Real-time monitoring ß-lactam/ß-lactamase inhibitor (BL/BLI) mixture towards the bacteria porin pathway at single molecule level.

Multidrug-resistant bacteria are a great concern and a problem that must be addressed. Extended-spectrum ß-lactamases are a common defence mechanism of bacteria to make ß-lactam (BL) antibiotics ineffective. ß-Lactamase inhibitors (BLIs) are consequently designed and are often clinically prescribed with a BL antibiotic to hinder degradation. Current studies focusing on how BL antibiotics or BLIs interact solely with the bacterial outer membrane nanopores (porins) on reaching the periplasmic side using a nanopore-based sensing technique. In electrochemical studies, the bias voltage allows real-time monitoring of BL antibiotics, BLIs and their mixture through the porin pathway at the single-molecule level. Here we consider the most abundant membrane protein from Escherichia coli (i.e. OmpF), purify and reconstitute the membrane protein in an artificial lipid bilayer and then study its ex vivo electrochemical behaviour. We show the piperacillin/tazobactam mixture interacts with OmpF, whereas the substrate interacts under the maximum bandwidth. The power spectrum analysis of the ionic current trace demonstrates the ampicillin/sulbactram mixture requires more energy than ampicillin alone to pass through the porin pathway. Our results demonstrate that clinically relevant combinations (e.g. piperacillin/tazobactam and ampicillin/sulbactam) interact more strongly with OmpF than either the BL antibiotic or the BLI alone. We suggest a quick and relatively cheap screening method to test the ability of BL antibiotics/BLIs to cross the bacterial cellular membrane.

Antibacterial mechanisms of cinnamon and its constituents: A review.

BACKGROUND: In the current healthcare environment, an alarming rise in multi-drug resistant bacterial infections has led to a global health threat. The lack of new antibiotics has created a need for developing alternative strategies. OBJECTIVE: Understanding the antibacterial mechanisms of cinnamon and its constituents is crucial to enhance it as a potential new source of antibiotic. The objective of this review is to provide a compilation of all described mechanisms of antibacterial action of cinnamon and its constituents and synergism with commercial antibiotics in order to better understand how cinnamon and its constituents can collaborate as alternative treatment to multi-drug resistant bacterial infections. METHODS: The relevant references on antibacterial activities of cinnamon and its constituents were searched. Meanwhile, the references were classified according to the type of mechanism of action against bacteria. Relationships of cinnamon or its constituents and antibiotics were also analyzed and summarized. RESULTS: Cinnamon extracts, essential oils, and their compounds have been reported to inhibit bacteria by damaging cell membrane; altering the lipid profile; inhibiting ATPases, cell division, membrane porins, motility, and biofilm formation; and via anti-quorum sensing effects. CONCLUSION: This review describes the antibacterial effects of cinnamon and its constituents, such as cinnamaldehyde and cinnamic acid, against pathogenic Gram-positive and Gram-negative bacteria. The review also provides an overview of the current knowledge of the primary modes of action of these compounds as well as the synergistic interactions between cinnamon or its constituents with known antibacterial agents. This information will be useful in improving the effectiveness of therapeutics based on these compounds.

Novel cationic peptide TP359 down-regulates the expression of outer membrane biogenesis genes in Pseudomonas aeruginosa: a potential TP359 anti-microbial mechanism.

BACKGROUND: Antimicrobial peptides (AMPs) are a class of antimicrobial agents with broad-spectrum activities. Several reports indicate that cationic AMPs bind to the negatively charged bacterial membrane causing membrane depolarization and damage. However, membrane depolarization and damage may be insufficient to elicit cell death, thereby suggesting that other mechanism(s) of action could be involved in this phenomenon. In this study, we investigated the antimicrobial activity of a novel antimicrobial peptide, TP359, against two strains of Pseudomonas aeruginosa, as well as its possible mechanisms of action. RESULTS: TP359 proved to be bactericidal against P. aeruginosa as confirmed by the reduced bacteria counts, membrane damage and cytoplasmic membrane depolarization. In addition, it was non-toxic to mouse J774 macrophages and human lung A549 epithelial cells. Electron microscopy analysis showed TP359 bactericidal effects by structural changes of the bacteria from viable rod-shaped cells to those with cell membrane damages, proceeding into the efflux of cytoplasmic contents and emergence of ghost cells. Gene expression analysis on the effects of TP359 on outer membrane biogenesis genes underscored marked down-regulation, particularly of oprF, which encodes a major structural and outer membrane porin (OprF) in both strains studied, indicating that the peptide may cause deregulation of outer membrane genes and reduced structural stability which could lead to cell death. CONCLUSION: Our data shows that TP359 has potent antimicrobial activity against P aeruginosa. The correlation between membrane damage, depolarization and reduced expression of outer membrane biogenesis genes, particularly oprF may suggest the bactericidal mechanism of action of the TP359 peptide.

Doripenem MICs and ompK36 porin genotypes of sequence type 258, KPC-producing Klebsiella pneumoniae may predict responses to carbapenem-colistin combination therapy among patients with bacteremia.

Treatment failures of a carbapenem-colistin regimen among patients with bacteremia due to sequence type 258 (ST258), KPC-2-producing Klebsiella pneumoniae were significantly more likely if both agents were inactive in vitro, as defined by a colistin MIC of >2 µg/ml and the presence of either a major ompK36 porin mutation (guanine and alanine insertions at amino acids 134 and 135 [ins aa 134-135 GD], IS5 promoter insertion [P = 0.007]) or a doripenem MIC of >8 µg/ml (P = 0.01). Major ompK36 mutations among KPC-K. pneumoniae strains are important determinants of carbapenem-colistin responses in vitro and in vivo.

Electrostatic interactions drive the nonsteric directional block of OmpF channel by La3+.

Ion channels regulate the transport of molecules and the electric signal transduction in living cells by means of complex and even highly sophisticated mechanisms. We focus here on the crucial role that polyvalent ions, well-known modulators of many biological nanosystems, play in ion channel function. In particular, we show that trace amounts of lanthanum are able to block the bacterial porin OmpF, a large biological pore of Escherichia coli wide enough to exchange antibiotics and other larger molecules. The underlying mechanism has a strong directional character: it is sensitive to the sign of the applied voltage and to the side of the blocker addition. We explore these channel features by combining planar lipid bilayer electrophysiology at the single channel level, site-directed mutagenesis, and inductively coupled plasma mass spectrometry (ICP-MS). In contrast to other well-described channel blockers, which seem to occlude the narrower part of the pore, we envisage a nonsteric mechanism based on electrostatic interactions.

Channel blocking of MspA revisited.

Porin A from Mycobacterium smegmatis (MspA) is a highly stable, octameric channel protein, which acts as the main transporter of electrolytes across the cell membrane. MspA features a narrow, negatively charged constriction zone, allowing stable binding of various analytes thereby blocking the channel. Investigation of channel blocking of mycobacterial porins is of significance in developing alternate treatment methods for tuberculosis. The concept that ruthenium(II)quaterpyridinium complexes have the capability to act as efficient channel blockers for MspA and related porins, emerged after very high binding constants were measured by high-performance liquid chromatography and steady-state luminescence studies. Consequently, the interactions between the ruthenium(II) complex RuC2 molecules and MspA, leading to RuC2@MspA assemblies, have been studied utilizing time-resolved absorption/emission, atomic force microscopy, dynamic light scattering, ζ potential measurements, and isothermal titration calorimetry. The results obtained provide evidence for the formation of clusters/large aggregates of RuC2 and MspA. The results are of interest with respect to utilizing prospective channel blockers in porins. The combination of results from conceptually different techniques shed some light onto the chemical nature of MspA-channel blocker interactions thus contributing to the development of a paradigm for channel blocking.

A novel negative regulation mechanism of bacterial outer membrane proteins in response to antibiotic resistance.

Although some outer membrane (OM) proteins involved in antibiotic resistance have been previously reported, the OM proteins regulating chlortetracycline (CTC) resistance are largely unknown. In this study, we employed a subproteomics approach to identify altered OM proteins of Escherichia coli in response to CTC exposure. Upregulation of TolC and downregulation of LamB, FadL, OmpC, OmpT, and OmpW were found in E. coli strains exposed to CTC at a high concentration that was increased suddenly and at a half-minimum inhibitory concentration (MIC) that was kept constant in the culture medium. These changes in the level of protein expression were validated using Western blotting. In addition, the possible roles of these altered proteins and their regulation mechanisms in response to CTC exposure were investigated using genetically modified strains with gene deletion of these altered proteins. It was found that deletion of tolC, fadL, ompC, ompT, or ompW resulted in a decrease in the MICs and survival capabilities of the gene-deleted strains, whereas the absence of lamB led to an improvement of the two abilities. The downregulation of LamB expression in the CTC-resistant E. coli strain and the increased antibiotic resistance in its gene-deleted strain suggested a negative regulation mechanism in E. coli in response to CTC exposure. Meanwhile, the direction of the regulation pattern in response to CTC exposure was different from that in E. coli in response to exposure to other antibiotics. These findings uncover a novel antibiotic-resistant mechanism in which bacteria respond to exposure to antibiotics through alteration of the direction of regulation of OM proteins.

Gonococcal invasion of epithelial cells driven by P.IA, a bacterial ion channel with GTP binding properties.

The neisserial porin P.I is a GTP binding protein that forms a voltage-gated channel that translocates into mammalian cell membranes and modulates host cell signaling events. Here, we report that P.I confers invasion of the bacterial pathogen Neisseria gonorrhoeae into Chang epithelial cells and that this event is controlled by GTP, as well as other phosphorus-containing compounds. Bacterial invasion was observed only for strains carrying the P.IA subtype of porin, which is typically associated with the development of disseminated neisserial disease, and did not require opacity outer membrane proteins, previously recognized as gonococcal invasins. Allelic replacement studies showed that bacterial invasiveness cotransferred with the P.IA (por1A) gene. Mutation of the P.I-associated protein Rmp did not alter the invasive properties. Cross-linking of labeled GTP to the porin revealed more efficient GTP binding to the P.IA than P.IB porin subtype. GTP binding was inhibited by an excess of unlabeled GTP, ATP, and GDP, as well as inorganic phosphate, but not by UTP or beta-glycerophosphate, fully in line with the respective invasion-inhibitory activities observed for these compounds. The P.IA-mediated cellular invasion may explain the more invasive behavior of P.IA strains in the natural infection and may broaden the basis for the development of a P.I-based gonococcal vaccine.

Molecular mechanisms associated with quinolone resistance in Enterobacteriaceae: review and update.

BACKGROUND: Quinolones are broad-spectrum antibiotics, which are used for the treatment of different infectious diseases associated with Enterobacteriaceae. During recent decades, the wide use as well as overuse of quinolones against diverse infections has led to the emergence of quinolone-resistant bacterial strains. Herein, we present the development of quinolone antibiotics, their function and also the different quinolone resistance mechanisms in Enterobacteriaceae by reviewing recent literature. METHODS: All data were extracted from Google Scholar search engine and PubMed site, using keywords; quinolone resistance, Enterobacteriaceae, plasmid-mediated quinolone resistance, etc. RESULTS AND CONCLUSION: The acquisition of resistance to quinolones is a complex and multifactorial process. The main resistance mechanisms consist of one or a combination of target-site gene mutations altering the drug-binding affinity of target enzymes. Other mechanisms of quinolone resistance are overexpression of AcrAB-tolC multidrug-resistant efflux pumps and downexpression of porins as well as plasmid-encoded resistance proteins including Qnr protection proteins, aminoglycoside acetyltransferase (AAC(6')-Ib-cr) and plasmid-encoded active efflux pumps such as OqxAB and QepA. The elucidation of resistance mechanisms will help researchers to explore new drugs against the resistant strains.

Effects of novel antituberculosis agents on OmpF channel activity.

Nanopore forming proteins spanning the outer membrane mediate in the diffusion of hydrophilic chemicals through the hydrophobic bacterial cell wall. In this study, the effects of two novel anti-TB derivatives, ethyl alpha-[5-(5-nitro-2-thienyl)-1,3,4-thiadiazole-2-ylthio] acetates and propyl alpha-[5-(5-nitro-2-thienyl)-1,3,4-thiadiazole-2-ylthio] acetates, on OmpF channel reconstituted in artificial bilayers were evaluated by voltage clamp technique. Surprisingly, ethyl derivative (MIC > or = 6.75 microg/ml) showed no effects on OmpF channel activity but the propyl derivative (MIC=0.39 microg/ml) reduced the channel conductance considerably and changed the gating pattern of the channel. The findings obtained here at molecular level, might shed light on better understanding of the actual mechanism(s) by which the novel anti-TB agents permeate through the cell wall of the Mycobacterium tuberculosis.

Mechanisms of multidrug resistance in Acinetobacter species and Pseudomonas aeruginosa.

Acinetobacter species and Pseudomonas aeruginosa are noted for their intrinsic resistance to antibiotics and for their ability to acquire genes encoding resistance determinants. Foremost among the mechanisms of resistance in both of these pathogens is the production of beta -lactamases and aminoglycoside-modifying enzymes. Additionally, diminished expression of outer membrane proteins, mutations in topoisomerases, and up-regulation of efflux pumps play an important part in antibiotic resistance. Unfortunately, the accumulation of multiple mechanisms of resistance leads to the development of multiply resistant or even "panresistant" strains.

Effects of spontaneous bilayer curvature on influenza virus-mediated fusion pores.

Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a "flat" structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.

The orientation of porin OmpF in the outer membrane of Escherichia coli.

The in vivo orientation of the channel forming porin OmpF from the outer membrane of Escherichia coli was assessed by immunological, biochemical and structural techniques. Porin OmpF exists as a trimer of channels formed by 16 antiparallel beta-strands. These are connected by long hydrophilic loops on one side of the bilayer and short loops or beta-turns on the other. The former constitute the rough side of the porin channel, the latter the smooth side. Epitopes at the cell surface have all been mapped within the long loops, suggesting a rough-side-out orientation of OmpF in the membrane. We analyzed detergent solubilized OmpF trimers, reconstituted 2-D OmpF crystals, OmpF containing outer membranes (sacculi) and intact cells of an E. coli strain overexpressing OmpF. Both solubilized OmpF and OmpF containing sacculi were exposed to proteases, and distinct cleavage sites were identified by protein sequencing. Solubilized OmpF, reconstituted 2-D OmpF crystals and detergent extracted sacculi were tested for their capacity to adsorb colicin N. We used antibodies directed against surface exposed epitopes for immunogold labeling of reconstituted 2-D OmpF crystals and sacculi. The surfaces of intact cells and extracted sacculi were analyzed by electron microscopy and image processing. Finally, a full 3-D reconstruction of negatively stained OmpF containing sacculi revealed the OmpF trimer in its native conformation within the outer membrane. Colicin N and antibody experiments, as well as the 3-D map of the sacculi demonstrated that OmpF exposes the long loops to the extracellular space. In contrast, reconstituted crystalline OmpF vesicles and double layered sheets were found to be in an inside-out conformation, hence hiding colicin or antibody binding epitopes. Two proteinase K cleavage sites were identified, one on a protruding loop and the other inside the channel on the loop penetrating the pore.

Interaction between quinolones antibiotics and bacterial outer membrane porin OmpF.

In these work, we try to establish a relation between the hydrophobicity of some quinolones and their interaction with OmpF. In order to do that, the values of the binding constant of some quinolones of different "generations" with OmpF were determined by UV-visible spectrophotometry and by fluorimetry. Our results show that there is a strong interaction between all the drugs and the protein and that it becomes larger for the last "generation" fluoroquinolones. These results were compared with previous ones obtained for the interaction of these drugs with simpler biomembrane models (liposomes) and it is possible to conclude that some of the quinolones associate preferably with the protein than with these models. This suggests that an interaction drug/porin is, probably, the preferentially used for the latest fluoroquinolones what makes reasonable to believe that a strong affinity for OmpF means a better capacity to transpose the barrier formed by the outer membrane.

Bromoageliferin and dibromoageliferin, secondary metabolites from the marine sponge Agelas conifera, inhibit voltage-operated, but not store-operated calcium entry in PC12 cells.

Two alkaloids isolated from the marine sponge Agelas conifera were tested for interactions with cellular calcium homeostasis. Bromoageliferin and dibromoageliferin reduced voltage-dependent calcium entry in PC12 cells as measured with Fura II as calcium indicator. The half maximal concentration of both alkaloids to reduce voltage-dependent calcium entry was only slightly different: bromoageliferin showed a half maximal concentration of 6.61+/-0.33 microM, dibromoageliferin of 4.44+/-0.59 microM. Removal of calcium from extracellular solution for 10 min leads to an, at least, partial depletion of intracellular calcium stores, which induces a store-operated calcium entry after re-supplementation of calcium to the buffer. The store-operated calcium entry was unchanged by dibromoageliferin at a concentration of 30 microM, which fully blocks voltage-dependent calcium entry. The store-operated calcium entry induced by application of 5 microM thapsigargin was similarly not altered by 30 microM bromoageliferin. Both alkaloids reduce voltage-dependent calcium entry, but not store-operated calcium entry. The inhibition of voltage-operated calcium entry by bromoageliferin is shown in whole-cell patch clamp experiments.

Opposite Effects of Lysophosphatidylethanolamines on Conformation of OmpF-like Porin from Yersinia pseudotuberculosis.

Lysophosphatidyletnolamine (LPE) is one of enigmatic lipids of bacteria. It is generated from major membrane lipid - phosphatidylethanolamine at severe changes of the bacterial growth conditions. Accumulation of this phospholipid in cells of Gram-negative enterobacterium Yersinia pseudotuberculosis results in the enhanced thermostability of OmpF-like porin (YOmpF) from the same bacteria. The respective integral conformational rearrangements may disturb the channel permeability of protein under stress conditions. However, role of fatty acid composition of LPE in this effect remained unclear. Present work demonstrated that the level of unsaturated LPE is 3.5 times higher than saturated one in total LPE of bacterial cells exposed to stress (phenol treatment). Unsaturated 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (MOPE) and saturated LPE 1-palmitoyl-2- hydroxy-sn-glycero-3-phosphoethanolamine (MPPE) oppositely affect the conformation of YOmpF. MOPE increases the protein thermal stability due to more dense packing of monomers in porin and preserves its trimeric form at elevated temperature, while MPPE weakens the contact between monomers and promotes dissociation of the protein.

High-conductance channel induced by the interaction of phage lambda with its receptor maltoporin.

Bacteriophage lambda that binds to liposomes bears its receptor maltoporin (LamB) and is able to inject its DNA into the internal space. During this process, the liposomes are permeabilized, suggesting that a transmembrane channel has formed (Roessner and Ihler (1986) J. Biol. Chem. 261, 386-390). This pore possibly constitutes the pathway used by lambda DNA to cross the membrane. We reconstituted purified LamB from Shigella in liposomes that were incubated with lambda phages. Addition of this mixture to a bilayer chamber resulted in the incorporation in planar bilayers of high-conductance channels whose conductance, kinetics and voltage dependence were totally different from those of maltoporin channels.

Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF.

The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current-voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by approximately 60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.

Specific interaction of the protein-D2 porin of Pseudomonas aeruginosa with antibiotics.

The protein-D2 porin of Pseudomonas aeruginosa is lacking in carbapenem or fluoroquinolone-resistant strains and hence was thought to facilitate the diffusion of these antibiotics. We examined the effect of several antibiotics on the single channel conductivity of protein-D2 in planar lipid bilayers and found that fluoroquinolones and carbapenems at concentrations of around 1 mM caused closure of the protein-D2 channel. Tetracycline, ampicillin, piperacillin, and latamoxef did not exert any detectable effect on the protein-D2 channel activity.

Bacterial resistance to cephalosporins as a function of outer membrane permeability and access to their target.

Resistance of Gram-negative bacteria to cephalosporins, as with other beta-lactam antibiotics, is a function of a combination of outer-membrane permeability, affinity and stability to beta-lactamases, and their activity against target sites (penicillin-binding proteins). Permeation through the outer-membrane is largely governed by the presence and properties of porins, which are water-filled channels facilitating the movement of hydrophilic molecules across the membrane. The properties of porins vary considerably between wild-type bacterial species, and their numbers (and hence the ability of a bacterial cell to exclude the antibiotic) may be reduced in strains with acquired resistance. In the case of cephalosporins, ability to cross the outer-membrane is related to physico-chemical properties such as molecular size, hydrophobicity and the number and charge of ionised groups. Thus, for example, dianionic compounds have in general lower permeability rates than dipolar cephalosporins. These relationships are discussed in detail. The phenotypically expressed susceptibility of a particular bacterial strain to a cephalosporin is brought about by a dynamic combination of permeation, the ability of the agent to resist degradation or binding to the beta-lactamases in the periplasmic space which act upon the relatively low concentration of cephalosporin present there, and target affinity. The interplay of these factors is discussed.