Membrane Permeabilizing Effect Research Articles

Optimized Methods to Quantify Tumor Treating Fields (TTFields)-Induced Permeabilization of Glioblastoma Cell Membranes

Glioblastoma (GBM) is a lethal primary brain cancer with a 5.6% five-year survival rate. Tumor treating fields (TTFields) are alternating low-intensity electric fields that have demonstrated a GBM patient survival benefit. We previously reported that 0.5–24 h of TTFields exposure resulted in an increased uptake of FITC-dextran fluorescent probes (4–20 kDa) in human GBM cells. However, this approach, in which a fluorescence plate-based detector is used to evaluate cells attached to glass coverslips, cannot distinguish FITC-dextran uptake in live vs. dead cells. The goal of the study was to report the optimization and validation of two independent methods to quantify human GBM cell membrane permeabilization induced by TTFields exposure. First, we optimized flow cytometry by measuring mean fluorescence intensity at 72 h for 4 kDa (TTFields 6726 ± 958.0 vs. no-TTFields 5093 ± 239.7, p = 0.016) and 20 kDa (7087 ± 1137 vs. 5055 ± 897.8, p = 0.031) probes. Second, we measured the ratio of lactate dehydrogenase (LDH) to cell viability (measured using the CellTiter-Glo [CTG] viability assay); the LDH/CTG ratio was higher under TTFields (1.47 ± 0.15) than no-TTFields (1.08 ± 0.08) conditions, p < 0.0001. The findings using these two independent methods reproducibly demonstrated their utility for time-dependent evaluations. We also showed that these methods can be used to relate the cell membrane-permeabilizing effects of the non-ionizing radiation of TTFields to that of an established cell membrane permeabilizer, the non-ionic detergent Triton-X-100. Evaluating carboplatin ± TTFields, the LDH/CTG ratio was significantly higher in the TTFields vs. no-TTFields condition at each carboplatin concentration (0–30 µM), p = 0.014. We successfully optimized and validated two cost-effective methods to reproducibly quantify TTFields-induced human GBM cancer cell membrane permeabilization.

Indole phytochemical camalexin as a promising scaffold for AcrB efflux pump inhibitors against Escherichia coli.

Escherichia coli is amongst the most frequent causative agent of nosocomial infections and the overexpression of the efflux pump gene acrB plays a major role in its resistance to various antibiotics. In this study, we evaluated two indole phytochemicals, camalexin and brassinin, as potential AcrB efflux pump inhibitors. Among these two phytochemicals, camalexin increased the accumulation of ethidium in acrB proficient E.coli with no membrane permeabilization effect observed, indicating a direct interaction of camalexin with the pump. Camalexin also showed up to 64-fold MIC reduction for drugs in the acrB proficient strain. Brassinin was less effective, showing up to 4-fold MIC reduction for the same drugs. Camalexin did not potentiate drugs in the AcrB inactive strain D407N. Plate dilution assays in E. coli acrB variants further corroborated the effect of camalexin in diminishing pump activity. Blind docking results suggested that camalexin and brassinin may enter mainly via CH3, one of the channels present in AcrB, and camalexin showed a more stable binding mode than brassinin in the distal binding pocket of AcrB. Camalexin, therefore, holds potential as a scaffold for further development as a potent AcrB inhibitor to tackle antimicrobial resistance in the gram-negative bacterium E. coli.

PACAP sequence modifications modulate the peptide antimicrobial activity against bacterial pathogens affecting aquaculture

The global aquaculture industry has significant losses each year due to disease outbreaks. Antibiotics are one of the common methods to treat fish infections, but prolonged use can lead to the emergence of resistant strains. Aeromonas spp. Infections are a common and problematic disease in fish, and members of this genera can produce antibiotic resistant strains. Antimicrobial peptides (AMPs) have emerged as an alternative method to treat and prevent infections and pituitary adenylate cyclase activating polypeptide (PACAP) is a prominent member of this family. The objective of this research was to study PACAP's direct antimicrobial activity and its toxicity in fish cells. Four synthetic variants of the natural PACAP from Clarias gariepinus were tested in addition to the natural variant. The experimental results show a different antimicrobial activity against A. salmonicida and A. hydrophila of each PACAP variant, and for the first time show dependence on the culture broth used. Furthermore, the results suggest that the underlying mechanism of PACAP antimicrobial activity includes a bacterial membrane permeabilizing effect, classifying PACAP as a membrane disruptive AMP. This study also demonstrated that the five PACAP variants evaluated showed low toxicity in vitro, at concentrations relevant for in vivo applications. Therefore, PACAP could be a promising alternative to antibiotics in the aquaculture sector.

Klebicin E, a pore-forming bacteriocin of Klebsiella pneumoniae, exploits the porin OmpC and the Ton system for translocation

Bacteriocins, which have narrow-spectrum activity and limited adverse effects, are promising alternatives to antibiotics. In this study, we identified klebicin E (KlebE), a small bacteriocin derived from Klebsiella pneumoniae. KlebE exhibited strong efficacy against multidrug-resistant K. pneumoniae isolates and conferred a significant growth advantage to the producing strain during intraspecies competition. A giant unilamellar vesicle leakage assay demonstrated the unique membrane permeabilization effect of KlebE, suggesting it is a pore-forming toxin. In addition to a colicin A-like C-terminal domain, KlebE also has a disordered N-terminal domain and a globular central domain. Pulldown assays and soft agar overlay experiments revealed the essential role of the outer membrane porin OmpC and the Ton system in KlebE recognition and cytotoxicity. Strong binding between KlebE and both OmpC and TonB was observed. The TonB-box, a crucial component of the toxin-TonB interaction, was identified as the 7-amino acid sequence (E3ETLTVV9) located in the N-terminal region. Further studies showed that a region near the bottom of the central domain of KlebE plays a primary role in recognizing OmpC, with eight residues surrounding this region identified as essential for KlebE toxicity. Finally, based on the discrepancies in OmpC sequences between the KlebE-resistant and sensitive strains, it was found that the 91st residue of OmpC, an aspartic acid residue, is a key determinant of KlebE toxicity. The identification and characterization of this toxin will facilitate the development of bacteriocin-based therapies targeting multidrug-resistant K. pneumoniae infections.

Prenylated isoflavonoids from Fabaceae against the NorA efflux pump in Staphylococcus aureus

Overexpression of NorA efflux pumps plays a pivotal role in the multidrug-resistance mechanism in S. aureus. Here, we investigated the activities of prenylated isoflavonoids, present in the legume plant family (Fabaceae), as natural efflux pump inhibitors (EPIs) in fluoroquinolone-resistant S. aureus. We found that four prenylated isoflavonoids, namely neobavaisoflavone, glabrene, glyceollin I, and glyceollin III, showed efflux pump inhibition in the norA overexpressing S. aureus. At sub-inhibitory concentrations, neobavaisoflavone (6.25 µg/mL, 19 µM) and glabrene (12.5 µg/mL, 39 µM), showed up to 6 times more Eth accumulation in norA overexpressing S. aureus than in the control. In addition, these two compounds boosted the MIC of fluoroquinolones up to eightfold. No fluoroquinolone potentiation was observed with these isoflavonoids in the norA knockout strain, indicating NorA as the main target of these potential EPIs. In comparison to the reported NorA EPI reserpine, neobavaisoflavone showed similar potentiation of fluoroquinolone activity at 10 µM, higher Eth accumulation, and less cytotoxicity. Neobavaisoflavone and glabrene did not exhibit membrane permeabilization effects or cytotoxicity on Caco-2 cells. In conclusion, our findings suggest that the prenylated isoflavonoids neobavaisoflavone and glabrene are promising phytochemicals that could be developed as antimicrobials and resistance-modifying agents to treat fluoroquinolone-resistant S. aureus strains.

LL37-Derived Fragments Improve the Antibacterial Potential of Penicillin G and Ampicillin against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are a severe threat to public health. Antimicrobial peptides (AMPs) are novel and potential antimicrobials with specific antibacterial mechanisms. Our aim was to study the potential of LL37, FK16, and FK13 to enhance the anti-MRSA activity of antibiotics in vitro, particularly penicillin G and ampicillin. Our results showed that FK16 and FK13 have more synergistic inhibitory effects to MRSA strains when combined with penicillin G and ampicillin. In addition, AMPs exhibited strong membrane permeabilizing properties, and membrane permeabilizing effects can provide a possible explanation for the improved antibacterial effects of antibiotics, since permeabilizing AMPs have the potential to increase the access of antibiotics. To further study the electrostatic interactions among cationic AMPs with negatively charged bacteria, we measured the zeta potentials of three MRSA strains and also neutralized three MRSA strains with the addition of cationic AMPs. Further, we demonstrated the connection between membrane permeabilization and zeta potential neutralization. Finally, we treated MRSA strains with AMPs and characterized the MICs of penicillin G and ampicillin. FK16 was the most promising AMP among the three AMPs, since exposure to FK16 decreased the MICs of both penicillin G and ampicillin for all MRSA strains and also demonstrated more synergistic combinations when combined with antibiotics. AMP exposure and subsequent membrane permeabilization provide a possible pathway to re-sensitize drug-resistant bacteria to traditional antibiotics. Re-sensitization may help preserve the effectiveness of traditional antibiotics, thus providing a potential new strategy for fighting MRSA infections.

Heterogeneous Structural Disturbance of Cell Membrane by Peptides with Modulated Hydrophobic Properties.

Extensive effort has been devoted to developing new clinical therapies based on membrane-active peptides (MAPs). Previous models on the membrane action mechanisms of these peptides mostly focused on the MAP-membrane interactions in a local region, while the influence of the spatial heterogeneity of the MAP distribution on the membrane was much ignored. Herein, three types of natural peptide variants, AS4-1, AS4-5, and AS4-9, with similar amphiphilic α-helical structures but distinct hydrophobic degrees (AS4-1 < AS4-5 < AS4-9) and net charges (+9 vs. +7 vs. +5), were used to interact with a mixed phosphatidylcholine (PC) and phosphatidylglycerol (PG) membrane. A combination of giant unilamellar vesicle (GUV) leakage assays, atomic force microscopy (AFM) characterizations, and molecular dynamics (MD) simulations demonstrated the coexistence of multiple action mechanisms of peptides on a membrane, probably due to the spatially heterogeneous distribution of peptides on the membrane surface. Specifically, the most hydrophobic peptide (i.e., AS4-9) had the strongest membrane binding, perturbation, and permeabilization effects, leading to the formation of large peptide-lipid aggregates (10 ± 5 nm in height and 150 ± 50 nm in size), as well as continuous fragments and ridges on the supported membrane surface. The AS4-5 peptides, with a half-hydrophilic and half-hydrophobic structure, induced membrane lysis in addition to reconstruction. The most hydrophilic peptide AS4-1 only exhibited unstable binding on the supported membrane surface. These results demonstrate the heterogeneous structural disturbance of model cell membranes by amphiphilic α-helical peptides, which could be significantly strengthened by increasing the degree of hydrophobicity and/or local number density of peptides. This work provides support for the modulation of the membrane activity of MAPs by adjusting their hydrophobicity and local concentration.

Added Value of Biophysics to Study Lipid-Driven Biological Processes: The Case of Surfactins, a Class of Natural Amphiphile Molecules.

The role of membrane lipids is increasingly claimed to explain biological activities of natural amphiphile molecules. To decipher this role, biophysical studies with biomimetic membrane models are often helpful to obtain insights at the molecular and atomic levels. In this review, the added value of biophysics to study lipid-driven biological processes is illustrated using the case of surfactins, a class of natural lipopeptides produced by Bacillus sp. showing a broad range of biological activities. The mechanism of interaction of surfactins with biomimetic models showed to be dependent on the surfactins-to-lipid ratio with action as membrane disturber without membrane lysis at low and intermediate ratios and a membrane permeabilizing effect at higher ratios. These two mechanisms are relevant to explain surfactins' biological activities occurring without membrane lysis, such as their antiviral and plant immunity-eliciting activities, and the one involving cell lysis, such as their antibacterial and hemolytic activities. In both biological and biophysical studies, influence of surfactin structure and membrane lipids on the mechanisms was observed with a similar trend. Hence, biomimetic models represent interesting tools to elucidate the biological mechanisms targeting membrane lipids and can contribute to the development of new molecules for pharmaceutical or agronomic applications.

Influence of the Hydrophobicity of Pluronic Micelles Encapsulating Curcumin on the Membrane Permeability and Enhancement of Photoinduced Antibacterial Activity.

Apart from its well-known activity as an antimicrobial agent, Curcumin (CURC) has recently started to arouse interest as a photosensitizer in the photodynamic therapy of bacterial infections. The aim of the present study was to evidence the influence of the encapsulation of Curcumin into polymeric micelles on the efficiency of photoinduced microbial inhibition. The influence of the hydrophobicity of the selected Pluronics (P84, P123, and F127) on the encapsulation, stability, and antimicrobial efficiency of CURC-loaded micelles was investigated. In addition, the size, morphology, and drug-loading capacity of the micellar drug delivery systems have been characterized. The influence of the presence of micellar aggregates and unassociated molecules of various Pluronics on the membrane permeability was investigated on both normal and resistant microbial strains of E. coli, S. aureus, and C. albicans. The antimicrobial efficiency on the common pathogens was assessed for CURC-loaded polymeric micelles in dark conditions and activated by blue laser light (470 nm). Significant results in the reduction of the microorganism’s growth were found in cultures of C. albicans, even at very low concentrations of surfactants and Curcumin. Unlike the membrane permeabilization effect of the monomeric solution of Pluronics, reported in the case of tumoral cells, a limited permeabilization effect was found on the studied microorganisms. Encapsulation of the Curcumin in Pluronic P84 and P123 at very low, nontoxic concentrations for photosensitizer and drug-carrier, produced CURC-loaded micelles that prove to be effective in the light-activated inhibition of resistant species of Gram-positive bacteria and fungi.

New antimicrobial peptide-antibiotic combination strategy for Pseudomonas aeruginosa inactivation.

Novel antimicrobials or new treatment strategies are urgently needed to treat Pseudomonas aeruginosa (P. aeruginosa) related infections and especially to address the problem of antibiotic resistance. We propose a novel strategy that combines the human antimicrobial peptide (AMP) LL37 with different antibiotics to find synergistic AMP-antibiotic combinations against P. aeruginosa strains in vitro. Our results showed that LL37 exhibited synergistic inhibitory and bactericidal effects against P. aeruginosa strains PAO1 and PA103 when combined with the antibiotics vancomycin, azithromycin, polymyxin B, and colistin. In addition, LL37 caused strong outer membrane permeabilization, as demonstrated through measurement of an increased uptake of the fluorescent probe N-phenyl-1-naphthylamine. The membrane permeabilization effects appear to explain why it was easier to rescue the effectiveness of the antibiotic toward the bacteria because the outer membrane of P. aeruginosa exhibits barrier function for antibiotics. Furthermore, the change in the zeta potential was measured for P. aeruginosa strains with the addition of LL37. Zeta potentials for P. aeruginosa strains PAO1 and PA103 were -40.9 and -10.9 mV, respectively. With the addition of LL37, negative zeta potentials were gradually neutralized. We found that positively charged LL37 can interact with and neutralize the negatively charged bacterial outer membrane through electrostatic interactions, and the process of neutralization is believed to have contributed to the increase in outer membrane permeability. Finally, to further illustrate the relationship between outer membrane permeabilization and the uptake of antibiotics, we used LL37 to make the outer membrane of P. aeruginosa strains more permeable, and minimum inhibitory concentrations (MICs) for several antibiotics (colistin, gentamicin, polymyxin B, vancomycin, and azithromycin) were measured. The MICs decreased were twofold to fourfold, in general. For example, the MICs of azithromycin and vancomycin decreased more than fourfold when against P. aeruginosa strain PAO1, which were the greatest decrease of any of the antibiotics tested in this experiment. As for PA103, the MIC of polymyxin B2 decreased fourfold, which was the strongest decrease seen for any of the antibiotics tested in this experiment. The increased uptake of antibiotics not only demonstrates the barrier role of the outer membrane but also validates the mechanism of synergistic effects that we have proposed. These results indicate the great potential of an LL37-antibiotic combination strategy and provide possible explanations for the mechanisms behind this synergy.

Metallated Anticancer Peptides: An Expanded Mechanism that Encompasses Physical and Chemical Bilayer Disruption

In the search for novel broad‐spectrum therapeutics to fight chronic infections, inflammation, and cancer, host defense peptides (HDPs) have garnered increasing interest. Characterizing their biologically‐active conformations and minimum motifs for function represents a requisite step to developing them into efficacious and safe therapeutics. Here, we demonstrate that metallating HDPs is an effective chemical strategy to improve their cytotoxicity on cancer cells. Mechanistically, we find that the metallated peptides not only physically but also chemically damage lipid membranes. Our testing ground features piscidins 1 and 3 (P1/3), two amphipathic, histidine‐rich, membrane‐interacting, and cell‐penetrating HDPs that are α‐helical bound to membranes. To investigate their membrane location, permeabilization effects, and lipid‐oxidation capability, we employ neutron reflectometry, impedance spectroscopy, neutron diffraction, and UV spectroscopy. While P1‐apo is more potent than P3‐apo, metallation boosts their cytotoxicities by up to two‐ and seven‐fold, respectively. Remarkably, P3 is particularly effective at inserting its metallated motif in bilayers, causing water crevices in the hydrocarbon region and placing Cu2+ near the double bonds of the acyl chains, as needed to oxidize them. This study points at a new paradigm where metallating HDPs to expand their mechanistic reach could be explored to design more potent peptide‐based anticancer therapeutics.

Real-Time Fluorescence Microscopy on Living E. coli Sheds New Light on the Antibacterial Effects of the King Penguin β-Defensin AvBD103b.

(1) Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. Among AMPs, the disulfide-rich β-defensin AvBD103b, whose antibacterial activities are not inhibited by salts contrary to most other β-defensins, is particularly appealing. Information about the mechanisms of action is mandatory for the development and approval of new drugs. However, data for non-membrane-disruptive AMPs such as β-defensins are scarce, thus they still remain poorly understood. (2) We used single-cell fluorescence imaging to monitor the effects of a β-defensin (namely AvBD103b) in real time, on living E. coli, and at the physiological concentration of salts. (3) We obtained key parameters to dissect the mechanism of action. The cascade of events, inferred from our precise timing of membrane permeabilization effects, associated with the timing of bacterial growth arrest, differs significantly from the other antimicrobial compounds that we previously studied in the same physiological conditions. Moreover, the AvBD103b mechanism does not involve significant stereo-selective interaction with any chiral partner, at any step of the process. (4) The results are consistent with the suggestion that after penetrating the outer membrane and the cytoplasmic membrane, AvBD103b interacts non-specifically with a variety of polyanionic targets, leading indirectly to cell death.

Identification and Characterization of a Two-Peptide Class IIb Bacteriocin in Streptococcus pluranimalium Isolated from the Nasal Cavity of a Healthy Pig.

In addition to be an important zoonotic agent, Streptococcus suis serotype 2 causes severe infections in pigs. In this study, we characterized a new bacteriocin produced by Streptococcus pluranimalium 2N12 isolated from a pig nasal sample. The bacteriocin, termed pluranimalicin 2N12, was a two-peptide class IIb bacteriocin active against S. suis. The gene cluster responsible for the biosynthesis of pluranimalicin 2N12 by S. pluranimalium contained seven open reading frames, including putative genes for peptides (pluα, pluβ), export (pluA, pluB), and regulation (pluC, pluD, pluE). The deduced amino acid sequences of the peptides Pluα (33 amino acids) and Pluβ (29 amino acids) showed 73% and 69% identity in amino acid residues, respectively, with the peptides SthA and SthB of the streptocin produced by Streptococcus gordonii. The antibacterial activity of pluranimalicin 2N12 against S. suis was dependent on the presence of the two peptides Pluα and Pluβ that exhibited a membrane permeabilization effect. No activity was found against the other swine pathogens tested. Depending on the concentrations used, Pluα and Pluβ displayed no or low toxicity towards swine tracheal epithelial cells. The pluranimalicin peptides Pluα and Pluβ, either individually or in combination, exhibited anti-inflammatory activity since they attenuated IL-6 and TNF-α production by macrophages challenged with lipopolysaccharide. Given its dual action (antibacterial and anti-inflammatory), pluranimalicin 2N12 holds promise as a potential therapeutic agent for controlling S. suis infections.

Lipid Nanoparticles Loaded with Farnesol or Geraniol to Enhance the Susceptibility of E. coli MCR-1 to Colistin.

Resistance to colistin, one of the antibiotics of last resort against multidrug-resistant Gram-negative bacteria, is increasingly reported. Notably, MCR plasmids discovered in 2015 have now been reported worldwide in humans. To keep this antibiotic of last resort efficient, a way to tackle this mechanism seems essential. Terpene alcohols such as farnesol have been shown to improve the efficacy of some antibiotics. However, their high lipophilicity makes them difficult to use. This problem can be solved by encapsulating them in water-dispersible lipid nanoparticles (LNPs). The aim of this study was to discover, using checkerboard tests and time-kill curve experiments, an association between colistin and farnesol or geraniol loaded in LNPs, which would improve the efficacy of colistin against E. coli and, in particular, MCR-1 transconjugants. Then, the effect of the combination on E. coli inner membrane permeabilisation was evaluated using propidium iodide (PI) uptake and compared to human red blood cells plasma membrane permeabilisation. Both terpene alcohols were able to restore the susceptibility of E. coli J53 MCR-1 to colistin with the same efficacy (Emax = 16, i.e., colistin MIC was decreased from 8 to 0.5 mg/L). However, with an EC50 of 2.69 mg/L, farnesol was more potent than geraniol (EC50 = 39.49 mg/L). Time-kill studies showed a bactericidal effect on MCR-1 transconjugant 6 h after incubation, with no regrowth up to 30 h in the presence of 1 mg/L colistin (1/8 MIC) and 60 mg/L or 200 mg/L farnesol or geraniol, respectively. Colistin alone was more potent in increasing PI uptake rate in the susceptible strain (EC50 = 0.86 ± 0.08 mg/L) than in the MCR-1 one (EC50 = 7.38 ± 0.85 mg/L). Against the MCR-1 strain, farnesol-loaded LNP at 60 mg/L enhanced the colistin-induced inner membrane permeabilization effect up to 5-fold and also increased its potency as shown by the decrease in its EC50 from 7.38 ± 0.85 mg/L to 2.69 ± 0.25 mg/L. Importantly, no hemolysis was observed for LNPs loaded with farnesol or geraniol, alone or in combination with colistin, at the concentrations showing the maximum decrease in colistin MICs. The results presented here indicate that farnesol-loaded LNPs should be studied as combination therapy with colistin to prevent the development of resistance to this antibiotic of last resort.

Copper-binding anticancer peptides from the piscidin family: an expanded mechanism that encompasses physical and chemical bilayer disruption

In the search for novel broad-spectrum therapeutics to fight chronic infections, inflammation, and cancer, host defense peptides (HDPs) have garnered increasing interest. Characterizing their biologically-active conformations and minimum motifs for function represents a requisite step to developing them into efficacious and safe therapeutics. Here, we demonstrate that metallating HDPs with Cu2+ is an effective chemical strategy to improve their cytotoxicity on cancer cells. Mechanistically, we find that prepared as Cu2+-complexes, the peptides not only physically but also chemically damage lipid membranes. Our testing ground features piscidins 1 and 3 (P1/3), two amphipathic, histidine-rich, membrane-interacting, and cell-penetrating HDPs that are α-helical bound to membranes. To investigate their membrane location, permeabilization effects, and lipid-oxidation capability, we employ neutron reflectometry, impedance spectroscopy, neutron diffraction, and UV spectroscopy. While P1-apo is more potent than P3-apo, metallation boosts their cytotoxicities by up to two- and seven-fold, respectively. Remarkably, P3-Cu2+ is particularly effective at inserting in bilayers, causing water crevices in the hydrocarbon region and placing Cu2+ near the double bonds of the acyl chains, as needed to oxidize them. This study points at a new paradigm where complexing HDPs with Cu2+ to expand their mechanistic reach could be explored to design more potent peptide-based anticancer therapeutics.

Anti-Staphylococcal and cytotoxic activities of the short anti-microbial peptide PVP.

Over the past years, short anti-microbial peptides have drawn growing attention in the research and trade literature because they are usually capable of killing a broad spectrum of pathogens by employing unique mechanisms of action. This study aimed to evaluate the anti-bacterial effects of a previously designed peptide named PVP towards the clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) in vitro. Secondary structure, cytotoxicity, and membrane-permeabilizing effects of the peptide were also assessed. PVP had a tendency to adopt alpha-helical conformation based upon structural predictions and circular dichroism spectroscopy (in 50% trifluoroethanol). The peptide showed MIC values ranging from 1 to 16µg/mL against 10 strains of MRSA. In contrast to ciprofloxacin and gentamicin, PVP at sub-lethal concentration (1µg/mL) did not provoke the development of peptide resistance after 14 serial passages. Remarkably, 1h of exposure to 4 × MBC of PVP (8µg/mL) was sufficient for total bacterial clearance, whereas 4 × MBC of vancomycin (8µg/mL) failed to totally eradicate bacterial cells, even after 8h. PVP showed negligible cytotoxicity against human dermal fibroblasts at concentrations required to kill the MRSA strains. The results of flow cytometric analysis and fluorescence microscopy revealed that PVP caused bacterial membrane permeabilization, eventually culminating in cell death. Owing to the potent anti-bacterial activity, fast bactericidal kinetics, and negligible cytotoxicity, PVP has the potential to be used as a candidate antibiotic for the topical treatment of MRSA infections.

Abstract 6258: Combining the glioblastoma cell membrane-permeabilizing effect of tumor treating fields with chemotherapy

Abstract BACKGROUND Glioblastoma (GBM) is the most lethal primary brain cancer (median survival: 15-17 months, 5-year survival: 5.6%). Standard interventions consist of aggressive surgical resection, radiotherapy, and chemotherapy; however, GBM is heterogeneous and present therapies are ineffective. Tumor treating fields (TTFields) is a form of alternating electric field therapy that has been shown to prolong survival in patients with newly-diagnosed GBM when combined with standard chemotherapy. The mechanism of TTFields' potentiation of standard chemotherapy against GBM is not well understood. We hypothesized that TTFields increases access of chemotherapy to cancer cells by disrupting the cell membrane. METHODS Human and murine GBM cells (GBM2, GBM39, U87-MG, KR158B) were isolated from primary gliomas. Cells were engineered to stably express firefly or renilla luciferase (fLuc or rLuc, respectively). Cells were either exposed to TTFields (50-500 kHz, 1-4 V/cm) or control conditions. Proliferation was assessed by bioluminescence imaging (BLI) and cell counting. Dextran-FITC binding and influx of 5-aminolevulinic acid (5-ALA) were also assessed. Scanning electron microscopy (SEM) studies were used to probe effects on cellular membranes. All experiments were performed in at least triplicate and 2-way ANOVA or univariate Mann-Whitney test was performed to compare the groups. RESULTS TTFields significantly inhibited growth of cells (p≤0.02, no TTFields vs. TTFields). BLI suggested alterations in membrane configuration when cancer cells were exposed to TTFields. This was validated with observations of greater fluorescence of membrane-associating Dextran-FITC to U87-MG cells that were subjected to TTFields (p< 0.01, no TTFields vs. TTFields). In GBM39 cells, the optimal effect by TTFields on enhancing Dextran-FITC binding occurred in the range of 100-300 kHz (p<0.02, no TTFields vs. TTFields). TTFields also enhanced 5-ALA uptake into exposed GBM cells (p<0.001, no TTFields vs. TTFields). SEM revealed significantly greater and larger number of holes on the membrane surface of TTFields-exposed U87-MG cancer cells (53.5±19.1 holes per field of view and mean size=240.6±91.7 nm2) compared to unexposed cells (23.9±11.0 holes per field of view and mean size=129.8±31.9 nm2, p< 0.005: TTFields exposed vs. non-exposed). Morphologically, GBM cells unexposed to TTFields had matted and elongated projections from the cell membrane, which were replaced by short, bulbous, bleb-like structures upon TTFields exposure. All observed effects were reversed upon cessation of TTFields. CONCLUSION The findings suggest a permeabilization of the GBM cell membrane upon exposure to TTFields. This may explain the previously observed synergy between TTFields and standard (e.g., temozolomide) and emerging (Withaferin A) therapies, whereby increased permeability on membranes confers greater accessibility to drugs. Such a strategy is thus a promising candidate for future clinical translation in glioblastoma. Citation Format: Edwin Chang, Chirag Patel, Caroline J. Young, Thomas Anthony Flores, Lydia-Marie Joubert, Yitian Zeng, Robert Sinclair, Sanjiv Gambhir. Combining the glioblastoma cell membrane-permeabilizing effect of tumor treating fields with chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6258.

Hybrid Magnetic Nanovectors Promote Selective Glioblastoma Cell Death through a Combined Effect of Lysosomal Membrane Permeabilization and Chemotherapy.

Glioblastoma multiforme is the most aggressive brain tumor, due to its high invasiveness and genetic heterogeneity. Moreover, the blood–brain barrier prevents many drugs from reaching a therapeutic concentration at the tumor site, and most of the chemotherapeutics lack in specificity toward cancer cells, accumulating in both healthy and diseased tissues, with severe side effects. Here, we present in vitro investigations on lipid-based nanovectors encapsulating a drug, nutlin-3a, and superparamagnetic iron oxide nanoparticles, to combine the proapoptotic action of the drug and the hyperthermia mediated by superparamagnetic iron oxide nanoparticles stimulated with an alternating magnetic field. The nanovectors are functionalized with the peptide angiopep-2 to induce receptor-mediated transcytosis through the blood–brain barrier and to target a receptor overexpressed by glioma cells. The glioblastoma multiforme targeting efficiency and the blood–brain barrier crossing abilities were tested through in vitro fluidic models, where different human cell lines were placed to mimic the tumor microenvironment. These nanovectors successfully cross the blood–brain barrier model, maintaining their targeting abilities for glioblastoma multiforme with minimal interaction with healthy cells. Moreover, we showed that nanovector-assisted hyperthermia induces a lysosomal membrane permeabilization that not only initiates a caspase-dependent apoptotic pathway, but also enhances the anticancer efficacy of the drug.

Vacuolar H+ -ATPase is involved in preventing heavy metal-induced oxidative stress in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, vacuolar H+ -ATPase (V-ATPase) involved in the regulation of intracellular pH homeostasis has been shown to be important for tolerances to cadmium, cobalt and nickel. However, the molecular mechanism underlying the protective role of V-ATPase against these metals remains unclear. In this study, we show that cadmium, cobalt and nickel disturbed intracellular pH balance by triggering cytosolic acidification and vacuolar alkalinization, likely via their membrane permeabilizing effects. Since V-ATPase plays a crucial role in pumping excessive cytosolic protons into the vacuole, the metal-sensitive phenotypes of the Δvma2 and Δvma3 mutants lacking V-ATPase activity were supposed to result from highly acidified cytosol. However, we found that the metal-sensitive phenotypes of these mutants were caused by increased production of reactive oxygen species, likely as a result of decreased expression and activities of manganese superoxide dismutase and catalase. In addition, the loss of V-ATPase function led to aberrant vacuolar morphology and defective endocytic trafficking. Furthermore, the sensitivities of the Δvma mutants to other chemical compounds (i.e. acetic acid, H2 O2 , menadione, tunicamycin and cycloheximide) were a consequence of increased endogenous oxidative stress. These findings, therefore, suggest the important role of V-ATPase in preventing endogenous oxidative stress induced by metals and other chemical compounds.

Structural insights into the lipid transfer mechanism of a non-specific lipid transfer protein.

The non-specific lipid transfer proteins (nsLTPs) are multifunctional seed proteins engaged in several different physiological processes. The nsLTPs are stabilized by four disulfide bonds and exhibit a characteristic hydrophobic cavity, which is the primary lipid binding site. While these proteins are known to transfer lipids between membranes, the mechanism of lipid transfer has remained elusive. Four crystal structures of nsLTP from Solanum melongena, one in the apo-state and three myristic acid bound states were determined. Among the three lipid bound states, two lipid molecules were bound on the nsLTP surface at different positions and one was inside the cavity. The lipid-dependent conformational changes leading to opening of the cavity were revealed based on structural and spectroscopic data. The surface-bound lipid represented a transient intermediate state and the lipid ultimately moved inside the cavity through the cavity gate as revealed by molecular dynamics simulations. Two critical residues in the loop regions played possible 'gating' role in the opening and closing of the cavity. Antifungal activity and membrane permeabilization effect of nsLTP against Fusarium oxysporum suggested that it could possibly involve in bleaching out the lipids. Collectively, these studies support a model of lipid transfer mechanism by nsLTP via intermediate states.