Martinez-de-Tejada, G. (Guillermo)

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    A synthetic peptide sensitizes multi-drug resistant pseudomonas aeruginosa to antibiotics for more than two hours and permeabilizes its envelope for twenty hours
    (Springer, 2020) Shahrour, H. (Hawraa); Martinez-de-Tejada, G. (Guillermo); Razquin-Olazaran, I. (Iosu)
    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 β-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.
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    New antiseptic peptides to protect against endotoxin-mediated shock
    (American Society for Microbiology, 2010) Howe, J. (Jörg); Bartels, R. (Rainer); Brandenburg, K. (Klaus); Kowalski, I. (Ina); Moriyon, I. (Ignacio); Kaconis, Y. (Yani); Gutsmann, T. (Thomas); Sánchez-Gómez, S. (Susana); Rossle, M. (Manfred); Martinez-de-Tejada, G. (Guillermo); Razquin-Olazaran, I. (Iosu); Schürholz, T. (Tobias); Hornef, M. (Mathias)
    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.
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    Structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against Pseudomonas aeruginosa in vitro and in vivo
    (American Society for Microbiology, 2011) Brandenburg, K. (Klaus); Moriyon, I. (Ignacio); Leiva, J. (José); Jerala, R. (Roman); Sánchez-Gómez, S. (Susana); Fernández-Alonso, M. (Miriam); Andrä, J. (Jörg); Martinez-de-Tejada, G. (Guillermo); Blondelle, S.E. (Sylvie E.); Lohner, K. (Karl); Japelj, B. (Bostjan)
    Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. 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.
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    A comparison between SARS-CoV-2 and Gram-negative bacte- 1 ria-induced hyperinflammation and sepsis
    (MDPI, 2023) Fukuoka, S. (Satoshi); Brandenburg, K. (Klaus); Ferrer-Espada, R. (Raquel); Nehls, C. (Christian); Martinez-de-Tejada, G. (Guillermo); Mauss, K. (Karl); Weindl, G. (Gunther); Garidel, P. (Patrick)
    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.
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    The antimicrobial peptide cathelicidin and polymyxin B neutralize endotoxins by a multifactorial mechanism including not only direct LPS-interaction but also targeting of host cell membrane domains
    (PNAS, 2021) Brandenburg, K. (Klaus); Kopp, F. (Franziska); Kaconis, Y. (Yani); Donoghue, A. (Annemarie); Gutsmann, T. (Thomas); Nehls, C. (Christian); Koistinen, M. (Max); Sánchez-Gómez, S. (Susana); Wernecke, J. (Julia); Sevcsik, E. (Eva); Andrä, J. (Jörg); Martinez-de-Tejada, G. (Guillermo); Schütz, G.J. (Gerhard J.); Paulowski, L. (Laura); Brameshuber, M. (Mario); Lohner, K. (Karl); Keese, S. (Susanne); Garidel, P. (Patrick); Schromm, A.B. (Andra B.)
    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.
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    Comparative analysis of selected methods for the assessment of antimicrobial and membrane-permeabilizing activity: a case study for lactoferricin derived peptides
    (BioMed Central, 2008) Brandenburg, K. (Klaus); Moriyon, I. (Ignacio); Leiva, J. (José); Jerala, R. (Roman); Sánchez-Gómez, S. (Susana); Andrä, J. (Jörg); Martinez-de-Tejada, G. (Guillermo); Blondelle, S.E. (Sylvie E.); Lohner, K. (Karl); Lamata, M. (M.)
    Growing concerns about bacterial resistance to antibiotics have prompted the development of alternative therapies like those based on cationic antimicrobial peptides (APs). These compounds not only are bactericidal by themselves but also enhance the activity of antibiotics. Studies focused on the systematic characterization of APs are hampered by the lack of standard guidelines for testing these compounds. We investigated whether the information provided by methods commonly used for the biological characterization of APs is comparable, as it is often assumed. For this purpose, we determined the bacteriostatic, bactericidal, and permeability-increasing activity of synthetic peptides (n = 57; 9-13 amino acid residues in length) analogous to the lipopolysaccharide-binding region of human lactoferricin by a number of the most frequently used methods and carried out a comparative analysis. RESULTS: While the minimum inhibitory concentration determined by an automated turbidimetry-based system (Bioscreen) or by conventional broth microdilution methods did not differ significantly, bactericidal activity measured under static conditions in a low-ionic strength solvent resulted in a vast overestimation of antimicrobial activity. Under these conditions the degree of antagonism between the peptides and the divalent cations differed greatly depending on the bacterial strain tested. In contrast, the bioactivity of peptides was not affected by the type of plasticware (polypropylene vs. polystyrene). Susceptibility testing of APs using cation adjusted Mueller-Hinton was the most stringent screening method, although it may overlook potentially interesting peptides. Permeability assays based on sensitization to hydrophobic antibiotics provided overall information analogous - though not quantitatively comparable- to that of tests based on the uptake of hydrophobic fluorescent probes. CONCLUSION: We demonstrate that subtle changes in methods for testing cationic peptides bring about marked differences in activity. Our results show that careful selection of the test strains for susceptibility testing and for screenings of antibiotic-sensitizing activity is of critical importance. A number of peptides proved to have potent permeability-increasing activity at subinhibitory concentrations and efficiently sensitized Pseudomonas aeruginosa both to hydrophilic and hydrophobic antibiotics.
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    Antimicrobial peptides in the battle against orthopedic implant-related infections: A review
    (MDPI, 2021) Costa, F. (Fabiola); Gomes, P.A.C. (Paula A. C.); Martins, M.C.L. (M. Cristina L.); Costa, B. (Bruna); Martinez-de-Tejada, G. (Guillermo)
    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.
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    The outer membranes of Brucella spp. are not barriers to hydrophobic permeants
    (American Society for Microbiology, 1993) Moriyon, I. (Ignacio); Martinez-de-Tejada, G. (Guillermo)
    The patterns of susceptibility to hydrophobic and hydrophilic drugs and the uptake of the fluorescent probe N-phenyl-naphthylamine in Brucella spp., Haemophilus influenzae, Escherichia coli, and deep rough Salmonella minnesota mutants were compared. The results show that the outer membranes of smooth and naturally rough Brucella spp. do not represent barriers to hydrophobic permeants and that this absence of a barrier relates at least in part to the properties of Brucella lipopolysaccharide.
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    Inhibition of lipopolysaccharide- and lipoprotein-induced inflammation by antitoxin peptide Pep 19-2.5
    (Frontiers, 2018) Brandenburg, K. (Klaus); Gutsmann, T. (Thomas); Sánchez-Gómez, S. (Susana); Martinez-de-Tejada, G. (Guillermo); Heinbockel, L. (Lena); Weindl, G. (Gunther); Goldmann, T. (Torsten); Correa, W. (Wilmar); Bárcena-Varela, S. (Sergio); Garidel, P. (Patrick)
    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.
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    An antibiotic potentiator retains its activity after being immobilized on silicone and prevents growth of multidrug-resistant Pseudomonas aeruginosa biofilms
    (Elsevier, 2021) González-Gaitano, G. (Gustavo); Shahrour, H. (Hawraa); Martinez-de-Tejada, G. (Guillermo); López, A. (Andrés); Chokr, A. (Ali); Dandache, I. (Israa)
    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 for- mation 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