Molecular mechanism of MBX2319 inhibition of Escherichia coli AcrB multidrug efflux pump and comparison with other inhibitors

Efflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus, in silico struc- ture-based analysis is essential for elucidating the interaction between other inhibitors and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps of Enterobacteriaceae species, as well as other known inhibitors (D13-9001, 1-[1-naphthylmethyl]-piperazine, and phenylalanylarginine-ß-naphthyl-amide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a sub- strate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic por- tion of D13-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-ß-naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates

Sox10 contributes to the balance of fate choice in dorsal root ganglion progenitors

The development of functional peripheral ganglia requires a balance of specification of both neuronal and glial components. In the developing dorsal root ganglia (DRGs), these compo- nents form from partially-restricted bipotent neuroglial precursors derived from the neural crest. Work in mouse and chick has identified several factors, including Delta/Notch signal- ing, required for specification of a balance of these components. We have previously shown in zebrafish that the Sry-related HMG domain transcription factor, Sox10, plays an unex- pected, but crucial, role in sensory neuron fate specification in vivo. In the same study we described a novel Sox10 mutant allele, sox10baz1, in which sensory neuron numbers are elevated above those of wild-types. Here we investigate the origin of this neurogenic pheno- type. We demonstrate that the supernumerary neurons are sensory neurons, and that enteric and sympathetic neurons are almost absent just as in classical sox10 null alleles; peripheral glial development is also severely abrogated in a manner similar to other sox10 mutant alleles. Examination of proliferation and apoptosis in the developing DRG reveals very low levels of both processes in wild-type and sox10baz1, excluding changes in the bal- ance of these as an explanation for the overproduction of sensory neurons. Using chemical inhibition of Delta-Notch-Notch signaling we demonstrate that in embryonic zebrafish, as in mouse and chick, lateral inhibition during the phase of trunk DRG development is required to achieve a balance between glial and neuronal numbers. Importantly, however, we show that this mechanism is insufficient to explain quantitative aspects of the baz1 phenotype. The Sox10(baz1) protein shows a single amino acid substitution in the DNA binding HMG domain; structural analysis indicates that this change is likely to result in reduced flexibility in the HMG domain, consistent with sequence-specific modification of Sox10 binding to DNA. Unlike other Sox10 mutant proteins, Sox10(baz1) retains an ability to drive neurogenin1 transcription. We show that overexpression of neurogenin1 is sufficient to produce supernu- merary DRG sensory neurons in a wild-type background, and can rescue the sensory neu- ron phenotype of sox10 morphants in a manner closely resembling the baz1 phenotype. We conclude that an imbalance of neuronal and glial fate specification results from the Sox10 (baz1) protein\u2019s unique ability to drive sensory neuron specification whilst failing to drive glial development. The sox10baz1 phenotype reveals for the first time that a Notch-dependent lat- eral inhibition mechanism is not sufficient to fully explain the balance of neurons and glia in the developing DRGs, and that a second Sox10-dependent mechanism is necessary. Sox10 is thus a key transcription factor in achieving the balance of sensory neuronal and glial fates

Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process

The porin and the permeating antibiotic: A selective diffusion barrier in gram-negative bacteria

Gram-negative bacteria are responsible for a large proportion of antibiotic resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channel

Self-etch Adhesive Systems: A Literature Review

This paper presents the state of the art of self-etch adhesive systems. Four topics are shown in this review and included: the historic of this category of bonding agents, bonding mechanism, characteristics/properties and the formation of acid-base resistant zone at enamel/dentin-adhesive interfaces. Also, advantages regarding etch-and-rinse systems and classifications of self-etch adhesive systems according to the number of steps and acidity are addressed. Finally, issues like the potential durability and clinical importance are discussed. Self-etch adhesive systems are promising materials because they are easy to use, bond chemically to tooth structure and maintain the dentin hydroxyapatite, which is important for the durability of the bonding.261310Brudevold, F., Buonocore, M., Wileman, W., A report on a resin composition capable of bonding to human dentin surfaces (1956) J Dent Res, 35, pp. 846-851Kramer, J., McLan, J.W., Alterations in the staining reactions of dentine resulting from a constituent of a new self-polymerizing resin (1952) Brit Dent J, 93, pp. 150-153Chigira, H., Yukitani, W., Hasegawa, T., Manabe, A., Itoh, K., Hayakawa, T., Debari, K., Hisamitsu, H., Self-etching dentin primers containing Phenyl-P (1994) J Dent Res, 73, pp. 1088-1095Watanabe, I., Nakabayashi, N., Pashley, D.H., Bonding to ground dentin by a phenyl-P self-etching primer (1994) J Dent Res, 73, pp. 1212-1220Van Landuyt, K.L., Snauwaert, J., De Munck, J., Peumans, M., Yoshida, Y., Poitevin, A., Systematic review of the chemical composition of contemporary dental adhesives (2007) Biomaterials, 28, pp. 3757-3785Tay, F.R., King, N.M., Chan, K., Pashley, D.H., How can nanoleakage occur in self-etching adhesive systems that demineralize and infiltrate simultaneously? 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Bonding effectiveness of a new ‘multi-mode’ adhesive to enamel and dentine (2012) J Dent, 40, pp. 475-484Perdigao, J., Loguercio, A.D., (2014) Universal Or Multi-Mode Adhesives: Why and How? J Adhes Dent, 16, pp. 193-194Van Meerbeek, B., Peumans, M., Poitevin, A., Mine, A., Van Ende, A., Neves, A., Relationship between bond-strength tests and clinical outcomes (2010) Dent Mater, 26, pp. e100-e121Moszner, N., Salz, U., Zimmermann, J., Chemical aspects of selfetching enamel-dentin adhesives: A systematic review (2005) Dent Mater, 21, pp. 895-910Perdigao, J., Lopes, M.M., Gomes, G., In vitro bonding performance of self-etch adhesives (2008) Ii--Ultramorphological Evaluation. Oper Dent, 33, pp. 534-549Van Landuyt, K.L., Kanumilli, P., De Munck, J., Peumans, M., Lambrechts, P., Van Meerbeek, B., Bond strength of a mild self-etch adhesive with and without prior acid-etching (2006) J Dent, 34, pp. 77-85Nazari, A., Shimada, Y., Sadr, A., Tagami, J., Pre-etching vs. Grinding in promotion of adhesion to intact enamel using self-etch adhesives (2012) Dent Mater J, 31, pp. 394-400Yoshida, Y., Nagakane, K., Fukuda, R., Nakayama, Y., Okazaki, M., Shintani, H., Comparative study on adhesive performance of functional monomers (2004) J Dent Res, 83, pp. 454-458Koshiro, K., Sidhu, S.K., Inoue, S., Ikeda, T., Sano, H., New concept of resindentin interfacial adhesion: The nanointeraction zone (2006) J Biomed Mater Res B Appl Biomater, 77, pp. 401-408Lima, G.S., Ogliari, F.A., Da Silva, E.O., Ely, C., Demarco, F.F., Carreno, N.L., . 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pH Modulation of Efflux Pump Activity of Multi-Drug Resistant Escherichia coli: Protection During Its Passage and Eventual Colonization of the Colon

BACKGROUND:Resistance Nodulation Division (RND) efflux pumps of Escherichia coli extrude antibiotics and toxic substances before they reach their intended targets. Whereas these pumps obtain their energy directly from the proton motive force (PMF), ATP-Binding Cassette (ABC) transporters, which can also extrude antibiotics, obtain energy from the hydrolysis of ATP. Because E. coli must pass through two pH distinct environments of the gastrointestinal system of the host, it must be able to extrude toxic agents at very acidic and at near neutral pH (bile salts in duodenum and colon for example). The herein described study examines the effect of pH on the extrusion of ethidium bromide (EB). METHODOLOGY/PRINCIPAL FINDINGS:E. coli AG100 and its tetracycline induced progeny AG100(TET) that over-expresses the acrAB efflux pump were evaluated for their ability to extrude EB at pH 5 and 8, by our recently developed semi-automated fluorometric method. At pH 5 the organism extrudes EB without the need for metabolic energy (glucose), whereas at pH 8 extrusion of EB is dependent upon metabolic energy. Phe-Arg beta-naphtylamide (PAbetaN), a commonly assumed inhibitor of RND efflux pumps has no effect on the extrusion of EB as others claim. However, it does cause accumulation of EB. Competition between EB and PAbetaN was demonstrated and suggested that PAbetaN was preferentially extruded. A K(m) representing competition between PAbetaN and EB has been calculated. CONCLUSIONS/SIGNIFICANCE:The results suggest that E. coli has two general efflux systems (not to be confused with a distinct efflux pump) that are activated at low and high pH, respectively, and that the one at high pH is probably a putative ABC transporter coded by msbA, which has significant homology to the ABC transporter coded by efrAB of Enterococcus faecalis, an organism that faces similar challenges as it makes its way through the toxic intestinal system of the host

Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibitiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack