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.

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 Peptides as Anti-biofilm Agents in Medical Implants.

Biofilm-associated infections constitute a daunting threat to human health, since these pathologies increase patient mortality and morbidity, resulting in prolonged hospitalization periods and heavy economic losses. Moreover, these infections contribute to the increasing emergence and dissemination of antibiotic resistance in hospitals and in the community. Although biofilm-associated microorganisms can proliferate in healthy tissue, abiotic surfaces like those of medical implants greatly increase the likelihood of biofilm formation in the host. Due to their broad spectrum of bactericidal activity against multi-drug resistant microorganisms including metabolically inactive cells, antimicrobial peptides (AMPs) have great potential as anti-biofilm agents. In fact, a clinically available AMP, polymyxin E (colistin), frequently constitutes the drug of last recourse in biofilm-associated infections (e.g. cystic fibrosis) when resistance to all the other drugs arises. In this article, we outline the main strategies under development to combat biofilm-associated infections with an emphasis in the prevention of microbial colonization of medical implants. These approaches include the use of AMPs both for the development of anti-adhesive surface coatings and to kill biofilm-forming cells either on contact or via controlled release (leaching surfaces). Although in vitro results for all these applications are very encouraging, further research is needed to improve the anti-biofilm activity of these coatings in vivo. The possibility of exploiting the antibiotic potentiating activity of some AMPs and to combine several anti-biofilm mechanisms in tandem targeting the biofilm formation process at different stages is also discussed.

Lipoproteins/peptides are sepsis-inducing toxins from bacteria that can be neutralized by synthetic anti-endotoxin peptides.

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(®) 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.

Preclinical investigations reveal the broad-spectrum neutralizing activity of peptide Pep19-2.5 on bacterial pathogenicity factors.

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.

Structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against Pseudomonas aeruginosa in vitro and in vivo.

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.

New antiseptic peptides to protect against endotoxin-mediated shock.

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.

Comparative analysis of selected methods for the assessment of antimicrobial and membrane-permeabilizing activity: a case study for lactoferricin derived peptides.

BACKGROUND: 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.