Arbing M A, Hanrahan J W, Coulton J W
Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada, H3A 2B4.
J Membr Biol. 2002 Sep 15;189(2):131-41. doi: 10.1007/s00232-002-1008-6.
Changes in amino-acid sequence of the unique pore-forming protein of H. influenzae (OmpP2; porin) have been associated with increased antimicrobial resistance in H. influenzae strains isolated from cystic fibrosis patients. From patients who were subjected to long-term antimicrobial therapy, H. influenzae strains 67d and 69a (patient 27) and strains 77a and 77f (patient 30) were isolated. Strains 67d and 77a were previously shown to have elevated values for minimal inhibitory concentrations of antibiotics compared to strains 69a and 77f. Porins were extracted from all four H. influenzae strains by detergent treatment and purified to homogeneity by ion exchange chromatography. By reconstitution of the clinical Hi porins into planar lipid bilayers, single-channel conductance, ionic selectivity, and voltage-gating characteristics were assessed. Porins 77a and 77f displayed similar single-channel conductance and ionic selectivity. Current-voltage relationships were determined for the different porins: porin 77f displayed substantial voltage gating at both positive and negative polarity; porin 77a gated at negative polarity only. Porins 67d and 69a showed substantial differences in their pore-forming properties: the single-channel conductance of porin 69a was significantly increased (1.05 nS) relative to porin 67d (0.73 nS). Porin 67d was twice as permeable to cations as porin 69a, and at both positive and negative polarities the extent of voltage gating was greater for porin 67d relative to porin 69a. Expression of the porins in an isogenic, porin-deleted H. influenzae background allowed for assessment of the contribution of each porin to the minimum inhibitory concentrations of various antimicrobial compounds. Porin 67d was found to have a decreased susceptibility to the antimicrobials novobiocin and streptomycin. This decreased susceptibility of porin 67d to novobiocin and streptomycin correlates with its decrease in single-channel conductance.
流感嗜血杆菌独特的成孔蛋白（OmpP2；孔蛋白）氨基酸序列的变化与从囊性纤维化患者中分离出的流感嗜血杆菌菌株抗菌耐药性增加有关。从接受长期抗菌治疗的患者中，分离出了流感嗜血杆菌菌株67d和69a（患者27）以及菌株77a和77f（患者30）。与菌株69a和77f相比，先前已显示菌株67d和77a的抗生素最低抑菌浓度值升高。通过去污剂处理从所有四种流感嗜血杆菌菌株中提取孔蛋白，并通过离子交换色谱法纯化至同质。通过将临床流感嗜血杆菌孔蛋白重组到平面脂质双分子层中，评估了单通道电导、离子选择性和电压门控特性。孔蛋白77a和77f表现出相似的单通道电导和离子选择性。测定了不同孔蛋白的电流-电压关系：孔蛋白77f在正负极性下均表现出显著的电压门控；孔蛋白77a仅在负极性下门控。孔蛋白67d和69a在其成孔特性上表现出显著差异：孔蛋白69a的单通道电导（1.05 nS）相对于孔蛋白67d（0.73 nS）显著增加。孔蛋白67d对阳离子的通透性是孔蛋白69a的两倍，并且在正负极性下，孔蛋白67d的电压门控程度相对于孔蛋白69a更大。在同基因、缺失孔蛋白的流感嗜血杆菌背景中表达孔蛋白，可评估每种孔蛋白对各种抗菌化合物最低抑菌浓度的贡献。发现孔蛋白67d对新霉素和链霉素的敏感性降低。孔蛋白67d对新霉素和链霉素敏感性的降低与其单通道电导的降低相关。
