Concentrations Of Antimicrobial Peptides Research Articles

Antimicrobial Peptide Reduces Cytotoxicity and Inflammation in Canine Epidermal Keratinocyte Progenitor Cells Induced by Pseudomonas aeruginosa Infection.

The direct effects and antimicrobial activity of synthetic antimicrobial peptides (AMPs) obtained from dogs, including cBD, cBD103, and cCath, against P. aeruginosa wild-type strain PAO1 and canine keratinocytes were analyzed. Antibacterial effects on planktonic bacteria were assessed by determining the minimum bactericidal concentrations (MBCs) of AMPs and by a time-kill assay. Antibiofilm effects were assessed using the microtiter plate assay. We also evaluated the effects of AMPs on cell cytotoxicity and host immune response induced by stimulating canine epidermal keratinocyte progenitor (CPEK) cells with PAO1 and its LPS. cBD, cBD103, and cCath all exhibited dose-dependent antimicrobial and antibiofilm effects. In particular, 25 μg/mL cBD103 showed rapid bactericidal activity within 60 min and inhibited biofilm formation. In addition, pretreatment with cBD103 (25 µg/mL) and cCath (50 µg/mL) 1 h before stimulation significantly reduced the cytotoxicity of the CPEK cells by PAO1 and LPS-induced IL-6 and TNF-a expressions. cBD had little effect on the response to PAO1 and LPS in the cells. These results indicate the therapeutic potential of AMPs in P. aeruginosa skin infections. However, further studies on the mechanism of action of AMPs in keratinocytes and clinical trials are needed.

Levels of Lysozyme and SLPI in Bronchoalveolar Lavage: Exploring Their Role in Interstitial Lung Disease.

Interstitial lung diseases (ILDs) are characterized by inflammation or fibrosis of the pulmonary parenchyma. Despite the involvement of immune cells and soluble mediators in pulmonary fibrosis, the influence of antimicrobial peptides (AMPs) remains underexplored. These effector molecules display a range of activities, which include immunomodulation and wound repair. Here, we investigate the role of AMPs in the development of fibrosis in ILD. We compare the concentration of different AMPs and different cytokines in 46 fibrotic (F-ILD) and 17 non-fibrotic (NF-ILD) patients by ELISA and using peripheral blood mononuclear cells from in vitro stimulation in the presence of lysozyme or secretory leukocyte protease inhibitor (SLPI) from 10 healthy donors. We observed that bronchoalveolar lavage (BAL) levels of AMPs were decreased in F-ILD patients (lysozyme: p < 0.001; SLPI: p < 0.001; LL-37: p < 0.001; lactoferrin: p = 0.47) and were negatively correlated with levels of TGF-β (lysozyme: p = 0.02; SLPI: p < 0.001) and IL-17 (lysozyme: p < 0.001; SLPI: p < 0.001). We observed that lysozyme increased the percentage of CD86+ macrophages (p < 0.001) and the production of TNF-α (p < 0.001). We showed that lysozyme and SLPI were associated with clinical parameters (lysozyme: p < 0.001; SLPI: p < 0.001) and disease progression (lysozyme: p < 0.001; SLPI: p = 0.01). These results suggest that AMPs may play an important role in the anti-fibrotic response, regulating the effect of pro-fibrotic cytokines. In addition, levels of lysozyme in BAL may be a potential biomarker to predict the progression in F-ILD patients.

Antibacterial activity of the antimicrobial peptide PMAP-36 in combination with tetracycline against porcine extraintestinal pathogenic Escherichia coli in vitro and in vivo

The increase in the emergence of antimicrobial resistance has led to great challenges in controlling porcine extraintestinal pathogenic Escherichia coli (ExPEC) infections. Combinations of antimicrobial peptides (AMPs) and antibiotics can synergistically improve antimicrobial efficacy and reduce bacterial resistance. In this study, we investigated the antibacterial activity of porcine myeloid antimicrobial peptide 36 (PMAP-36) in combination with tetracycline against porcine ExPEC PCN033 both in vitro and in vivo. The minimum bactericidal concentrations (MBCs) of AMPs (PMAP-36 and PR-39) against the ExPEC strains PCN033 and RS218 were 10 μM and 5 μM, respectively. Results of the checkerboard assay and the time-kill assay showed that PMAP-36 and antibiotics (tetracycline and gentamicin) had synergistic bactericidal effects against PCN033. PMAP-36 and tetracycline in combination led to PCN033 cell wall shrinkage, as was shown by scanning electron microscopy. Furthermore, PMAP-36 delayed the emergence of PCN033 resistance to tetracycline by inhibiting the expression of the tetracycline resistance gene tetB. In a mouse model of systemic infection of PCN033, treatment with PMAP-36 combined with tetracycline significantly increased the survival rate, reduced the bacterial load and dampened the inflammatory response in mice. In addition, detection of immune cells in the peritoneal lavage fluid using flow cytometry revealed that the combination of PMAP-36 and tetracycline promoted the migration of monocytes/macrophages to the infection site. Our results suggest that AMPs in combination with antibiotics may provide more therapeutic options against multidrug-resistant porcine ExPEC.

Concentration of novel urinary tract infection biomarkers in neonates

Urinary tract infections (UTIs) are a common comorbidity in hospitalized neonates. The current UTI diagnostics have several limitations including invasive collection of urinary samples to ensure sterility, risk of contamination and lack of consensus definitions of UTI based on urine culture. Antimicrobial peptides (AMPs) have been recently utilized as novel biomarkers that can efficiently and accurately diagnose pediatric UTI. However, the concentration of AMPs in neonatal urine is not well-defined. Urine from neonates admitted to a single level IV neonatal intensive care unit was obtained to determine baseline concentration of two AMPs, Ribonuclease 7 (RNase 7) and Beta Defensin-1 (BD-1) and to define the relationship between AMP concentration and gestational age (GA). AMP levels were normalized to urine creatinine. RNase 7 and BD-1 were expressed in neonatal urine (n = 66) regardless of GA and as early as 22 weeks gestation. Urinary concentrations of both AMPs decreased as GA and birthweight increased. The overall median urinary RNase 7/UCr and BD-1/UCr values were 271 ng/mg, and 116 ng/mg, respectively. Median urinary concentrations of RNase 7/UCr for infants born at < 27, 27–32, 33–35 and ≥ 36 weeks were 569, 308, 254, and 124 ng/mg respectively. Similarly, the concentrations of BD-1/UCr at these GA were 166, 115, 108, and 14 ng/mg, respectively. Baseline neonatal urinary concentration of two AMPs (RNase 7 and BD-1) and the variation by GA were identified. This is an essential first step toward the potential utilization of AMPs in improving neonatal UTI diagnostics.

Vernix caseosa reveals mechanistic clues linking maternal obesity to atopic dermatitis pathogenesis

Maternal overweight and obesity have been associated with an increased risk of atopic dermatitis (AD) in the offspring, but the underlying mechanisms are unclear. Vernix caseosa (VC) is a proteolipid material covering the fetus produced during skin development. However, whether maternal prepregnancy weight excess influences fetal skin development is unknown. Characterizing the VC of newborns from mothers with prepregnancy overweight and obesity might reveal AD-prone alterations during fetal skin development. We sought to explore AD biomarkers and staphylococcal loads in VC from the offspring of mothers who were overweight/obese (O/O) before pregnancy versus in those from offspring of normal weight mothers. The VC of newborns of 14 O/O and 12 normal weight mothers were collected immediately after birth. Biomarkers were determined by ELISA and staphylococcal species by quantitative PCR. The VC from the O/O group showed decreased expression of skin barrier proteins (filaggrin and loricrin) and increased levels of proinflammatory biomarkers (IgA, thymic stromal lymphopoietin [TSLP], S100A8, IL-25, and IL-33). No differences in concentrations of antimicrobial peptides and enzymes were detected. The VC from the O/O group had a lower Staphylococcus epidermidis and Staphylococcus hominis commensal bacterial load, whereas Staphylococcus aureus bacterial load was not significantly different between the 2 groups. Maternal body mass index was negatively correlated with VC filaggrin expression and S epidermidis load and was positively associated with TSLP concentration. One-year follow-up established that the offspring of O/O mothers had a higher incidence of AD that was specifically linked with decreased VC filaggrin expression and lower S epidermidis load. VC from neonates of mothers with prepregnancy overweight and obesity exhibit skin barrier molecular alterations and staphylococcal dysbiosis that suggest earlymechanistic clues to this population's increased risk of AD.

Relationship between antimicrobial peptides-induced cell membrane damage and bactericidal activity

Most antimicrobial peptides (AMPs) act by killing bacterial cells. However, there is little information regarding the required interaction time between AMPs and bacterial cells to exert the bactericidal activity. One of the causes of the bactericidal activity is considered to be cell membrane damage, although little direct evidence is available. Here, we investigated the relationship between AMP-induced cell membrane damage in Escherichia coli and AMP-induced cell death at the single-cell level. Magainin 2, lactoferricin B, and PGLa were selected as the AMPs. First, we examined the interaction time (t) of AMPs with cells required to induce cell death using the single-cell analysis. The fraction of microcolonies containing only a single cell, Psingle (t), which indicates the fraction of dead cells, increased with time to reach ∼1 in a short time (≤5 min). Then, we examined the interaction between AMPs and single cells using confocal laser scanning microscopy in the presence of membrane-impermeable SYTOX green. Within a short time interaction, the fluorescence intensity of the cells due to SYTOX green increased, indicating that AMPs induced cell membrane damage through which the dye entered the cytoplasm. The fraction of cells in which SYTOX green entered the cytoplasm among all examined cells after the interaction time (t), Pentry (t), increased with time, reaching ∼1 in a short time (≤5 min). The values of Psingle (t) and Pentry (t) were similar at t ≥ 3 min for all AMPs. The bindings of AMPs to cells were largely reversible, whereas the AMP-induced cell membrane damages were largely irreversible because SYTOX green entered the cells after dilution of AMP concentration. Based on these results, we conclude that the rapid, substantial membrane permeabilization of cytoplasmic contents after a short interaction time with AMPs and the residual damage after dilution induce cell death.

Modification of microfiltration membranes by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/n-hydroxy sulfosuccinimide coupling mediated antimicrobial peptides grafting for biofouling mitigation

Microfiltration membrane separation is widely applied for water and wastewater but biofouling is a challenging task in real applications. In this work, we fabricated a novel antibiofouling polyvinylidene fluoride (PVDF) membrane by grafting non-drug-resistant antimicrobial peptides (AMPs) on the membranes via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxy sulfosuccinimide (EDC/Sulfo-NHS) chemistry. Effects of AMPs concentration on the membrane basic properties, antibacterial and antibiofouling behaviors of the modified membranes were systematically investigated. Membrane surface characterization demonstrated that the AMPs were successfully grafted onto the base PVDF membranes. The AMPs modified membrane showed enhanced hydrophilicity, consistent permeability and prominent antimicrobial ability (i.e., >90% of antibacterial efficiency against both Escherichia coli and Staphylococcus aureus). Batch filtration tests exhibited that the modified membranes with the concentration of AMPs less than or equal to 0.7 mg/mL had lower flux decline rate compared to the pristine membrane (M0). According to the images from confocal laser scanning microscopy analysis, less total cells as well as more dead cells were observed on the AMPs modified membrane compared to M0. Furthermore, the grafted AMPs on the membranes also displayed favorable stability suffered from a cycle involving biofouling and chemical cleaning. This grafting protocol for AMPs highlights a robust way to fabricate antibiofouling membranes for water and wastewater treatment.

A deep learning method for predicting the minimum inhibitory concentration of antimicrobial peptides against Escherichia coli using Multi-Branch-CNN and Attention.

Antimicrobial peptides (AMPs) are a promising alternative to antibiotics to combat drug resistance in pathogenic bacteria. However, the development of AMPs with high potency and specificity remains a challenge, and new tools to evaluate antimicrobial activity are needed to accelerate the discovery process. Therefore, we proposed MBC-Attention, a combination of a multi-branch convolution neural network architecture and attention mechanisms to predict the experimental minimum inhibitory concentration of peptides against Escherichia coli. The optimal MBC-Attention model achieved an average Pearson correlation coefficient (PCC) of 0.775 and a root mean squared error (RMSE) of 0.533 (log μM) in three independent tests of randomly drawn sequences from the data set. This results in a 5-12% improvement in PCC and a 6-13% improvement in RMSE compared to 17 traditional machine learning models and 2 optimally tuned models using random forest and support vector machine. Ablation studies confirmed that the two proposed attention mechanisms, global attention and local attention, contributed largely to performance improvement. IMPORTANCE Antimicrobial peptides (AMPs) are potential candidates for replacing conventional antibiotics to combat drug resistance in pathogenic bacteria. Therefore, it is necessary to evaluate the antimicrobial activity of AMPs quantitatively. However, wet-lab experiments are labor-intensive and time-consuming. To accelerate the evaluation process, we develop a deep learning method called MBC-Attention to regress the experimental minimum inhibitory concentration of AMPs against Escherichia coli. The proposed model outperforms traditional machine learning methods. Data, scripts to reproduce experiments, and the final production models are available on GitHub.

Antimicrobial peptide-loaded decellularized placental sponge as an excellent antibacterial skin substitute against XDR clinical isolates.

Post-wound infections have remained a serious threat to society and healthcare worldwide. Attempts are still being made to develop an ideal antibacterial wound dressing with high wound-healing potential and strong antibacterial activity against extensively drug-resistant bacteria (XDR). In this study, a biological-based sponge was made from decellularized human placenta (DPS) and then loaded with different concentrations (0, 16µg/mL, 32µg/mL, 64µg/mL) of an antimicrobial peptide (AMP, CM11) to optimize an ideal antibacterial wound dressing. The decellularization of DPS was confirmed by histological evaluations and DNA content assay. The DPS loaded with different contents of antimicrobial peptides (AMPs) showed uniform morphology under a scanning electron microscope (SEM) and cytobiocompatibility for human adipose tissue-derived mesenchymal stem cells. Antibacterial assays indicated that the DPS/AMPs had antibacterial behavior against both standard strain and XDR Acinetobacter baumannii in a dose-dependent manner, as DPS loaded with 64µg/mL showed the highest bacterial growth inhibition zone and elimination of bacteria under SEM than DPS alone and DPS loaded with 16µg/mL and 32µg/mL AMP concentrations. The subcutaneous implantation of all constructs in the animal model demonstrated no sign of acute immune system reaction and graft rejection, indicating in vivo biocompatibility of the scaffolds. Our findings suggest the DPS loaded with 64µg/mL as an excellent antibacterial skin substitute, and now promises to proceed with pre-clinical and clinical investigations.

Antimicrobial peptides alter lipid bilayer properties before disrupting membrane barrier property.

Antimicrobial peptides (AMPs) have been the subject of inquiry since the initial search for antibiotics in the ­first half of 20th century; the first antibiotic used in humans was the linear pentadecapeptide gramicidin. Much of this interest comes from the modest frequency of bacterial resistance against AMPs. Though AMPs differ in sequence and structure, the majority share three core physicochemical properties; they are short, cationic and amphipathic. Moreover, enantiomers of AMPs tend to have similar potencies. These common features have led to a focus on the bacterial membrane as a target for AMPs. High concentrations of AMPs cause gross bilayer defects, through pore formation or micellar breakdown. At lower concentrations, AMPs have effects on bacterial physiology that appear unrelated to membrane permeabilization, suggesting that they may act through bilayer-mediated regulation (BMR) of membrane protein function. If correct, this offers a mechanism of action that integrates the known membrane-modifying effects of AMPs with their ability to broadly alter bacterial physiology at sub-pore forming concentrations. We tested this hypothesis using melittin, gramicidin S, and indolicidin, three AMPs with distinct structures, that indeed alter bilayer properties at concentrations below those required for pore formation. Contrary to expectation, AMP induced BMR is attenuated in anionic bilayers despite increased AMP binding, suggesting an organizing effect of membrane-bound counterions. We further found that amphiphilic bilayer modifiers promote membrane permeabilization, suggesting that BMR may be responsible for the cooperativity of AMP-induced permeabilization. Two membrane-impermeant detergents, CHAPS and zwittergent, promote pore formation despite inhibiting AMP binding, suggesting a role for leaflet-specific lateral pressure differences. These results suggest a new mechanism by which AMPs exert their bactericidal effect, which may guide antibiotic design and provide a path for circumventing bacterial resistance.

Antibacterial Potential Analysis of Novel α-Helix Peptides in the Chinese Wolf Spider Lycosa sinensis.

The spider Lycosa sinensis represents a burrowing wolf spider (family Lycosidae) widely distributed in the cotton region of northern China, whose venom is rich in various bioactive peptides. In previous study, we used a combination strategy of peptidomic and transcriptomic analyses to systematically screen and identify potential antimicrobial peptides (AMPs) in Lycosa sinensis venom that matched the α-helix structures. In this work, the three peptides (LS-AMP-E1, LS-AMP-F1, and LS-AMP-G1) were subjected to sequence analysis of the physicochemical properties and helical wheel projection, and then six common clinical pathogenic bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) with multiple drug-resistance were isolated and cultured for the evaluation and analysis of antimicrobial activity of these peptides. The results showed that two peptides (LS-AMP-E1 and LS-AMP-F1) had different inhibitory activity against six clinical drug-resistant bacteria; they can effectively inhibit the formation of biofilm and have no obvious hemolytic effect. Moreover, both LS-AMP-E1 and LS-AMP-F1 exhibited varying degrees of synergistic therapeutic effects with traditional antibiotics (azithromycin, erythromycin, and doxycycline), significantly reducing the working concentration of antibiotics and AMPs. In terms of antimicrobial mechanisms, LS-AMP-E1 and LS-AMP-F1 destroyed the integrity of bacterial cell membranes in a short period of time and completely inhibited bacterial growth within 10 min of action. Meanwhile, high concentrations of Mg2+ effectively reduced the antibacterial activity of LS-AMP-E1 and LS-AMP-F1. Together, it suggested that the two peptides interact directly on bacterial cell membranes. Taken together, bioinformatic and functional analyses in the present work sheds light on the structure-function relationships of LS-AMPs, and facilitates the discovery and clinical application of novel AMPs.

In vitro synergistic action of TAT-RasGAP317-326 peptide with antibiotics against Gram-negative pathogens

ObjectivesMultidrug-resistant (MDR) bacteria are a continuously increasing threat for medicine, causing infections recalcitrant to antibiotics. Antimicrobial peptides (AMPs) were identified as alternatives to antibiotics, being naturally occurring short peptides and part of the innate immune system of a vast majority of organisms. However, the clinical application of AMPs is limited by suboptimal pharmacokinetic properties and relatively high toxicity. Combinatorial treatments using AMPs and classical antibiotics may decrease the concentrations of AMPs required for bacterial eradication, thus lowering the side effects of these peptides. MethodsHere, we investigate the in vitro efficiency of combinations of the recently described antimicrobial peptide TAT-RasGAP317-326 with a panel of commonly used antimicrobial agents against three Gram-negative bacteria, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii, using checkerboard and time-kill assays. ResultsWe identified synergistic combinations towards all three bacteria and demonstrated that these combinations had an increased bactericidal effect compared to individual drugs. Moreover, combinations were also effective against clinical isolates of A. baumannii. Finally, combination of TAT-RasGAP317-326 and meropenem had a promising antibiofilm effect towards A. baumannii. ConclusionsTaken together, our results indicate that combinations of TAT-RasGAP317-326 with commonly used antimicrobial agents may lead to the development of new treatment protocols against infections caused by MDR bacteria.

The Levels of the Human-β-Defensin-2 and LL-37 in the Sputum of Children with Cystic Fibrosis: A Case–control Study and Literature Review

BACKGROUND: Cystic fibrosis (CF) is a genetic disorder with an autosomal-recessive type of inheritance. Based on their host-defending and pro-inflammatory functions, antimicrobial peptides (AMPs) likely have one of the central roles in the pathogenesis of lung disease in CF. AIM: The purpose of the study was to measure the concentration of AMPs in the sputum of children with CF and evaluate any correlation with a bacterial profile of the lungs. METHODS: Lung colonization was evaluated using a culture-dependent method, sputum was utilized. A sandwich-ELISA was used to measure hBD-2 and hCAP-18/LL-37 in the sputum. RESULTS: There were 27 children enrolled in the study group, median age of inclusion was 11.4 (8.5; 14.8) years old. The control group consisted of 14 children, 11.6 (8.6; 12.6) years old. The concentration of AMPs was not correlating with participants` age (rs = −0.286, p = 0.148 – defensin hDB-2; rs = −0.084, p = 0.676 – cathelicidin hCAP-18/LL-37). The concentration of hBD-2 was from 64.01 to 813.61 pg/mL. The concentration of hCAP-18/LL-37 was from 3.24 to 35.98 ng/mL. There were significant differences in the content of AMPs on respiratory samples between study and control group (U = 976.5, p = 0.001 – for hBD-2; U = 1080.5, p &lt; 0.001). The correlation between current infection Pseudomonas aeruginosa and concentration of hBD-2 (rs = 0.167; p = 0.406) was not found. However, the presence of P. aeruginosa correlated with density of neutrophilic infiltration (rs = 0.622; p = 0.001). The concentration of hBD-2 showed direct medium correlation with total cells count (rs = 0.881, p &lt; 0.001). Correlation between current infection P. aeruginosa and concentration of hCAP-18/LL-37 (rs = 0.788; p &lt; 0.001) was observed. With increases in total cell count and relative neutrophils count, the concentration of hCAP-18/LL-37 was increased and the power of the association was medium (rs = 0.453; p = 0,018; rs = 0,592; p = 0,001). The correlation between concentrations of hBD-2 and hCAP-18/LL-37 (rs = 0.316, p &gt; 0.1) was not found. CONCLUSIONS: Measured AMPs correlated with cellular inflammatory markers and, probably, their overexpression is dedicated to stimulating a cellular component of innate immune response; there was no correlation between bacterial colonization of lungs and levels of hBD-2, so our findings sustain that P. aeruginosa is a leading but non-single contributor to persistent local inflammation in polymicrobial lungs.

Roles of Antimicrobial Peptides in Gynecological Cancers.

Antimicrobial peptides (AMPs) are essential components of the mucosal barrier of the female reproductive tract (FRT) and are involved in many important physiological processes, including shaping the microbiota and maintaining normal reproduction and pregnancy. Gynecological cancers seriously threaten women’s health and bring a heavy burden to society so that new strategies are needed to deal with these diseases. Recent studies have suggested that AMPs also have a complex yet intriguing relationship with gynecological cancers. The expression level of AMPs changes during tumor progression and they may act as promising biomarkers in cancer detection and prognosis prediction. Although AMPs have long been considered as host protective, they actually play a “double-edged sword” role in gynecological cancers, either tumorigenic or antitumor, depending on factors such as AMP and cancer types, as well as AMP concentrations. Moreover, AMPs are associated with chemoresistance and regulation of AMPs’ expression may alter sensitivity of cancer cells to chemotherapy. However, more work is needed, especially on the identification of molecular mechanisms of AMPs in the FRT, as well as the clinical application of these AMPs in detection, diagnosis and treatment of gynecological malignancies.

Single-Cell Analysis of the Antimicrobial and Bactericidal Activities of the Antimicrobial Peptide Magainin 2.

ABSTRACTAntimicrobial peptides (AMPs) inhibit the proliferation of or kill bacterial cells. To measure these activities, several methods have been used, which provide only the average value of many cells. Here, we report the development of a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis) and apply this strategy to examine the interaction of an AMP, magainin 2 (Mag), with Escherichia coli cells. Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. For method A, we incubated cells in the presence of various concentrations of AMPs for 3 h. The fraction of microcolonies containing only a single cell, Psingle, increased with the Mag concentration and reached 1 at a specific concentration, which corresponded to the MIC. For method B, after the interaction of a cell suspension with an AMP for a specific time, an aliquot was diluted to stop the interaction, and the proliferation of single cells then was monitored after a 3-h incubation; this method permits the definition of Psingle(t), the fraction of dead cells after the interaction. For the interaction of Mag with E. coli cells, Psingle(t) increased with the interaction time, reaching ~1 at 10 and 20 min for 25 and 13 μM Mag, respectively. Thus, these results indicate that a short interaction time between Mag and E. coli cells is sufficient to induce bacterial cell death.IMPORTANCE To elucidate the activity of antimicrobial peptides (AMPs) against bacterial cells, it is important to estimate the interaction time that is sufficient to induce cell death. We have developed a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis). Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. We found that during the interaction of magainin 2 (Mag) with E. coli cells, the fraction of dead cells, Psingle(t), increased with the interaction time, rapidly reaching 1 (e.g., 10 min for 25 μM Mag). This result indicates that Mag induces cell death after a short time of interaction.

Lactoferricins impair the cytosolic membrane of Escherichia coli within a few seconds and accumulate inside the cell.

We report the real-time response of Escherichia coli to lactoferricin-derived antimicrobial peptides (AMPs) on length scales bridging microscopic cell sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multiscale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than 3 s-much faster than previously considered. Final intracellular AMP concentrations of ∼80-100 mM suggest an efficient obstruction of physiologically important processes as the primary cause of bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.

Regulation of Antimicrobial Peptide Activity via Tuning Deformation Fields by Membrane-Deforming Inclusions.

Antimicrobial peptides (AMPs) are considered prospective antibiotics. Some AMPs fight bacteria via cooperative formation of pores in their plasma membranes. Most AMPs at their working concentrations can induce lysis of eukaryotic cells as well. Gramicidin A (gA) is a peptide, the transmembrane dimers of which form cation-selective channels in membranes. It is highly toxic for mammalians as being majorly hydrophobic gA incorporates and induces leakage of both bacterial and eukaryotic cell membranes. Both pore-forming AMPs and gA deform the membrane. Here we suggest a possible way to reduce the working concentrations of AMPs at the expense of application of highly-selective amplifiers of AMP activity in target membranes. The amplifiers should alter the deformation fields in the membrane in a way favoring the membrane-permeabilizing states. We developed the statistical model that allows describing the effect of membrane-deforming inclusions on the equilibrium between AMP monomers and cooperative membrane-permeabilizing structures. On the example of gA monomer-dimer equilibrium, the model predicts that amphipathic peptides and short transmembrane peptides playing the role of the membrane-deforming inclusions, even in low concentration can substantially increase the lifetime and average number of gA channels.

A rapid one-step process for the isolation of antibacterial peptides by silica-decorated Fe3O4nanoparticles

Antimicrobial peptides (AMPs), which are widely used as food preservatives, were purified to control the spoilage microorganism Alicyclobacillus acidoterrestris. The AMPs were produced by Pediococcus acidilactici CICC10344 and purified by silica-decorated Fe3O4 nanoparticles (SMNPs). The SMNPs were synthesized and characterized by transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FT-IR). The diameter of the SMNPs was 31 ± 7 nm. The VSM measurements indicated that the saturation magnetization value (Ms) of the SMNPs was 30 emu/g. We monitored AMP binding to the surface of Fe3O4 nanoparticles by FT-IR, showing that the amide had been successfully formed. The optimal condition for purification by SMNPs was an AMP concentration of 0.5 mg/mL for 30 min of exposure. The AMP purified by SMNPs retained good antimicrobial activity in pH 4–8 and temperature of 80 °C and 100 °C for 20 min. Scanning electron microscope (SEM) images indicated that a likely antibacterial mechanism is pore formation in the cell membrane. The one-step process proposed in the present work is convenient and effective for the purification of antibacterial peptides that may be widely used for the purification of other peptides.

Evaluation of Three Antimicrobial Peptides Mixtures to Control the Phytopathogen Responsible for Fire Blight Disease.

Fire blight is a severe bacterial plant disease that affects important chain-of-value fruit trees such as pear and apple trees. This disease is caused by Erwinia amylovora, a quarantine phytopathogenic bacterium, which, although highly distributed worldwide, still lacks efficient control measures. The green revolution paradigm demands sustainable agriculture practices, for which antimicrobial peptides (AMPs) have recently caught much attention. The goal of this work was to disclose the bioactivity of three peptides mixtures (BP100:RW-BP100, BP100:CA-M, and RW-BP100:CA-M), against three strains of E. amylovora representing distinct genotypes and virulence (LMG 2024, Ea 630 and Ea 680). The three AMPs’ mixtures were assayed at eight different equimolar concentrations ranging from 0.25 to 6 μM (1:1). Results showed MIC and MBC values between 2.5 and 4 μM for every AMP mixture and strain. Regarding cell viability, flow cytometry and alamarBlue reduction, showed high reduction (>25%) of viable cells after 30 min of AMP exposure, depending on the peptide mixture and strain assayed. Hypersensitive response in tobacco plants showed that the most efficient AMPs mixtures and concentrations caused low to no reaction of the plant. Altogether, the AMPs mixtures studied are better treatment solutions to control fire blight disease than the same AMPs applied individually.

Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag.

Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to limitations associated with large-scale production. The use of recombinant DNA technology for the production of AMPs fused with polymer tags presents the advantage of high-yield production and cost-efficient purification processes at high recovery rates. Owing to their unique properties, we explored the use of an elastin-like recombinamer (ELR) as a fusion partner for the production and isolation of two different AMPs (ABP-CM4 and Synoeca-MP), with an interspacing formic acid cleavage site. Recombinant AMP-ELR proteins were overproduced in Escherichia coli and efficiently purified by temperature cycles. The introduction of a formic acid cleavage site allowed the isolation of AMPs, resorting to a two-step methodology involving temperature cycles and a simple size-exclusion purification step. This simple and easy-to-implement purification method was demonstrated to result in high recovery rates of bioactive AMPs. The minimum inhibitory concentration (MIC) of the free AMPs was determined against seven different bacteria of clinical relevance (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two Burkholderia cenocepacia strains), in accordance with the EUCAST/CLSI antimicrobial susceptibility testing standards. All the bacterial strains (except for Pseudomonas aeruginosa) were demonstrated to be susceptible to ABP-CM4, including a resistant Burkholderia cenocepacia clinical strain. As for Synoeca-MP, although it did not inhibit the growth of Pseudomonas aeruginosa or Klebsiella pneumoniae, it was demonstrated to be highly active against the remaining bacteria. The present work provides the basis for the development of an efficient and up-scalable biotechnological platform for the production and purification of active AMPs against clinically relevant bacteria.