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

Dynamic metabolic modeling of CHO cell metabolism coupled with N-glycosylation in the industrial pharmaceutical production

Cellular metabolism consists of a complex network of biochemical reactions and thus presents a challenging modeling problem. Experimental and modeling work, described in this article is focused on the metabolic pathway of Chinese hamster ovary (CHO) cells, which are the preferred expression system for monoclonal antibody (mAb) protein production. CHO cells are one of the primary hosts for mAbs production, which have extensive applications in multiple fields like biochemistry, biology and medicine. Here, an approach to explain cellular metabolism with in-silico modeling of a microkinetic reaction network is presented and validated with unique experimental results. Experimental data of 25 different fed-batch bioprocesses included the variation of multiple process parameters, such as pH, agitation speed, oxygen and carbon dioxide content, and dissolved oxygen. 151 metabolites were involved in our proposed metabolic network, which consisted of 132 chemical reactions that describe the reaction pathways, and include 25 reactions describing N-glycosylation and additional reactions for the accumulation of the produced glycoforms. Additional 8 reactions are considered for accumulation of the N-glycosylation products in the extracellular environment and 1 reaction to correlate cell degradation. The following pathways were considered: glycolysis, pentose phosphate pathway, nucleotide synthesis, tricarboxylic acid cycle, lipid synthesis, protein synthesis, biomass production, anaplerotic reactions and membrane transport. Our contribution to this field is the comparison of unique experimental data to our model, which is coupled with biomass production and N-glycosylation. The effect of various operational conditions was assessed and their effect on the cell production process. The modeling performed is a complementary tool to experimentation, nevertheless, with the applied modeling procedure, different operational scenarios and fed-batch techniques can be tested without the need for long-term experimental campaigns

N-acylated peptides derived from human lactoferricin perturb organization of cardiolipin and phosphatidylethanolamine in cell membranes and induce defects in Escherichia coli cell division.

Two types of recently described antibacterial peptides derived from human lactoferricin, either nonacylated or N-acylated, were studied for their different interaction with membranes of Escherichia coli in vivo and in model systems. Electron microscopy revealed striking effects on the bacterial membrane as both peptide types induced formation of large membrane blebs. Electron and fluorescence microscopy, however demonstrated that only the N-acylated peptides partially induced the generation of oversized cells, which might reflect defects in cell-division. Further a different distribution of cardiolipin domains on the E. coli membrane was shown only in the presence of the N-acylated peptides. The lipid was distributed over the whole bacterial cell surface, whereas cardiolipin in untreated and nonacylated peptide-treated cells was mainly located at the septum and poles. Studies with bacterial membrane mimics, such as cardiolipin or phosphatidylethanolamine revealed that both types of peptides interacted with the negatively charged lipid cardiolipin. The nonacylated peptides however induced segregation of cardiolipin into peptide-enriched and peptide-poor lipid domains, while the N-acylated peptides promoted formation of many small heterogeneous domains. Only N-acylated peptides caused additional severe effects on the main phase transition of liposomes composed of pure phosphatidylethanolamine, while both peptide types inhibited the lamellar to hexagonal phase transition. Lipid mixtures of phosphatidylethanolamine and cardiolipin revealed anionic clustering by all peptide types. However additional strong perturbation of the neutral lipids was only seen with the N-acylated peptides. Nuclear magnetic resonance demonstrated different conformational arrangement of the N-acylated peptide in anionic and zwitterionic micelles revealing possible mechanistic differences in their action on different membrane lipids. We hypothesized that both peptides kill bacteria by interacting with bacterial membrane lipids but only N-acylated peptides interact with both charged cardiolipin and zwitterionic phosphatidylethanolamine resulting in remodeling of the natural phospholipid domains in the E. coli membrane that leads to defects in cell division

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

Primary structure, hydrophobicity and biological activity of LF11 [<i>2</i>]; [<i>3</i>] derived peptides and N-acylations thereof.

a<p>Minimal inhibitory concentration (MIC) against <i>E. coli</i> ATCC 25922 were determined as peptide concentration resulting in less than 2% growth following an overnight incubation in Mueller Hinton medium at 37°C in the presence of 5×10⁵ CFU/ml.</p

Cardiolipin domains – NAO staining after peptide treatment.

<p>Fluorescence microscopy of <i>E. coli</i> strain W3110 stained with 10-N-nonyl acridine orange (NAO) demonstrating localization of CL domains. NAO staining was performed after incubation of the cells with peptides O-LF11-215 and LF11-215, and controls consisting of aliquots of 0.1% acetic acid. For details, see “Experimental Procedures”. Cells were immobilized on a microscope slide cover glass with poly-L-lysine and viewed using Olympus BX60 microscope with a 100× oil-immersion objective and FITC filter. White bar, 2 µm. Arrows indicate altered CL domain formation in the treated by N-acylated peptide O-LF11-215 mainly elongated or filamentous cells.</p

Model studies with TMCL and POPE.

<p>DSC thermograms of TMCL, POPE/TMCL 80:20 (w/w) and POPE in the absence and presence of peptides (lipid-to-peptide molar ratio of 25:1). For clarity, the DSC curves were displayed on the ordinate by arbitrary units. Scan rate was 30°C/h. N-acylated peptides are shown in gray. For analyzed data see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090228#pone-0090228-t002" target="_blank">Table 2</a>.</p

Thermodynamic parameters of TMCL, POPE/TMCL 80:20 and POPE in the absence and presence of LF11-derived peptides at a lipid-to-peptide molar ratio of 25:1.

<p>Thermodynamic parameters of TMCL, POPE/TMCL 80:20 and POPE in the absence and presence of LF11-derived peptides at a lipid-to-peptide molar ratio of 25:1.</p

NMR-structures of micelles in presence of N-acylated peptide.

<p>NMR structures of O-LF11-215 in complex with SDS (A,B,C) and DPC (D,E,F) micelles. Ensembles of structures are shown in A and D, average structures in B and E and backbone folds in C and F for SDS and DPC micelles, respectively.</p