Publication: Chıp based electrophysıology on membrane proteın : ınvestıgatıon of antıbıotıc translocatıon through ompf
Abstract
CHIP BASED ELECTROPHYSIOLOGY on MEMBRANE PROTEIN: INVESTIGATION of ANTIBIOTIC TRANSLOCATION through OmpF Özet Gram-negatif bakteriler, hastane ve kliniklerde dünya genelinde problem oluşturan enfeksiyonların yarısının sebebi olan antibiyotik direnci geliştirmiş bakterilerdir. Gram-negatif bakterinin dış hücre zarı, antibiyotiklerin hedeflerine ulaşmak için geçmeleri gereken ilk savunma hattıdır. Escherichia coli (Gram negatif bakteri) ´nin dış hücre zarında bulunan “genel difüzyon porinleri” (OmpF, OmpC ve PHoE), hücre içine antibiyotik alımı için esas yolak olarak düşünülmektedirler ve bu proteinlerden çözünmüş madde alımları genellikle difüzyon tarafından yönetilir. Bu çalışmada, kanal formlu bir protein olan, dış hücre zarı F proteini (OmpF), yeniden yapılandırma (reconstution) deneylerinde model olarak kullanılmıştırÇalışmanın amacı, antibiyotiklerin OmpF kanalı ile olan yüksek çözünürlüklü etkileşimlerini yapay lipid çift-tabakalı (artificial lipid bilayer) deneylerinde incelemektir. OmpF kanalları, lipozomlar (GUVs) içinde yeniden yapılandırıldığında, bu lipozomlar, proteo-lipozom (proteo-GUVs) olarak adlandırılır. Bu çalışmada, Port-a-Patch, düzlemsel çip bazlı otomatik patch clamp sistem, çözücüsüz-düzlemsel lipid çift katmanlılarını hazırlamak için kullanıldı. Proteo-lipozomların hafif bir vakumla micro-şekilli aralık üzerinde pozisyon aldırılıp, ardından patlatılmasi ile çözücüsüz-düzlemsel lipid çift-tabakalar elde edildi. OmpF´in karakterizasyonu, çözücüsüz-düzlemsel lipid çift-tabakalar deneyleri ile gerçekleştirildi. OmpF´in iletkenliği regresyon analizi ile 4,05±0,02nS (SE, n=3) olarak hesaplandı. Bu çalışmada, dört farklı antibiyotik sınıfının OmpF kanallarından geçisleri izlendi, Ampicillin (ß-lactams), Norfloxacin (Quinolones), Imipenem (Cerbapenems), Cefotaxime (Cephalosporins). Hücre zarına uygulanan voltajın (holding potential) ve antibiyotik konsantrasyonunun antibiyotik geçişleri üzerindeki etkisi incelendi. Antibiyotik moleküllerinin OmpF kanallarından geçişleri sırasında oluşan ilaç-kaynaklı akım dalgalanmaları izlendi. Bu ilaç-kaynaklı akım dalgalanmaları Dwell time analizi kullanılarak incelendi ve antibiyotik translokasyon parametreleri hem tek-kanal (single channel) hemde çoklu-kanal (multi-channel) seviyesinde hesaplandı. Bunun yanında antibiyotik bağlanma kinetikleride hesaplandı. antibiyotik translokasyonu, OmpF, yapay lipid çift-katmanlılar, otomatik patch clamp, Port-a-Patch. CHIP BASED ELECTROPHYSIOLOGY on MEMBRANE PROTEIN: INVESTIGATION of ANTIBIOTIC TRANSLOCATION through OmpF Serap Guinot
Gram-negative bacteria represent half of the number of antibiotic resistant bacteria which are a world-wide problem in hospital and clinic infections. The outer cell membrane of Gram-negative bacteria is the first defense barrier which the antibiotic has to cross. The general diffusion porins (OmpF, OmpC and PHoE) from the outer cell membrane of Escherichia coli (E.coli, Gram-negative bacteria) are considered to be the main pathway for the antibiotic uptake which is generally conducted by diffusion. Outer membrane protein F (OmpF), which is a channel-forming protein, was used as a model protein in this study. The aim of this study was to screen time-resolved interactions of antibiotics with OmpF reconstituted into artificial lipid bilayer and study the kinetics of the antibiotic translocation through OmpF. OmpF channels were reconstituted into GUVs, so-called proteo-GUVs. The Port-a-Patch, a planar chip based automated patch clamp system, was used to form solvent-free planar lipid bilayers by positioning and subsequently bursting of proteo-GUVs by slightly suction on a micro-structured aperture. The characterization of OmpF was performed in the solvent-free planar lipid bilayer experiments. The regression analysis showed a conductance of OmpF of 4,05±0,02nS (SE, n=3). In this study, the translocation of four different antibiotic classes through the OmpF was screened, Ampicillin (ß-lactams), Norfloxacin (Quinolones), Imipenem (Cerbapenems) and Cefotaxime (Cephalosporins). The effect of the transmembrane voltage (holding potential) and the concentration on the antibiotic translocation was investigated. During the translocation of antibiotics through OmpF, drug-induced ion current fluctuations were observed through OmpF. These fluctuations were analyzed using dwell time analysis and translocation parameters were calculated on both single channel and multi-channel levels as well as antibiotic binding kinetics were calculated Key Words: antibiotic translocation, OmpF, artificial lipid bilayers, automated patch clamp, Port-a-Patch.
Gram-negative bacteria represent half of the number of antibiotic resistant bacteria which are a world-wide problem in hospital and clinic infections. The outer cell membrane of Gram-negative bacteria is the first defense barrier which the antibiotic has to cross. The general diffusion porins (OmpF, OmpC and PHoE) from the outer cell membrane of Escherichia coli (E.coli, Gram-negative bacteria) are considered to be the main pathway for the antibiotic uptake which is generally conducted by diffusion. Outer membrane protein F (OmpF), which is a channel-forming protein, was used as a model protein in this study. The aim of this study was to screen time-resolved interactions of antibiotics with OmpF reconstituted into artificial lipid bilayer and study the kinetics of the antibiotic translocation through OmpF. OmpF channels were reconstituted into GUVs, so-called proteo-GUVs. The Port-a-Patch, a planar chip based automated patch clamp system, was used to form solvent-free planar lipid bilayers by positioning and subsequently bursting of proteo-GUVs by slightly suction on a micro-structured aperture. The characterization of OmpF was performed in the solvent-free planar lipid bilayer experiments. The regression analysis showed a conductance of OmpF of 4,05±0,02nS (SE, n=3). In this study, the translocation of four different antibiotic classes through the OmpF was screened, Ampicillin (ß-lactams), Norfloxacin (Quinolones), Imipenem (Cerbapenems) and Cefotaxime (Cephalosporins). The effect of the transmembrane voltage (holding potential) and the concentration on the antibiotic translocation was investigated. During the translocation of antibiotics through OmpF, drug-induced ion current fluctuations were observed through OmpF. These fluctuations were analyzed using dwell time analysis and translocation parameters were calculated on both single channel and multi-channel levels as well as antibiotic binding kinetics were calculated Key Words: antibiotic translocation, OmpF, artificial lipid bilayers, automated patch clamp, Port-a-Patch.