Escherichia coli Cell Surface Perturbation and Disruption Induced by Antimicrobial Peptides BP100 and pepR

The potential of antimicrobial peptides (AMPs) as an alternative to conventional therapies is well recognized. Insights into the biological and biophysical properties of AMPs are thus key to understanding their mode of action. In this study, the mechanisms adopted by two AMPs in disrupting the Gram-negative Escherichia coli bacterial envelope were explored. BP100 is a short cecropin A-melittin hybrid peptide known to inhibit the growth of phytopathogenic Gram-negative bacteria. pepR, on the other hand, is a novel AMP derived from the dengue virus capsid protein. Both BP100 and pepR were found to inhibit the growth of E. coli at micromolar concentrations. Zeta potential measurements of E. coli incubated with increasing peptide concentrations allowed for the establishment of a correlation between the minimal inhibitory concentration (MIC) of each AMP and membrane surface charge neutralization. While a neutralization-mediated killing mechanism adopted by either AMP is not necessarily implied, the hypothesis that surface neutralization occurs close to MIC values was confirmed. Atomic force microscopy (AFM) was then employed to visualize the structural effect of the interaction of each AMP with the E. coli cell envelope. At their MICs, BP100 and pepR progressively destroyed the bacterial envelope, with extensive damage already occurring 2 h after peptide addition to the bacteria. A similar effect was observed for each AMP in the concentration-dependent studies. At peptide concentrations below MIC values, only minor disruptions of the bacterial surface occurred.     

The effects of subminimal inhibitory concentrations of beta-lactam antibiotics against Clostridium perfringens.

The effects of subminimal inhibitory concentrations (sub-MIC) of four beta-lactam antibiotics [penicillin-G (PCG), ampicillin (AMP), cephaloridine (CER), cephalothin (CET)] were tested against Clostridium perfringens type A PB6K, after determining the minimum inhibitory concentrations (MIC) of 29 different Clostridium strains. The majority of the strains were sensitive to all beta-lactam antibiotics. Morphological changes, such as filamentous development and lysis, occurred at concentrations considerably lower than the MIC of CER and CET in C. perfringens. Clear cooperation of AMP and CER with rabbit polymorphonuclear leucocytes (PMNL) against C. perfringens was observed. The filamentous bacteria produced as a result of exposure to sub-MIC of each antibiotic, were phagocytosed easily. The ratios between the drug concentrations (microg/ml) at which the morphological changes began to occur, the minimum antibiotic concentrations (MAC), and the MIC values (microg/ml), were calculated. A large ratio indicated a wide range of effective concentrations below the MIC value for the antibiotics.     

Mechanisms and Fitness Costs of Resistance to Antimicrobial Peptides LL-37, CNY100HL and Wheat Germ Histones

Antimicrobial peptides (AMPs) represent a potential new class of antimicrobial drugs with potent and broad-spectrum activities. However, knowledge about the mechanisms and rates of resistance development to AMPs and the resulting effects on fitness and cross-resistance is limited. We isolated antimicrobial peptide (AMP) resistant Salmonella typhimurium LT2 mutants by serially passaging several independent bacterial lineages in progressively increasing concentrations of LL-37, CNY100HL and Wheat Germ Histones. Significant AMP resistance developed in 15/18 independent bacterial lineages. Resistance mutations were identified by whole genome sequencing in two-component signal transduction systems (pmrB and phoP) as well as in the LPS core biosynthesis pathway (waaY, also designated rfaY). In most cases, resistance was associated with a reduced fitness, observed as a decreased growth rate, which was dependent on growth conditions and mutation type. Importantly, mutations in waaY decreased bacterial susceptibility to all tested AMPs and the mutant outcompeted the wild type parental strain at AMP concentrations below the MIC for the wild type. Our data suggests that resistance to antimicrobial peptides can develop rapidly through mechanisms that confer cross-resistance to several AMPs. Importantly, AMP-resistant mutants can have a competitive advantage over the wild type strain at AMP concentrations similar to those found near human epithelial cells. These results suggest that resistant mutants could both be selected de novo and maintained by exposure to our own natural repertoire of defence molecules.     

Neurohypophyseal peptide action on andenylate cyclase and hydro-osmotic properties of toad urinary bladder epithelium

The relationship between increase in water permeability in intact urinary bladder of toad, Bufo marinus, and activation of its membrane-bound adenylate cyclase preparation in response to neurohypophyseal peptides was examined. All of the peptides tested produced a maximal hydro-osmotic response in the intact bladder; in the adenylate cyclase assay the intrinsic activities of peptides varied greatly. While there is a positive qualitative correlation (r_s = 0.85, P < 0.01) between the apparent affinity parameters (pD₂) of the peptides obtained in the adenylate cyclase and hydro-osmotic assays, there is a difference in peptide sensitivity between the two assays of one to four orders of magnitude. The magnitude of this difference increases as the intrinsic activity of the peptides increases.The differences in peptide affinity observed in the two assay systems as well as the lack of correlation between intrinsic activities as measured in subcellular and membrane preparations can in part be explained on the basis that maximal hormonal response in intact tissue may require only a small franction (5% or less) of the cyclic AMP produced in response to maximally stimulating concentrations of hormone or analog. On this basis, the greater the potency of a peptide in generating cyclic AMP, the greater will be the discrepancy between the peptide concentration required to produce half-maximal responsens in the two assays.     

Antimicrobial activity of an abiotic host defense peptide mimic

Bacterial drug resistance is emerging as one of the most significant challenges to human health. Antimicrobial peptides (AMPs), which are produced by many tissues and cell types of invertebrates, insects, and humans, as part of their innate immune system, have attracted considerable interest as alternative antibiotics. Interest in novel mimics of AMPs has increased greatly over the last few years. This report details a new AMP mimic, based on phenylene ethynylene, with improved antimicrobial activity and selectivity. Screening against a large set of bacterial and other organisms demonstrates broad spectrum antimicrobial activity including activity against antibiotic resistant bacterial like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) as well as activity against yeast (Candida albicans) and fungus (Stachybotrys chartarum). Bacterial resistance development studies using Staphylococcus aureus show a rapid increase in MIC for conventional antibiotics, ciprofloxacin and norfloxacin. In sharp contrast, no change in MIC was observed for the AMP mimic. Cytotoxicity experiments show that the AMP mimic acts preferentially on microbes as opposed to mammalian red blood cells, 3T3 fibroblasts, and HEPG2 cells. In vivo experiments determined the maximum tolerated dose (MTD) to be 10 mg/kg suggesting a therapeutic window is available. These studies indicate that nonpeptidic amphiphilic AMP mimics could be developed as potential new treatments for antibiotic-resistant bacterial infections.     

Antimicrobial properties of membrane-active dodecapeptides derived from MSI-78

Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino acid (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa).The short 12-mer peptide CEM1 (GIGKFLKKAKKF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKKFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition.We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier.     

Antimicrobial activity of an abiotic host defense peptide mimic

Bacterial drug resistance is emerging as one of the most significant challenges to human health. Antimicrobial peptides (AMPs), which are produced by many tissues and cell types of invertebrates, insects, and humans, as part of their innate immune system, have attracted considerable interest as alternative antibiotics. Interest in novel mimics of AMPs has increased greatly over the last few years. This report details a new AMP mimic, based on phenylene ethynylene, with improved antimicrobial activity and selectivity. Screening against a large set of bacterial and other organisms demonstrates broad spectrum antimicrobial activity including activity against antibiotic resistant bacterial like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) as well as activity against yeast (Candida albicans) and fungus (Stachybotrys chartarum). Bacterial resistance development studies using Staphylococcus aureus show a rapid increase in MIC for conventional antibiotics, ciprofloxacin and norfloxacin. In sharp contrast, no change in MIC was observed for the AMP mimic. Cytotoxicity experiments show that the AMP mimic acts preferentially on microbes as opposed to mammalian red blood cells, 3T3 fibroblasts, and HEPG2 cells. In vivo experiments determined the maximum tolerated dose (MTD) to be 10 mg/kg suggesting a therapeutic window is available. These studies indicate that nonpeptidic amphiphilic AMP mimics could be developed as potential new treatments for antibiotic-resistant bacterial infections.     

Mode of action of cationic antimicrobial peptides defines the tethering position and the efficacy of biocidal surfaces

Covalent immobilization of cationic antimicrobial peptides (CAPs) at sufficient density and distance from the solid matrix has been suggested as a successful strategy for the generation of biocidal surfaces. To test the hypothesis that the mode of peptide action is decisive for the selection of an appropriate tethering position on solid surfaces, melittin (MEL), a channel-forming peptide, buforin 2 (BUF2), a peptide able to translocate bacterial membranes without permeabilization and targeting nucleic acids, and tritrpticin (TP), described to be membrane-lytic and to have intracellular targets, were C- and N-terminally immobilized on TentaGel S NH(2) resin beads as model surface. The peptide termini were modified with aminooxyacetic acid (AOA) and coupled via oxime-forming ligation. The comparison of the activities of the three peptides and their AOA-modified analogues with a KLAL model peptide which permeabilizes membranes by a so-called "carpet-like" mode provided the following results: The peptides in solution state were active against Bacillus subtilis and Escherichia coli at micromolar concentrations. MEL and TP but not BUF2-derived peptides permeabilized the inner and outer membrane of E. coli and enhanced the permeability of lipid bilayers at concentrations around their antimicrobial values (MICs). Immobilization reduced peptide activity to millimolar MICs. The activity reduction for KLAL was independent of the tethering position and comparably low, as reflected by a low ratio of MIC(tethered)/MIC(free). In contrary, the pore-forming MEL was much less active when immobilized at the N-terminus compared with the C-terminally tethered peptide. C- and N-terminal TP tethering caused an identical but much pronounced activity decrease. The tethered BUF2 peptides were inactive at the tested concentrations suggesting that the peptides could not reach the intracellular targets. In conclusion, membrane active peptides seem to be most suitable for the generation of antimicrobial surfaces, but knowledge about their mode of membrane insertion and positioning is required to identify optimal tethering positions. The relationship between the mechanism of action and position of immobilization is highly relevant for the establishment of a general approach to obtain efficient biocidal solid matrices loaded with CAPs.     

Cyclic AMP does not mediate the action of synthetic hypertrehalosemic peptides from the corpus cardiacum of Periplaneta americana

Two synthetic peptides identical to those present in the corpus cardiacum of the American cockroach, Periplaneta americana, were tested for their effect on the production of cyclic AMP and the activation of glycogen phosphorylase in cockroach fat body. The peptides activate glycogen phosphorylase and promote trehalose production in incubated tissue when calcium is included in the incubation medium, but have no obvious effect on cyclic AMP levels. The lack of effect of the peptides on cyclic AMP production was confirmed in a fragmented membrane preparation. By contrast, an aqueous extract of corpus cardiacum activates glycogen phosphorylase, promotes trehalose production and elevates cyclic AMP levels in incubated tissue; the extract also enhances cyclic AMP production in the fragmented cell membrane preparation. Observations on the nature of cyclic AMP production in cockroach fat body indicate that the adenylate cyclase has a requirement for GTP and magnesium ions, is stimulated by fluoride and forskolin and, therefore, is similar to the adenylate cyclase complex of other eukaryotes.The results suggest that increases in intracellular calcium concentrations may mediate the expression of hypertrehalosemic effects by the synthetic peptides.     

Bicontinuous inverted cubic phase stabilization as an index of antimicrobial and membrane fusion peptide activity

Some antimicrobial peptides (AMPs) and membrane fusion-catalyzing peptides (FPs) stabilize bicontinuous inverted cubic (Q_II) phases. Previous authors proposed a topological rationale: since AMP-induced pores, fusion intermediates, and Q_II phases all have negative Gaussian curvature (NGC), peptides which produce NGC in one structure also do it in another. This assumes that peptides change the curvature energy of the lipid membranes. Here I test this with a Helfrich curvature energy model. First, experimentally, I show that lipid systems often used to study peptide NGC have NGC without peptides at higher temperatures. To determine the net effect of an AMP on NGC, the equilibrium phase behavior of the host lipids must be determined. Second, the model shows that AMPs must make large changes in the curvature energy to stabilize AMP-induced pores. Peptide-induced changes in elastic constants affect pores and Q_II phase differently. Changes in spontaneous curvature affect them in opposite ways. The observed correlation between Q_II phase stabilization and AMP activity doesn't show that AMPs act by lowering pore curvature energy. A different rationale is proposed. In theory, AMPs could simultaneously stabilize Q_II phase and pores by drastically changing two particular elastic constants. This could be tested by measuring AMP effects on the individual constants. I propose experiments to do that. Unlike AMPs, FPs must make only small changes in the curvature energy to catalyze fusion. It they act in this way, their fusion activity should correlate with their ability to stabilize Q_II phases.     

Corticotropin Peptides and Melanotropins Elevate the Level of Adenosine 3'': 5''-Cyclic Monophosphate in Cultured Murine Brain Cells

Cell cultures derived from mouse and rat brain and consisting mainly of astroblasts are known to respond to several hormones by increasing or decreasing their intracellular concentration of cyclic AMP. In the present study these cultures were analyzed for their susceptibility to various additional hormonal and other neuroactive compounds. Only the peptides of the corticotropin (ACTH)/melanotropin (MSH) family were found active. Their potency for elevating the intracellular level of cyclic AMP decreases in the sequence (values for the half-maximally stimulating concentrations, EC50, in parentheses) ACTH-(1-24) (10 m) greater than alpha-,beta-MSH (30 nm) greater than ACTH (greater than or equal to 100 nm) gamma-MSH, ACTH-(1-10), -(4-10), -(4-11) (greater than or equal to 0.5 microM). The lack of additivity of the maximal effects of the peptides suggests that they all act at the same receptor. The stimulation exerted by these peptides is partially suppressed by hormones known to inhibit cyclic AMP formation in that culture, i.e., noradrenaline (acting via an alpha-adrenergic receptor), adenosine (acting via an A1 receptor), and somatostatin. It is concluded that the receptors for the ACTH/MSH peptides and the inhibitory hormones are located on the same cells, presumably the astroblasts. The maximal response to ACTH and alpha- and beta-MSH depends strongly on the age of culture. The results are discussed in view of the facts that (1) peptides of the ACTH/MSH family affect behavior and learning in animals, and (2) ACTH and alpha-MSH occur in brain.     

Adenosine 3′:5′-cyclic monophosphate-dependent protein kinase(s) of rat ovarian cells. Gonadotropin regulation of adenosine 3′:5′-cyclic monophosphate-receptor activity

Regulation of cyclic AMP-dependent protein kinase, cyclic AMP-receptor activity and intracellular cyclic AMP concentrations by choriogonadotropin was studied in ovarian cells prepared from 26-day-old rats. A close correlation was observed between phospho-transferase activity and cyclic AMP-receptor activity in 12000g supernatant fractions from rat ovarian homogenate. The apparent activation constant (K(a)) and I(50) (concentration required to produce 50% inhibition) of different cyclic nucleotides for phosphotransferase and cyclic AMP receptor activities respectively were also determined. Cyclic AMP and 8-bromo cyclic AMP were most effective, giving K(a) values of 0.08 and 0.09mum and I(50) of 0.12 and 0.16mum respectively. Other nucleotides were also effective, but required higher concentrations to give a comparable effect. An increased concentration of cyclic AMP produced by choriogonadotropin (1mug/ml) treatment was accompanied by decreased cyclic AMP binding as early as 5min after hormone addition. Choriogonadotropin also stimulated the protein kinase activity ratio (-cyclic AMP/+cyclic AMP) under identical experimental conditions. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated the action of choriogonadotropin on the three parameters measured in a dose- and time-dependent manner. The maximal cyclic AMP-binding capacity, as determined by cyclic AMP-exchange assay, remained unchanged before and after hormone addition. The endogenously bound cyclic AMP was determined from the difference between the maximal binding capacity and the exogenously bound cyclic AMP. With different choriogonadotropin concentrations, a quantitative correlation was established between maximal binding capacity, exogenous binding and endogenous binding activities. Approx. 60% of total binding sites were endogenously occupied in untreated cells, and choriogonadotropin (1mug/ml) treatment fully saturated available binding sites with a parallel 10-fold increase in cellular cyclic AMP. The present results provide evidence for a probable intracellular compartmentalization of cyclic AMP in the ovarian cell, and suggest that in the unstimulated state all cyclic AMP present in the ovarian cell may not be available for protein kinase activation.     

Antimicrobial peptides containing unnatural amino acid exhibit potent bactericidal activity against ESKAPE pathogens

A series of 36 synthetic antimicrobial peptides containing unnatural amino acids were screened to determine their effectiveness to treat Enterococcus faecium, Staphylococcus aureus, Klebsiella pnemoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) pathogens, which are known to commonly infect chronic wounds. The primary amino acid sequences of these peptides incorporate either three or six dipeptide units consisting of the unnatural amino acids Tetrahydroisoquinolinecarboxylic acid (Tic) and Octahydroindolecarboxylic acid (Oic). The Tic-Oic dipeptide units are separated by SPACER amino acids with specific physicochemical properties that control how these peptides interact with bacterial cell membranes of different chemical compositions. These peptides exhibited minimum inhibitory concentrations (MIC) against these pathogens in the range from >100 to 6.25 μg/mL. The observed diversity of MIC values for these peptides against the various bacterial strains are consistent with our hypothesis that the complementarity of the physicochemical properties of the peptide and the lipid of the bacteria’s cell membrane determines the resulting antibacterial activity of the peptide.     

Detecting antimicrobial peptides by exploring the mutual information of their sequences

Abstract

The rise of antibiotic resistance in pathogenic bacteria is a growing concern for every part of the world. The present study shows the prediction efficiency of mutual information for the classification of antimicrobial peptides. The proven role of antimicrobial peptides (AMPs) to fight against multidrug-resistant pathogens and AMP’s low toxic properties laid the foundation of computational methods to play their role in detecting AMPs from non-AMPs. Mutual information vectors (MIV) were created for AMP/non-AMP sequences and then fed to different machine learning classifiers out of which a random forest (RF) classifier showed best results for predicting AMPs. Random forest classifiers were evaluated on benchmark datasets by 10-fold cross-validation. The proposed MIV-RF method showed better prediction accuracy, MCC (Matthews correlation coefficient), and AUC-ROC (Area Under The Curve-Receiver Operating Characteristics) than available methods for detecting AMPs.

Communicated by Ramaswamy H. Sarma     

Coal Fly Ash Impairs Airway Antimicrobial Peptides and Increases Bacterial Growth

Air pollution is a risk factor for respiratory infections, and one of its main components is particulate matter (PM), which is comprised of a number of particles that contain iron, such as coal fly ash (CFA). Since free iron concentrations are extremely low in airway surface liquid (ASL), we hypothesize that CFA impairs antimicrobial peptides (AMP) function and can be a source of iron to bacteria. We tested this hypothesis in vivo by instilling mice with Pseudomonas aeruginosa (PA01) and CFA and determine the percentage of bacterial clearance. In addition, we tested bacterial clearance in cell culture by exposing primary human airway epithelial cells to PA01 and CFA and determining the AMP activity and bacterial growth in vitro. We report that CFA is a bioavailable source of iron for bacteria. We show that CFA interferes with bacterial clearance in vivo and in primary human airway epithelial cultures. Also, we demonstrate that CFA inhibits AMP activity in vitro, which we propose as a mechanism of our cell culture and in vivo results. Furthermore, PA01 uses CFA as an iron source with a direct correlation between CFA iron dissolution and bacterial growth. CFA concentrations used are very relevant to human daily exposures, thus posing a potential public health risk for susceptible subjects. Although CFA provides a source of bioavailable iron for bacteria, not all CFA particles have the same biological effects, and their propensity for iron dissolution is an important factor. CFA impairs lung innate immune mechanisms of bacterial clearance, specifically AMP activity. We expect that identifying the PM mechanisms of respiratory infections will translate into public health policies aimed at controlling, not only concentration of PM exposure, but physicochemical characteristics that will potentially cause respiratory infections in susceptible individuals and populations.     

Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli.

Antimicrobial cationic peptides are prevalent throughout nature as part of the intrinsic defenses of most organisms, and have been proposed as a blueprint for the design of novel antimicrobial agents. They are known to interact with membranes, and it has been frequently proposed that this represents their antibacterial target. To see if this was a general mechanism of action, we studied the interaction, with model membranes and the cytoplasmic membrane of Escherichia coli, of 12 peptides representing all 4 structural classes of antimicrobial peptides. Planar lipid bilayer studies indicated that there was considerable variance in the interactions of the peptides with model phospholipid membranes, but generally both high concentrations of peptide and high transmembrane voltages (usually -180 mV) were required to observe conductance events (channels). The channels observed for most peptides varied widely in magnitude and duration. An assay was developed to measure the interaction with the Escherichia coli cytoplasmic membrane employing the membrane potential sensitive dye 3,5-dipropylthiacarbocyanine in the outer membrane barrier-defective E. coli strain DC2. It was demonstrated that individual peptides varied widely in their ability to depolarize the cytoplasmic membrane potential of E. coli, with certain peptides such as the loop peptide bactenecin and the alpha-helical peptide CP26 being unable to cause depolarization at the minimal inhibitory concentration (MIC), and others like gramicidin S causing maximal depolarization below the MIC. We discuss the mechanism of interaction with the cytoplasmic membrane in terms of the model of Matsuzaki et al. [(1998) Biochemistry 37, 15144-15153] and the possibility that the cytoplasmic membrane is not the target for some or even most cationic antimicrobial peptides.     

Comparison of in vitro antibacterial activities of two cationic peptides CM15 and CM11 against five pathogenic bacteria: Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio cholerae, Acinetobacter baumannii, and Escherichia coli

In recent years, the widespread use of antibiotics has caused many bacterial pathogens resistance to conventional antibiotics. Therefore, generation of new antibiotics to control and reduce the effects of these pathogens is urgently needed. Antimicrobial peptides and proteins are important members of the host defense system in eukaryotes. These peptides are potent, broad-spectrum antibiotics that demonstrate potential as novel and alternative therapeutic agents for the treatment of drug-resistant infections. Accordingly, we evaluated two hybrid peptides CM11 (WKLFKKILKVL-NH2) and CM15 (KWKLFKKIGAVLKVL-NH2) on five important pathogenic bacteria. These peptides are short cecropin?Cmelittin hybrid peptides obtained through a sequence combination approach, which are highly effective to inhibit the growth of important pathogenic bacteria. The activity of these two cationic peptides (CM11 and CM15) in different concentrations (2?C64?mg/L) was investigated against standard and clinical isolates of important hospital infection bacteria by measuring MIC, MBC, and bactericidal assay. These peptides demonstrated the same ranges of inhibitory values: The organisms in early 24?h were more susceptible to polycationic peptides (MIC: 8?mg/L and MBC 32?mg/L), but after 48?h the MIC and MBC remained constant for the CM11 peptide. Bactericidal assay showed that all bacteria strains did not have any growth in agar plates after 40?min. The result showed that these two peptides are more effective than other peptides.     

Potentiation of the 5-hydroxytryptamine-induced increases in myocardial contractility in Mercenaria mercenaria ventricle by forskolin

Forskolin (10(-5) M), an activator of adenylate cyclase in many mammalian cell types, potentiated the positive inotropic effects of 5-hydroxytryptamine (5-HT) on the bivalve, Mercenaria mercenaria, myocardium. Forskolin (10(-5) M) also significantly increased intracellular cyclic AMP concentrations when compared to 5-HT controls. Ro 20-1724 (10(-5) M), a putative inhibitor of cyclic AMP phosphodiesterase, had no effect on myocardial contractility or cyclic AMP concentration. A positive correlation between intracellular cyclic AMP concentration and the efficacy of 5-HT was obtained.     

Encapsulin carrier proteins for enhanced expression of antimicrobial peptides

Antimicrobial peptides (AMPs) are regarded as attractive alternatives to conventional antibiotics, but their production in microbes remains challenging due to their inherent bactericidal nature. To address these limitations, we have developed a novel AMP fusion protein system based on an encapsulin nanocompartment protein and have demonstrated its utility in enhancing expression of HBCM2, an AMP with activity against Gram-negative bacteria. Here, HBCM2 was fused to the N-terminus of several Encapsulin monomer (Enc) variants engineered with multiple TEV protease recognition site insertions to facilitate proteolytic release of the fused HBCM2. Fusion of HBCM2 to the Enc variants, but not other common carrier proteins, enabled robust overexpression in Escherichia coli C43(DE3) cells. Interestingly, variants with a TEV site insertion following residue K71 in Enc exhibited the highest overexpression and HBCM2 release efficiencies compared to other variants but were deficient in cage formation. HBCM2 was purified from the highest expressing variant following TEV protease digestion and was found to be highly active in inhibiting E. coli growth (MIC = 5 μg/ml). Our study demonstrates the potential use of the Enc system to enhance expression of AMPs for biomanufacturing and therapeutic applications.