Membrane-damaging Activity Research Articles

Unlocking the therapeutic potential of Nigella sativa extract: phytochemical analysis and revealing antimicrobial and antioxidant marvels

The growing global threat of antimicrobial resistance endangers both human and animal life, necessitating the urgent discovery of novel antimicrobial solutions. Medicinal plants hold promise as sources of potential antimicrobial compounds. In this study, we investigated the phytochemical constituents and microbicidal capabilities of the ethanolic extract from Nigella sativa (black seed). Gas chromatography analysis (GC) identified 11 compounds, among them thymoquinone, and thymol, contributing to antimicrobial and antioxidant properties. Antimicrobial assays demonstrated notable inhibition zones against broad spectra of bacteria, including Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Staphylococcus aureus, Enterobacter, and Bacillus subtilis, along with potent antifungal activity against Aspergillus niger, Penicillium, and Candida albicans. Notably, when combined with antibiotics, the extract displayed exceptional synergistic antimicrobial efficacy. The black seed extract demonstrated membrane-damaging activity and disrupted virulence factors that protect microbes from antimicrobial agents, including the formation of bacterial biofilm and protease secretion. Thymoquinone, the primary active constituent of the extract, exhibited similar antimicrobial and ant virulence properties. In silico analysis targeting key regulators of quorum sensing and biofilm formation in P. aeruginosa, such as RhlG, LasR, and PqsR, showed a remarkable affinity of thymol and thymoquinone for these targets. Moreover, the N. sativa extract exhibited dose-dependent cytotoxicity against both the promastigote and amastigote forms of Leishmania tropica parasites, hinting at potential antiparasitic activity. In addition to its antimicrobial properties, the extract displayed potential antioxidant activity at a concentration of 400 μg/mL.

Zn(II) and Cu(II) Coordination Enhances the Antimicrobial Activity of Piscidin 3, but Not That of Piscidins 1 and 2.

Piscidins, antimicrobial peptides isolated from fish, are potent against a variety of human pathogens; they show minimum inhibitory concentration values comparable to those of commercially used antimicrobials. Piscidins 1 and 2 are generally more effective than piscidin 3 when applied alone; the contrary is observed for their metal complexes: Zn(II) and Cu(II) coordination does not enhance the efficacy of piscidins 1 and 2, while a moderate enhancement is observed for piscidin 3. All three piscidins bind Cu(II) in a so-called albumin-like binding mode, while for Zn(II) complexes, two coordination modes are observed: piscidins 1 and 2 bind Zn(II) by imidazole nitrogens from His4, His11, and His17 side chains; piscidin 3 coordinates Zn(II) by His3, His4, and His11 imidazole nitrogens and additionally supports the interaction, formed by carbonyl oxygen from His4. Most likely, the high antimicrobial activity of piscidin complexes is due to neither the stability of their complexes nor the change in their secondary structure. Copper(II) complexes with piscidins 1 and 2 can form hydroxyl radicals, which could be responsible for the antimicrobial membrane damaging activity of these complexes. Clearly, a different mechanism (most likely an intercellular targeted one) is observed for piscidin 3 metal complexes; in most cases, the coordination of Cu(II) and Zn(II) enhances the antimicrobial potency of piscidin 3, showing that not only piscidin 3 alone but also its metal complexes have a different mode of action than piscidins 1 and 2.

Hundreds of antimicrobial peptides create a selective barrier for insect gut symbionts

The spatial organization of gut microbiota is crucial for the functioning of the gut ecosystem, although the mechanisms that organize gut bacterial communities in microhabitats are only partially understood. The gut of the insect Riptortus pedestris has a characteristic microbiota biogeography with a multispecies community in the anterior midgut and a monospecific bacterial population in the posterior midgut. We show that the posterior midgut region produces massively hundreds of specific antimicrobial peptides (AMPs), the Crypt-specific Cysteine-Rich peptides (CCRs) that have membrane-damaging antimicrobial activity against diverse bacteria but posterior midgut symbionts have elevated resistance. We determined by transposon-sequencing the genetic repertoire in the symbiont Caballeronia insecticola to manage CCR stress, identifying different independent pathways, including AMP-resistance pathways unrelated to known membrane homeostasis functions as well as cell envelope functions. Mutants in the corresponding genes have reduced capacity to colonize the posterior midgut, demonstrating that CCRs create a selective barrier and resistance is crucial in gut symbionts. Moreover, once established in the gut, the bacteria differentiate into a CCR-sensitive state, suggesting a second function of the CCR peptide arsenal in protecting the gut epithelia or mediating metabolic exchanges between the host and the gut symbionts. Our study highlights the evolution of an extreme diverse AMP family that likely contributes to establish and control the gut microbiota.

Synthesis and antibacterial activities of baulamycin A inspired derivatives

SbnE is an essential enzyme for staphyloferrin B biosynthesis in Staphylococcus aureus. An earlier study showed that natural product baulamycin A has in vitro inhibitory activity against SbnE and antibacterial potency. A SAR study with analogues of baulamycin A was conducted to identify potent inhibitors of SbnE and/or effective antibiotics against MRSA. The results show that selected analogues, including 11, 18, 21, 24a, 24c, 24m and 24n, exhibit single-digit micromolar inhibitory potencies against SbnE (IC50s = 1.81–8.94 μM) and 11, 24m, 24n possess significant activities against both SbnE (IC50s = 4.12–6.12 μM) and bacteria (MICs = 4–32 μg/mL). Biological investigations revealed that these substances possess potent cell wall disruptive activities and that they inhibit siderophore production in MRSA. Among the selected analogues, 7 has excellent antibiotic activities both gram-positive and –negative bacteria (0.5–4 μg/mL). Moreover, these analogues significantly impede biofilm formation in a concentration-dependent manner. Taken together, the results of the investigation provide valuable insight into the nature of novel baulamycin A analogues that have potential efficacy against MRSA owing to their membrane damaging activity and/or inhibitory efficacy against siderophore production.

Activity of Lipid Loaded Lectin against co-infection of Candida albicans and Staphylococcus aureus using the Zebrafish model.

Nosocomial infection caused by mixed species of methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans (CA) is difficult to manage with existing antimicrobials, particularly in the presence of mixed-species biofilm. This study evaluates the activity of cationic lipid, specifically functionalized with lectin, against mixed biofilms of MRSA and CA and their effectiveness in vivo using the zebrafish model. The present study demonstrates for the first time the antimicrobial activity of 2-((N-[2-hydroxyethyl]palmitamido)methyl)-1-methylpyridin-1-ium iodide (cN16E) against MRSA and mixed species of MRSA + CA. The cN16E functionalized with Butea monosperma seed lectin (BMSL) showed a lower minimum inhibitory concentration (MIC) as compared with cN16E. BMSL-cN16E (BcN16E) exhibited strong membrane-damaging activity at a lower concentration than cN16E. Crystal violet assay showed that BcN16E inhibits mixed-species biofilm at the concentration of 15.63 µM, which is four-fold lower than the MIC. Especially, BcN16E was found to be effective in disturbing mature mixed biofilm at 31.25 µM, which is two-fold lower than the MIC, suggesting true antibiofilm activity without pressurizing the microorganisms. The treatment with BcN16E significantly reduced the exopolysaccharide synthesis (> 78%), cell surface hydrophobicity (> 70%), hyphae formation, staphyloxanthin biosynthesis (> 41%), and antioxidant enzyme and hemolysin activity (> 70%). Notably, BcN16E was efficient in reducing the in vivo colonization of bacterial and fungal burden in the blood and muscle tissues of zebrafish. Antimicrobial and antibiofilm efficacy of BcN16E against MRSA, and mixed species of MRSA + CA were demonstrated. Importantly, BcN16E treatment rescued Zebrafish coinfected with mixed species of MRSA + CA.

Curcumin Inhibits Membrane-Damaging Pore-Forming Function of the β-Barrel Pore-Forming Toxin Vibrio cholerae Cytolysin.

Vibrio cholerae cytolysin (VCC) is a β-barrel pore-forming toxin (β-PFT). Upon encountering the target cells, VCC forms heptameric β-barrel pores and permeabilizes the cell membranes. Structure-function mechanisms of VCC have been extensively studied in the past. However, the existence of any natural inhibitor for VCC has not been reported yet. In the present study, we show that curcumin can compromise the membrane-damaging activity of VCC. Curcumin is known to modulate a wide variety of biological processes and functions. However, the application of curcumin in the physiological scenario often gets limited due to its extremely poor solubility in the aqueous environment. Interestingly, we find that VCC can associate with the insoluble fraction of curcumin in the aqueous medium and thus gets separated from the solution phase. This, in turn, reduces the availability of VCC to attack the target membranes and thus blocks the membrane-damaging action of the toxin. We also observe that the soluble aqueous extract of curcumin, generated by the heat treatment, compromises the pore-forming activity of VCC. Interestingly, in the presence of such soluble extract of curcumin, VCC binds to the target membranes and forms the oligomeric assembly. However, such oligomers appear to be non-functional, devoid of the pore-forming activity. The ability of curcumin to bind to VCC and neutralize its membrane-damaging activity suggests that curcumin has the potential to act as an inhibitor of this potent bacterial β-PFT.

Current Perspective on the Membrane-Damaging Action of Thermostable Direct Hemolysin, an Atypical Bacterial Pore-forming Toxin.

Thermostable direct hemolysin (TDH) is the major virulence determinant of the gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin (PFT). TDH shares remarkable structural similarity with the actinoporin family of eukaryotic PFTs produced by the sea anemones. Unlike most of the PFTs, it exists as tetramer in solution, and such assembly state is crucial for its functionality. Although the structure of the tetrameric assembly of TDH in solution is known, membrane pore structure is not available yet. Also, the specific membrane-interaction mechanisms of TDH, and the exact role of any receptor(s) in such process, still remain unclear. In this mini review, we discuss some of the unique structural and physicochemical properties of TDH, and their implications for the membrane-damaging action of the toxin. We also present our current understanding regarding the membrane pore-formation mechanism of this atypical bacterial PFT.

Staphylococcus aureus membrane-damaging activities of four phenolics.

The membrane-damaging activities of four phenolics chosen for their bactericidal activity against Staphylococcus aureus CNRZ3 were investigated: 5,7-dihydroxy-4-phenylcoumarin (DHPC), 5,8-dihydroxy-1,4-naphthoquinone (DHNQ), epigallocatechin gallate (EGCG) and isobutyl 4-hydroxybenzoate (IBHB). Staphylococcusaureus CNRZ3 cells, as well as model liposomes mimicking its membrane phospholipids composition, were treated with each phenolic at its minimal bactericidal concentration. Membrane integrity, intracellular pH and intracellular esterase activity were examined by flow cytometric analysis of S.aureus cells stained with propidium iodide and SYTO® 9, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, and 5(6)-carboxyfluorescein diacetate, respectively. While intracellular pH was affected by the foyr phenolics, only DHNQ and to a lesser extent EGCG, caused a loss of membrane integrity. Flow cytometric analysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and DPPC/POPG (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphoglycerol) liposomes stained with Coumarin 6 (which penetrates the lipid bilayer) or 5-N(octadecanoyl)-amino-fluorescein (which binds to the liposome shell) suggested that only EGCG and DHNQ penetrated the bilayer of phospholipids of liposomes. Taken together, these findings support the hypothesis that EGCG and DHNQ bactericidal activity results from their accumulation in the phospholipid bilayer of S.aureus cells membrane causing its disruption.

Membrane Disruption Potential of Doped Cuprous Oxide Nanoparticles Against blaNDM-1 and mcr-1 Positive Colistin Resistant E. coli

Bacterial resistance against the last-resort antibiotic “colistin” is a growing problem worldwide. Novel strategies in the field of nanotechnology for the development of suitable antibacterial drug with minimal toxicities are need of the time. Here we synthesized, Cu2O nanoparticles (Cu2O-NPs) doped with 5% Zn metal ions by adopting solution-phase method using hydrazine reduction in aqueous medium. Antibacterial assays showed a promising activity against colistin-resistant Escherichia coli isolates. Leakage assay, propidium iodide uptake assay, EtBr uptake assay, and LIVE/DEAD® BacLight™ assay showed membrane damaging activity of Cu2O-NPs. Imaging by scanning electron microscopy of NP-treated bacterial cells confirmed membrane damaging potential of used Cu2O-NPs. Toxicity assessment by cell viability assay showed no cytotoxic activity to the HEK cell line. In conclusion, our results suggest that Cu2O-NPs doped with 5% Zn may exert their antimicrobial action by disruption of the bacterial cell membrane and probably generating reactive oxygen species (ROS). We suggest that Cu2O-NPs doped with 5% Zn can be used as effective antibacterial agent.

Modification of carboxyl groups converts α-lactalbumin into an active molten globule state with membrane-perturbing activity and cytotoxicity

We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity.

Blocking of negative charged carboxyl groups converts Naja atra neurotoxin to cardiotoxin-like protein

Naja atra cobrotoxin and cardiotoxin 3 (CTX3) exhibit neurotoxicity and cytotoxicity, respectively. In the present study, we aimed to investigate whether the carboxyl groups of cobrotoxin play a role in structural constraints, thereby preventing cobrotoxin from exhibiting cytotoxic activity. Six of the seven carboxyl groups in cobrotoxin were conjugated with semicarbazide. Measurement of circular dichroism spectra and Trp fluorescence quenching showed that the gross conformation of semicarbazide-modified cobrotoxin (SEM-cobrotoxin) and cobrotoxin differed. In sharp contrast to cobrotoxin, SEM-cobrotoxin demonstrated membrane-damaging activity and cytotoxicity, which are feature more characteristic of CTX3. Furthermore, both SEM-cobrotoxin and CTX3 induced cell death through AMPK activation. Analyses of the interaction between polydiacetylene/lipid vesicles and fluorescence-labeled lipids revealed that SEM-cobrotoxin and cobrotoxin adopted different membrane-bound states. The structural characteristics of SEM-cobrotoxin were similar to those of CTX3, including trifluoroethanol (TFE)-induced structural transformation and membrane binding-induced conformational change. Conversely, cobrotoxin was insensitive to the TFE-induced effect. Collectively, the data of this study indicate that blocking negatively charged residues confers cobrotoxin with membrane-damaging activity and cytotoxicity. The findings also suggest that the structural constraints imposed by carboxyl groups control the functional properties of snake venom α-neurotoxins during the divergent evolution of snake venom neurotoxins and cardiotoxins.

Mycobacterium marinum phthiocerol dimycocerosates enhance macrophage phagosomal permeabilization and membrane damage

Phthiocerol dimycocerosates (PDIMs) are a class of mycobacterial lipids that promote virulence in Mycobacterium tuberculosis and Mycobacterium marinum. It has recently been shown that PDIMs work in concert with the M. tuberculosis Type VII secretion system ESX-1 to permeabilize the phagosomal membranes of infected macrophages. As the zebrafish-M. marinum model of infection has revealed the critical role of PDIM at the host-pathogen interface, we set to determine if PDIMs contributed to phagosomal permeabilization in M. marinum. Using an ΔmmpL7 mutant defective in PDIM transport, we find the PDIM-ESX-1 interaction to be conserved in an M. marinum macrophage infection model. However, we find PDIM and ESX-1 mutants differ in their degree of defect, with the PDIM mutant retaining more membrane damaging activity. Using an in vitro hemolysis assay—a common surrogate for cytolytic activity, we find that PDIM and ESX-1 differ in their contributions: the ESX-1 mutant loses hemolytic activity while PDIM retains it. Our observations confirm the involvement of PDIMs in phagosomal permeabilization in M. marinum infection and suggest that PDIM enhances the membrane disrupting activity of pathogenic mycobacteria and indicates that the role they play in damaging phagosomal and red blood cell membranes may differ.

Status of Asp29 and Asp40 in the Interaction of Naja atra Cardiotoxins with Lipid Bilayers.

It is widely accepted that snake venom cardiotoxins (CTXs) target the plasma membranes of cells. In the present study, we investigated the role of Asp residues in the interaction of Naja atra cardiotoxin 1 (CTX1) and cardiotoxin 3 (CTX3) with phospholipid bilayers using chemical modification. CTX1 contains three Asp residues at positions 29, 40, and 57; CTX3 contains two Asp residues at positions 40 and 57. Compared to Asp29 and Asp40, Asp57 was sparingly modified with semi-carbazide, as revealed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass and mass/mass analyses. Thus, semi-carbazide-modified CTX1 (SEM-CTX1) mainly contained modified Asp29 and Asp40, while SEM-CTX3 contained modified Asp40. Compared to that of native toxins, trifluoroethanol easily induced structural transition of SEM-CTX1 and SEM-CTX3, suggesting that the structural flexibility of CTXs was constrained by Asp40. Modification of Asp29 and Asp40 markedly promoted the ability of CTX1 to induce permeability of cell membranes and lipid vesicles; CTX3 and SEM-CTX3 showed similar membrane-damaging activity. Modification of Asp residues did not affect the membrane-binding capability of CTXs. Circular dichroism spectra of SEM-CTX3 and CTX3 were similar, while the gross conformation of SEM-CTX1 was distinct from that of CTX1. The interaction of CTX1 with membrane was distinctly changed by Asp modification. Collectively, our data suggest that Asp29 of CTX1 suppresses the optimization of membrane-bound conformation to a fully active state and that the function of Asp40 in the structural constraints of CTX1 and CTX3 is not important for the manifestation of membrane-perturbing activity.

N-Terminal Region of Vibrio parahemolyticus Thermostable Direct Hemolysin Regulates the Membrane-Damaging Action of the Toxin.

Thermostable direct hemolysin (TDH) of Vibrio parahemolyticus is a membrane-damaging pore-forming toxin with potent cytolytic/cytotoxic activity. TDH exists as a tetramer consisting of protomers with a core β-sandwich domain, flanked by an 11-amino acid long N-terminal region (NTR). This NTR could not be modeled in the previously determined crystal structure of TDH. Moreover, the functional implication of NTR for the membrane-damaging action of TDH remains unknown. In the present study, we have explored the implications of NTR for the structure-function mechanism of TDH. Our data show that the presence of NTR modulates the physicochemical property of TDH in terms of augmenting the amyloidogenic propensity of the protein. Deletion of NTR compromises the binding of TDH toward target cell membranes and drastically affects the membrane-damaging cytolytic/cytotoxic activity of the toxin. Mutations of aromatic/hydrophobic residues within NTR also confer compromised cell-killing activity. Moreover, covalent trapping of NTR, via an engineered disulfide bond, against the core β-sandwich domain also abrogates the cytolytic/cytotoxic activity of TDH. This observation suggests that an unrestrained configuration of NTR is crucial for the membrane-damaging action of TDH. On the basis of our study, we propose a model explaining the role of NTR in the membrane-damaging function of TDH.

PH‐Dependent Interaction of Four Different Bile Salts with POPG Liposomes Studied by Dynamic Light Scattering and Second Harmonic Spectroscopy

AbstractIn recent years the role of bile salts in colorectal cancer has been gaining importance. In addition, it has been observed that for diseases associated with gastrointestinal tract, the pH becomes slightly acidic. In this report we have investigated the pH dependent bile acid – bile salt transformation on the integrity and permeability of phosphatidyl glycerol (POPG) liposomes by dynamic light scattering and interfacial second harmonic generation spectroscopy. Using four different bile salts whose chemical properties vary widely we have observed that in their neutral form (i.e bile acids) they are able to alter the POPG membrane properties significantly higher compared to their ionic (i. e. bile salts) form. Our results indicate that out of the four bile salts, Sodium Deoxycholate and Sodium Cholate have the potential to damage membranes in the colon as their pKa is closer to the pH measured in the gastrointestinal tract. Therefore, one of the pathways of bile salt induced cytotoxicity in the gastrointestinal tract, may occur due to the potential membrane damaging action of these bile salts at acidic pH.

In vivo fluid accumulation-inhibitory, anticolonization and anti-inflammatory and in vitro biofilm-inhibitory activities of methyl gallate isolated from Terminalia chebula against fluoroquinolones resistant Vibrio cholerae

Acute Vibrio cholerae infection triggers significant inflammatory response and immense fluid secretion in the intestine. In the present study, methyl gallate (MG) isolated from Terminalia chebula was evaluated to determine the in vivo fluid accumulation-inhibitory, anticolonization and anti-inflammatory and in vitro biofilm-inhibitory activities against multi-drug resistant (MDR) V. cholerae.Bacterial membrane-damaging and biofilm-inhibitory activities were determined by membrane perturbation and transmission electron microscopy (TEM); and microdilution assays, respectively. Fluid accumulation-inhibitory and anticolonization activities of MG (23.80–95.23 mg/kg body weight) were determined in 4–5 days old BALB/c mice with an incubation time of 18 h. The effect of MG (1, 50 and 500 mg/kg body weight) on intestinal inflammatory reaction induced by V. cholerae was studied by performing histology in Swiss albino mice.MIC and MBC of MG against the test strains were 32–64 and 64–256 μg/ml, respectively. MG showed the fluid accumulation-inhibitory activity with inhibition values of 42.86–89.08% at doses between 23.80 and 95.23 mg/kg body weight and significant anticolonization activity (p < 0.0001) against V. choleare in the suckling mouse intestine. MG (500 mg/kg body weight) significantly inhibited the inflammatory reactions induced by V. cholerae compared to the vehicle control. MG exhibited 70% minimum biofilm inhibition concentration of 64 μg/ml and bacterial membrane damaging activity at 1 × MBC. The results obtained in the present study suggest that MG has potential as an effective agent for the treatment of severe secretory and inflammatory diarrheal disease caused by MDR V. cholerae.

Intracellular pHluorin as Sensor for Easy Assessment of Bacteriocin-Induced Membrane-Damage in Listeria monocytogenes.

Bacteriocins are antimicrobial peptides naturally produced by many bacteria and were shown to be effective against various pathogens including Listeria monocytogenes. L. monocytogenes is a food-borne pathogen that frequently causes disease outbreaks around the world with fatal outcomes in at-risk individuals. Thus, bacteriocins are a promising solution to prevent contaminations with L. monocytogenes and other microorganisms during food production and preservation. In the present study, we constructed L. monocytogenes EGD-e/pNZ-Phelp-pHluorin, a strain that constitutively expresses the pH-sensitive fluorescent protein pHluorin, as a sensor strain to detect disruption of the pH gradient by the membrane-damaging activity of bacteriocins. The ratiometric fluorescence properties of pHluorin were validated both in crude extracts and permeabilized cells of this sensor strain. L. monocytogenes EGD-e/pNZ-Phelp-pHluorin was used to assess membrane damaging activity of the bacteriocins nisin A and pediocin PA-1 and to determine the minimal concentrations required for full disruption of the pH gradient across the membrane. Moreover, the sensor strain proved useful to analyze the presence of compounds affecting membrane integrity in supernatants of a nisin Z-producing Lactococcus lactis strain at different timepoints during growth. Supernatants of this strain that were active in disrupting the pH gradient across the membrane were also shown to inhibit growth of L. monocytogenes. In summary, the presented results suggest that the generated sensor strain is a convenient, fast and reliable tool to identify and characterize novel bacteriocins and other compounds that target membrane integrity.

Revisiting the role of cholesterol in regulating the pore-formation mechanism of Vibrio cholerae cytolysin, a membrane-damaging β-barrel pore-forming toxin.

Vibrio cholerae cytolysin (VCC) is a β-barrel pore-forming toxin with potent membrane-damaging cell-killing activity. Previous studies employing the model membranes of lipid vesicles (liposomes) have shown that pore formation by VCC requires the presence of cholesterol in the liposome membranes. However, the exact role of cholesterol in the mode of action of VCC still remains unclear. Most importantly, implication of cholesterol, if any, in regulating the pore-formation mechanism of VCC in the biomembranes of eukaryotic cells remains unexplored. Here, we show that the presence of cholesterol promotes the interaction of VCC with the membrane lipid bilayer, when non-lipid-dependent interactions are absent. However, in the case of biomembranes of human erythrocytes, where accessory interactions are available, cholesterol appears to play a less critical role in the binding step. Nevertheless, in the absence of an optimal level of membrane cholesterol in the human erythrocytes, membrane-bound fraction of the toxin remains trapped in the form of abortive oligomeric assembly, devoid of functional pore-forming activity. Our study also shows that VCC exhibits a prominent propensity to associate with the cholesterol-rich membrane micro-domains of human erythrocytes. Interestingly, mutation of the cholesterol-binding ability of VCC does not block association with the cholesterol-rich membrane micro-domains on human erythrocytes. Based on these results, we propose that the specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin.

1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol Kills Pseudomonas aeruginosa through Extensive Membrane Damage

The ever increasing multidrug-resistance of clinically important pathogens and the lack of novel antibiotics have resulted in a true antibiotic crisis where many antibiotics are no longer effective. Further complicating the treatment of bacterial infections are antibiotic-tolerant persister cells. Besides being responsible for the recalcitrant nature of chronic infections, persister cells greatly contribute to the observed antibiotic tolerance in biofilms and even facilitate the emergence of antibiotic resistance. Evidently, eradication of these persister cells could greatly improve patient outcomes and targeting persistence may provide an alternative approach in combatting chronic infections. We recently characterized 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009), a novel anti-persister molecule capable of directly killing persisters from both Gram-negative and Gram-positive pathogens. SPI009 potentiates antibiotic activity in several in vitro and in vivo infection models and possesses promising anti-biofilm activity. Strikingly, SPI009 restores antibiotic sensitivity even in resistant strains. In this study, we investigated the mode of action of this novel compound using several parallel approaches. Genetic analyses and a macromolecular synthesis assays suggest that SPI009 acts by causing extensive membrane damage. This hypothesis was confirmed by liposome leakage assay and membrane permeability studies, demonstrating that SPI009 rapidly impairs the bacterial outer and inner membranes. Evaluation of SPI009-resistant mutants, which only could be generated under severe selection pressure, suggested a possible role for the MexCD-OprJ efflux pump. Overall, our results demonstrate the extensive membrane-damaging activity of SPI009 and confirm its clinical potential in the development of novel anti-persister therapies.

Membrane-damaging activities of mannosylated ovalbumin are involved in its antibacterial action

Mannosylated ovalbumin (Man-OVA) prepared by modification of carboxyl groups with p-aminophenyl α-d-mannopyranoside shows an increase of net positive charge, which may enhance its binding to bacterial membrane. Thus, we aimed to investigate whether Man-OVA exerts antibacterial activity on Escherichia coli and Staphylococcus aureus via membrane-perturbing effect. Man-OVA inhibited the growth of E. coli and S. aureus, whereas ovalbumin (OVA) did not show any antibacterial activity. Moreover, Man-OVA induced an increase in the membrane permeability of E. coli and S. aureus, which was positively correlated to its bactericidal action. Morphological examination using scanning electron microscopy revealed that Man-OVA disrupted the bacterial membrane integrity. Destabilization of the lipopolysaccharide (LPS) layer and inhibition of lipoteichoic acid (LTA) biosynthesis in the cell wall increased the bactericidal effect of Man-OVA. In contrast to OVA, Man-OVA also induced leakage of bacterial membrane-mimicking liposomes. Color transformation of phospholipid/polydiacetylene membrane assay revealed that the membrane-interaction mode of Man-OVA was distinct from that of OVA. LPS and LTA suppressed the membrane-damaging activity of Man-OVA, whereas an increase in the Man-OVA concentration attenuated the inhibitory action of LPS and LTA. Taken together, our data indicate that the bactericidal activity of Man-OVA depends strongly on its ability to induce membrane permeability.