation in antimicrobial activity. To assess the efficacy of compounds against planktonic bacteria, microdilution assays to determine the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill studies were conducted. The anti-biofilm activity of the agents was assessed on biofilms grown under static (on microplates) and dynamic (in a CDC-reactor) flow regimes.
RESULTS:
The antimicrobial activity of lipopeptides differed from that of non-acylated peptides in their killing mechanisms on planktonic and biofilm-forming cells. Thus, acylation enhanced the bactericidal activity of the parental peptides and resulted in lipopeptides that were uniformly bactericidal at their MIC. In contrast, acylation of the most potent anti-biofilm peptides resulted in compounds with lower anti-biofilm activity. Both peptides and lipopeptides displayed very rapid killing kinetics and all of them required less than 21 min to reduce 1,000 times the viability of planktonic cells when tested at 2 times their MBC. The peptides, LF11-215 (FWRIRIRIRR) and LF11-227 (FWRRFWRR), displayed the most potent anti-biofilm activity causing a 10,000 fold reduction in cell viability after 1 h of treatment at 10 times their MIC. At that concentration, these two compounds exhibited low cytotoxicity on human cells. In addition to its bactericidal activity, LF11-227 removed more than 50 % of the biofilm mass in independent assays. Peptide LF11-215 and two of the shortest and least hydrophobic lipopeptides, DI-MB-LF11-322 (2,2-dimethylbutanoyl-PFWRIRIRR) and DI-MB-LF11-215, penetrated deep into the biofilm structure and homogenously killed biofilm-forming bacteria.
CONCLUSION:
We identified peptides derived from human lactoferricin with potent antimicrobial activity against P. aeruginosa growing either in planktonic or in biofilm mode. Although further structure-activity relationship analyses are necessary to optimise the anti-biofilm activity of these compounds, the results indicate that lactoferricin derived peptides are promising anti-biofilm agents.
Magazine:
SCIENTIFIC REPORTS
ISSN:
2045-2322
Year:
2015
Vol:
5
Pgs:
14292
Sepsis, a life-threatening syndrome with increasing incidence worldwide, is triggered by an overwhelming inflammation induced by microbial toxins released into the bloodstream during infection. A well-known sepsis-inducing factor is the membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS), signalling via Toll-like receptor-4. Although sepsis is caused in more than 50% cases by Gram-positive and mycoplasma cells, the causative compounds are still poorly described. In contradicting investigations lipoproteins/-peptides (LP), lipoteichoic acids (LTA), and peptidoglycans (PGN), were made responsible for eliciting this pathology. Here, we used human mononuclear cells from healthy donors to determine the cytokine-inducing activity of various LPs from different bacterial origin, synthetic and natural, and compared their activity with that of natural LTA and PGN. We demonstrate that LP are the most potent non-LPS pro-inflammatory toxins of the bacterial cell walls, signalling via Toll-like receptor-2, not only in vitro, but also when inoculated into mice: A synthetic LP caused sepsis-related pathological symptoms in a dose-response manner. Additionally, these mice produced pro-inflammatory cytokines characteristic of a septic reaction. Importantly, the recently designed polypeptide Aspidasept (R) which has been proven to efficiently neutralize LPS in vivo, inhibited cytokines induced by the various non-LPS compounds protecting animals from the pro-inflammatory activity of synthetic LP.
Magazine:
ANALYST
ISSN:
0003-2654
Year:
2015
Vol:
140
N°:
2
Pp:
654 - 660
This paper describes the design, implementation and validation of a sensitive and integral technology solution for endotoxin detection. The unified and portable platform is based on the electrochemical detection of endotoxins using a synthetic peptide immobilized on a thin-film biosensor. The work covers the fabrication of an optimised sensor, the biofunctionalization protocol and the design and implementation of the measuring and signalling elements (a microfluidic chamber and a portable potentiostat-galvanostat), framed ad hoc for this specific application. The use of thin-film technologies to fabricate the biosensing device and the application of simple immobilization and detection methods enable a rapid, easy and sensitive technique for in situ and real time LPS detection.
Authors:
Heinbockel; Marwitz, S.; Bárcena-Varela, S.; et al.
Magazine:
PLOS ONE
ISSN:
1932-6203
Year:
2015
Vol:
10
N°:
7
Pp:
e0133291
Sepsis is still a major cause of death and many efforts have been made to improve the physical condition of sepsis patients and to reduce the high mortality rate associated with this disease. While achievements were implemented in the intensive care treatment, all attempts within the field of novel therapeutics have failed. As a consequence new medications and improved patient stratification as well as a thoughtful management of the support therapies are urgently needed. In this study, we investigated the simultaneous administration of ibuprofen as a commonly used nonsteroidal anti-inflammatory drug (NSAID) and Pep19-2.5 (Aspidasept), a newly developed antimicrobial peptide. Here, we show a synergistic therapeutic effect of combined Pep19-2.5-ibuprofen treatment in an endotoxemia mouse model of sepsis. In vivo protection correlates with a reduction in plasma levels of both tumor necrosis factor a and prostaglandin E, as a likely consequence of Pep19-2.5 and ibuprofen-dependent blockade of TLR4 and COX pro-inflammatory cascades, respectively. This finding is further characterised and confirmed in a transcriptome analysis of LPS-stimulated human monocytes. The transcriptome analyses showed that Pep19-2.5 and ibuprofen exerted a synergistic global effect both on the number of regulated genes as well as on associated gene ontology and pathway expression. Overall, ibuprofen potentiated the anti-inflammatory activity of Pep19-2.5 both in vivo and in vitro, suggesting that NSAIDs could be useful to supplement future anti-sepsis therapies.
Authors:
Zuzuarregui, A.; Morant-Minana, C.; Pérez, Eva ; et al.
Magazine:
IEEE SENSORS JOURNAL
ISSN:
1530-437X
Year:
2014
Vol:
14
N°:
1
Pp:
270 - 277
In this paper, the implementation and characterization of a hand-held and simple biosensor for in-situ endotoxin determination are described. The integrated biosensor developed here is based on the electrochemical detection of endotoxin using polymyxin B as bioreceptor immobilized onto gold electrodes via a self-assembled monolayer. The cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy were used to characterize the biosensor performance and properties throughout the functionalization process. In addition, a comparative analysis of the behavior and features of two alternative electrochemical techniques for endotoxin detection was carried out. The biosensing device fabricated by thin-film technologies provided a simple and robust method to detect low concentrations of endotoxin.
Magazine:
JOURNAL OF BIOTECHNOLOGY
ISSN:
0168-1656
Year:
2014
Vol:
186
Pgs.:
162 - 168
The current validated endotoxin detection methods, in spite of being highly sensitive, present several drawbacks in terms of reproducibility, handling and cost. Therefore novel approaches are being carried out in the scientific community to overcome these difficulties. Remarkable efforts are focused on the development of endotoxin-specific biosensors. The key feature of these solutions relies on the proper definition of the capture protocol, especially of the bio-receptor or ligand. The aim of the presented work is the screening and selection of a synthetic peptide specifically designed for LPS detection, as well as the optimization of a procedure for its immobilization onto gold substrates for further application to biosensors.
Magazine:
JOURNAL OF SENSORS AND SENSOR SYSTEMS
ISSN:
2194-8771
Year:
2013
Vol:
2
N°:
2
Pp:
157 - 164
In this paper an electrochemical endotoxin biosensor consisting of an immobilized lipopolysaccharide (LPS) ligand, polymyxin B (PmB), is presented. Several parameters involved both in the device fabrication and in the detection process were analyzed to optimize the ligand immobilization and the interaction between PmB and LPS, aiming at increasing the sensitivity of the sensor. Di?erent electrochemical pre-treatment procedures as well as the functionalization methods were studied and evaluated. The use of a SAM (self-assembled monolayer) to immobilize PmB and the quanti?cation of the interactions via cyclic voltammetry allowed the development of a robust and simple device for in situ detection of LPS. Thus, the biosensor proposed in this work intends an approach to the demanding needs of the market for an integrated, portable and simple instrument for endotoxin detection.
Magazine:
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
ISSN:
0066-4804
Year:
2013
Vol:
57
N°:
3
Pp:
1480-1487
Bacterial infections are known to cause severe health-threatening conditions, including sepsis. All attempts to get this disease under control failed in the past, and especially in times of increasing antibiotic resistance, this leads to one of the most urgent medical challenges of our times. We designed a peptide to bind with high affinity to endotoxins, one of the most potent pathogenicity factors involved in triggering sepsis. The peptide Pep19-2.5 reveals high endotoxin neutralization efficiency in vitro, and here, we demonstrate its antiseptic/anti-inflammatory effects in vivo in the mouse models of endotoxemia, bacteremia, and cecal ligation and puncture, as well as in an ex vivo model of human tissue. Furthermore, we show that Pep19-2.5 can bind and neutralize not only endotoxins but also other bacterial pathogenicity factors, such as those from the Gram-positive bacterium Staphylococcus aureus. This broad neutralization efficiency and the additive action of the peptide with common antibiotics makes it an exceptionally appropriate drug candidate against bacterial sepsis and also offers multiple other medication opportunities.
Magazine:
CURRENT DRUG TARGETS
ISSN:
1389-4501
Year:
2012
Vol:
13
N°:
9
Pp:
1121 - 1130
The first barrier that an antimicrobial agent must overcome when interacting with its target is the microbial cell wall. In the case of Gram-negative bacteria, additional to the cytoplasmic membrane and the peptidoglycan layer, an outer membrane (OM) is the outermost barrier. The OM has an asymmetric distribution of the lipids with phospholipids and lipopolysaccharide (LPS) located in the inner and outer leaflets, respectively. In contrast, Gram-positive bacteria lack OM and possess a much thicker peptidoglycan layer compared to their Gram-negative counterparts. An additional class of amphiphiles exists in Gram-positives, the lipoteichoic acids (LTA), which may represent important structural components. These long molecules cross-bridge the entire cell envelope with their lipid component inserting into the outer leaflet of the cytoplasmic membrane and the teichoic acid portion penetrating into the peptidoglycan layer. Furthermore, both classes of bacteria have other important amphiphiles, such as lipoproteins, whose importance has become evident only recently. It is not known yet whether any of these amphiphilic components are able to stimulate the immune system under physiological conditions as constituents of intact bacteria. However, all of them have a very high pro-inflammatory activity when released from the cell. Such a release may take place through the interaction with the immune system, or with antibiotics (particularly with those targeting cell wall components), or simply by the bacterial division. Therefore, a given antimicrobial agent must ideally have a double character, namely, it must overcome the bacterial cell wall barrier, without inducing the release of the pro-inflammatory amphiphiles. Here, new data are presented which describe the development and use of membrane-active antimicrobial agents, in particular antimicrobial peptides (AMPs) and lipopolyamines. In this way, essential progress was achieved, in particular with respect to the inhibition of deleterious consequences of bacterial infections such as severe sepsis and septic shock.
Authors:
Schuerholz, T; Dömming, S; Hornef, M., ; et al.
Magazine:
CURRENT DRUG TARGETS
ISSN:
1389-4501
Year:
2012
Vol:
13
N°:
9
Pp:
1131 - 1137
The bacterial cell wall represents the primary target for antimicrobial agents. Microbial destruction is accompanied by the release of potent immunostimulatory membrane constituents. Both Gram-positive and Gram-negative bacteria release a variety of lipoproteins and peptidoglycan fragments. Gram-positive bacteria additionally provide lipoteichoic acids, whereas Gram-negative bacteria also release lipopolysaccharide (LPS, endotoxin), essential component of the outer leaflet of the bacterial cell wall and one of the most potent immunostimulatory molecules known. Immune activation therefore can be considered as an adverse effect of antimicrobial destruction and killing during anti-infective treatment. In contrast to antibiotics, the use of cationic amphiphilic antimicrobial peptides allows both effective bacterial killing and inhibition of the immunostimulatory effect of the released bacterial membrane constituents. The administration of antimicrobial peptides alone or in combination with antibiotic agents thus represents a novel strategy in the antiinfective treatment with potentially important beneficial aspects. Here, data are presented which describe immunological and clinical aspects of the use of antimicrobial peptides (AMPs) as therapeutic agents to treat bacterial infection and neutralize the immunostimulatory activity of released cell wall constituents.
Authors:
Kaconis, Y; Kowalski, L; Howe, J; et al.
Magazine:
BIOPHYSICAL JOURNAL
ISSN:
0006-3495
Year:
2011
Vol:
100
N°:
11
Pp:
2652 - 2661
Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity.
Magazine:
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
ISSN:
0066-4804
Year:
2011
Vol:
55
N°:
1
Pp:
218 - 228
A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals.
Authors:
Gutsmann, T.; Howe, J.; Zahringer, U.; et al.
Magazine:
INNATE IMMUNITY
ISSN:
1940-3011
Year:
2010
Vol:
16
N°:
1
Pp:
36 - 47
The structural prerequisites for lipopolysaccharide (LPS) and its partial structures for the activation of the Limulus clotting cascade (Limulus amebocyte lysate [LAL] test) are described and compared with the corresponding requirements for the activation of human immune cells such as mononuclear cells. A necessary, but not sufficient, structural motif for this is the presence of the 40-phosphate-diglucosamine backbone recognition structure ('epitope') in lipid A. High activity is only expressed by assemblies of endotoxins, but this is largely independent of the type of supramolecular aggregate structure. A particular conformation of the epitope within the lipid A assembly must be present, which is influenced by addition of further saccharide units to the lipid A moiety, but also reacts slightly to the acylation pattern. In contrast, the cytokine production of human immune cells induced by LPS sensitively depends on the type of its aggregate structure. In the case of a hexa-acylated bisphosphorylated lipid A structure, high activity is only observed with cubic inverted aggregates. Furthermore, addition of antimicrobial agents (such as polymyxin B) leads to a nearly complete inhibition of cytokine production, whereas the reduction in the Limulus assay is much lower. These data are important since a reliable determination of endotoxin concentrations, in particular with respect to its ability to elicit severe infections, is of high interest.
Authors:
Rodríguez-Cuesta, Juan; Hernando, Fernando L.; Mendoza, Lorea; et al.
Magazine:
CLINICAL AND EXPERIMENTAL METASTASIS
ISSN:
0262-0898
Year:
2010
Vol:
27
N°:
1
Pp:
35 - 42
Candida albicans infections are very frequent in cancer patients, whose immune system is often compromised, but whether this fungal pathogen affects cancer progression is unknown. C. albicans infection involves endogenous production of inflammatory cytokines such as tumour necrosis factor alpha (TNF-alpha) and interleukin-18 (IL-18). Increased levels of these cytokines have already been correlated with metastasis of most common cancer types. In this study, a well-established model of IL-18-dependent hepatic melanoma metastasis was used to study whether C. albicans can alter the ability of murine B16 melanoma (B16M) cells to colonize the liver. First, we determined the ability of intrasplenically (IS) injected B16M cells to metastasize into the liver of mice challenged with 5 x 10(4) C. albicans cells by three different routes (intravenous, IV; intrasplenic, IS; or intraperitoneal, IP) 12 h prior to injection of B16M cells. We demonstrated that C. albicans significantly increased metastasis of B16M cells with all three fungal injection routes. Pro-metastatic effects occurred when hepatic colonization with B16M cells place after the peak of TNF-alpha and IL-18 levels had been reached in the hepatic blood of fungal challenged mice. In a second set of experiments, mice were fungal challenged 4 days after injection of B16M cells. In these mice, C. albicans also potentiated the growth of established micro-metastases. Significantly, the fungal challenge had pro-metastatic effects without the C. albicans being able to reach the liver, suggesting that soluble factors can promote metastasis in remote sites. Mouse treatment with antifungal ketoconazole abrogated hepatic TNF-alpha stimulation by C. albicans and prevented the enhancement of hepatic metastasis in fungal challenged-mice. Therefore, the pro-inflammatory microenvironment generated by the host's systemic response to C. albicans stimulates circulating cancer cells to metastasize in the liver.
Magazine:
ANTI-INFECTIVE AGENTS IN MEDICINAL CHEMISTRY
ISSN:
1871-5214
Year:
2010
Vol:
9
N°:
1
Pp:
9 - 22
Bacterial endotoxins (lipopolysaccharide, LPS) from the outer leaflet of the outer membrane of Gram-negative bacteria are among the most effective natural compounds in triggering the human innate immune system. This may be beneficial at low but pathophysiological at high LPS concentrations, the latter leading to the severe septic shock syndrome with still high mortality rates especially in intensity care units. One approach to inactivate compounds such as LPS is the use of cationic amphiphilic peptides based on natural endotoxin- binding proteins, which are designed to neutralize bacterial LPS and thus inhibit its interaction with relevant mammalian binding proteins/ receptors such as lipopolysaccharide-binding protein (LBP), CD14, and TLR4/MD2. We have designed and synthesized peptides based on human lactoferricin, and have applied in an iterative process (first to third generation) various experimental systems to test their ability to fight against bacterial sepsis. Here, four compounds from the third generation were selected, with and without a short hydrocarbon chain, and analysed in detail starting from biophysical over cell biological and microbiological assays up to animal experiments. Thus, we were able to characterize the parameters relevant in endotoxin deactivation, as part of the bacterial cell as well as in isolated form. The most important parameters include the peptide length, number of cationic amino acids, and hydrophobicity of the peptides.
Magazine:
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
ISSN:
0066-4804
Year:
2010
Vol:
54
N°:
9
Pp:
3817 - 3824
Systemic bacterial infections are associated with high mortality. The access of bacteria or constituents thereof to systemic circulation induces the massive release of immunomodulatory mediators, ultimately causing tissue hypoperfusion and multiple-organ failure despite adequate antibiotic treatment. Lipid A, the "endotoxic principle" of bacterial lipopolysaccharide (LPS), is one of the major bacterial immunostimuli. Here we demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the Limulus anti-LPS factor for systemic application. We show efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock in vivo, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. The molecular mechanism of LPS neutralization was elucidated by biophysical techniques. The lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure, and the binding affinity of the peptide to LPS exceeds those of known LPS-binding proteins, such as LPS-binding protein (LBP). Our results thus delineate a novel therapeutic strategy for the clinical management of patients with septic shock.
Authors:
Kowalski, i.; Kaconis, Y.; Andrä, J.; et al.
Magazine:
Protein & Peptide Letters
ISSN:
0929-8665
Year:
2010
Vol:
17
N°:
11
Pp:
1328 - 1333
We have synthesized a series of short peptides (17 to 20 amino acids), originally derived from Limulus antilipopolysaccharide factor LALF, which were primarily designed to act as antimicrobial agents as well as neutralizers of bacterial endotoxin (lipopolysaccharide, LPS), Here, two selected peptides, a 17- and a 19-mer, were characterized physicochemically and in biological test systems. The secondary structure of the peptides indicates essentially a ß-sheet including antiparallel strands, the latter being reduced when the peptides bind to LPS. A very strong exothermic binding due to attractive Coulomb interactions governs the LPS-peptide reaction, which additionally leads to a fluidization of the acyl chains of LPS. A comparison of the interaction of the peptide with negatively charged phosphatidylserine shows in contrast a rigidification of the acyl chains of the lipid. Finally, the biological assays reveal a diverging behaviour of the two peptides, with higher antibacterial activity of the 17-mer, but a much higher activity of the 19-mer in its ability to inhibit the LPS-induced cytokine production in human mononuclear cells.
