pVEC hydrophobic N-terminus is critical for antibacterial activity

Cell-penetrating peptides (CPPs) are commonly defined by their shared ability to be internalized into eukaryotic cells, without inducing permanent membrane damage, and to improve cargo delivery. Many CPPs also possess antimicrobial action strong enough to selectively lyse microbes in infected mammalian cultures. pVEC, a CPP derived from cadherin, is able to translocate into mammalian cells, and it is also antimicrobial. Structure-activity relationship and sequence alignment studies have suggested that the hydrophobic N-terminus (LLIIL) of pVEC is essential for this peptide's uptake into eukaryotic cells. In this study, our aim was to examine the contribution of these residues to the antimicrobial action and the translocation mechanism of pVEC. We performed antimicrobial activity and microscopy experiments with pVEC and with del5 pVEC (N-terminal truncated variant of pVEC) and showed that pVEC loses its antimicrobial effect upon deletion of the LLIIL residues, even though both peptides induce membrane permeability. We also calculated the free energy of the transport process using steered molecular dynamic simulations and replica exchange umbrella sampling simulations to compare the difference in uptake mechanism of the 2 peptides in atomistic detail. Despite the difference in experimentally observed antimicrobial activity, the simulations on the 2 peptides showed similar characteristics and the energetic cost of translocation of pVEC was higher than that of del5 pVEC, suggesting that pVEC uptake mechanism cannot be explained by simple passive transport. Our results suggest that LLIIL residues are key contributors to pVEC antibacterial activity because of irreversible membrane disruption

Monte Carlo sampling and principal component analysis of flux distributions yield topological and modular information on metabolic networks

The work presented here uses Monte Carlo random sampling combined with flux balance analysis and linear programming to analyse the steady-state flux distributions on the surface of the glucose-ammonia phenotypic phase plane of an Escherichia coli system grown oil glucose-minimal medium. The distribution of allowable glucose and ammonia uptake rates showed a triangular shape, the apex corresponding to maximum growth rate. The exact shape, e.g. the diagonal boundary is determined by the relative amounts of nutrients required for growth. The logarithm of flux values has a normal distribution, e.g. there is a log normal distribution, and most of the reactions have an order of magnitude between 10(-1) and 1. The increase in the number of blocked reactions as growth switched from aerobic to micro-aerobic phase and the presence of alternate networks for a single optimal solution were both reflections of the variability of pathway utilization for survival and growth. Principal component analysis (PCA) provided us with significant clues on the correlations between individual reactions and correlations between sets of reactions. Furthermore, PCA identified the most influential reactions of the system. The PCA score plots clearly distinguish two different growth phases, micro-aerobic and aerobic. The loading plots for each growth phase showed both the impact of the reactions on the model and the clustering of reactions that are highly correlated. These results have proved that PCA is a promising way to analyse correlations in high-dimensional solution spaces and to detect modular patterns among reactions in a network. (c) 2006 Elsevier Ltd. All rights reserved

A novel chimeric peptide with antimicrobial activity

Beta-lactamase-mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co-administration of beta-lactam type antibiotics and beta-lactamase inhibitors. Antimicrobial peptides are promising broad-spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46-Tyr51 beta-hairpin loop of beta-lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta-lactamase. Here, our goal was to modify this peptide for improved beta-lactamase inhibition and cellular uptake. Motivated by the cell-penetrating pVEC sequence, which includes a hydrophobic stretch at its N-terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N-terminus to facilitate uptake. Activity measurements of the peptide based on the 45-53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta-lactamase inhibitor with a K-i value of 58M. Incubation of beta-lactam-resistant cells with peptide decreased the number of viable cells, while it had no effect on beta-lactamase-free cells, indicating that this peptide had antimicrobial activity via beta-lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N-terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta-lactamase but also for other intracellular targets. Copyright (c) 2015 European Peptide Society and John Wiley & Sons, Ltd

Tyrosinase-based production of L-DOPA by Corynebacterium glutamicum

An increase in the number of elderly people suffering from the symptoms of Parkinson's disease is leading to an expansion in the market size of 3,4-dihydroxyphenyl-l-alanine (l-DOPA), which is the most commonly used drug for the treatment of this disease. Need for better quality products through economically feasible and sustainable processes makes biotechnological approaches attractive. The current study is focused on heterologous expression of Ralstonia solanacearum tyrosinase in Corynebacterium glutamicum cells to produce l-DOPA during growth on glucose or glucose/xylose mixtures. Whole-cells pre-grown on glucose were further exploited for biotransformation of l-tyrosine to l-DOPA. To prevent l-DOPA oxidation, not only the most commonly used agent, ascorbic acid, but also for the first time, thymol was evaluated. The highest l-DOPA titer was 0.26 +/- 0.02 g/L at the end of growth on a mixture of 1% xylose and 3% glucose in the presence of 200 mu M thymol as the oxidation inhibitor. The ability to co-utilize glucose and xylose to reach this titer could make these cells ideal for l-DOPA production using hydrolyzed lignocellulosic biomass. When the pre-grown cells were further used for biotransformation, the highest l-DOPA yield was 0.61 +/- 0.02 g/gDCW with 4 mM ascorbic acid. Since l-tyrosine biotransformation is primarily dependent on tyrosinase activity, yield in this route could be improved by optimizing reaction conditions. As the industrial workhorse for amino acid production, these C. glutamicum cells will clearly benefit from strain development efforts and bioprocess optimization towards sustainable and economically feasible l-DOPA production

Repurposing bioactive aporphine alkaloids as efflux pump inhibitors

Extrusion of drugs or drug-like compounds through bacterial efflux pumps is a serious health issue that leads to loss in drug efficacy. Combinatorial therapies of low-efficacy drugs with efflux pump inhibitors may help to restore the activities of such drugs. In this quest, natural products are attractive molecules, since in addition to their wide range of bioactivities they may inhibit efflux pumps. The current work repurposed the bioactive alkaloid roemerine as a potential efflux pump inhibitor. In Bacillus subtilis, both Bmr and BmrA, belonging to the major facilitator and the ATP-binding cassette superfamilies, respectively, were found to be inhibited by roemerine. Scanning electron microscopy and RNA-Seq analyses showed that it potentiated the effect of berberine. Growth rates and checkerboard assays confirmed the synergy of roemerine and berberine and that roemerine prevented berberine efflux by inhibiting Bmr. Transport assays with inverted membrane vesicles prepared from Escherichia cob overexpressing BmrA showed that increasing roemerine concentration decreased the transport of doxorubicin, the BmrA substrate, confirming that roemerine may also be considered as an inhibitor of BmrA. Thus, these findings suggest that conjugation of roemerine to substrates of efflux pumps, Bmr and BmrA, may help to potentiate the activity of their drug substrates

Cellular distribution of activity for three enzymes with maltose binding protein as fusion partner and the structural implications

The bacterial SEC pathway is commonly used for secretion of heterologous proteins in E. coli by fusing them to transported proteins to facilitate downstream processing. While some proteins are translocated very efficiently, some reside in the cytoplasm. In this work, maltose binding protein (MBP) was fused to 3 cytoplamic enzymes from Thermus thermophilus (serine protease, 251 residues; glucose isomerase, 381 residues; pullulanase, 718 residues) to study the protein transport from the cytoplasm by quantifying the distribution of activities in different cellular compartments. Pullulanase activity was harvested exclusively in the periplasm; however, glucose isomerase activity was harvested exclusively in the cytoplasm. Considerable serine protease activity was found in the periplasm, but after 10 h of induction activity dropped sharply and no activity was found thereafter in either compartment. This was attributed to the instability of the plasmid probably caused by the proteolytic activity of the protease Computations of hypothetical folding rates and secondary structure contents of the proteins showed that folding rates, in addition to alpha-helix and beta-sheet contents of proteins, could be important determinants for efficient translocation by the SEC pathway. These results may give clues to predict whether a protein would be a suitable fusion tail for periplasmic transport with MBP

Expression and translocation of glucose isomerase as a fusion protein in E. coli

Glucose isomerase of Thermus thermophilus was fused to maltose binding protein to use its signal sequence and slow folding characteristic for transport to the periplasm in Escherichia coli. The product was mostly retained in the cytoplasm and 1.6% of the total glucose isomerase activity was detected in the periplasm as a fusion protein. The effect of inducer concentration on translocation was insignificant, however induction at 23°C increased periplasmic glucose isomerase fusion by 50%. Growth medium was supplemented with amino acids to investigate their effect on translocation. Addition of 0.5% (w/v) alanine, the most abundant amino acid in the glucose isomerase sequence, increased expression by 24%, and induced extracellular secretion of the fusion protein by 18%. On the other hand, glycine retarded growth and caused lysis. The elevated pH of cultures with alanine indicated its possible effect of on translocation, but no significant change was observed by externally imposed pH variations. These results indicate that the secretion efficiency of a fusion protein depends on the characteristics of the system used. © 2004 Elsevier Inc. All rights reserved