Involvement of bacterial membranes in drug resistance

细菌膜参与耐药性

• 批准号: 05304028
• 负责人: NAKAE Taiji
• 金额: $ 12.74万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Co-operative Research (A)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05304028/
• 关键词: drug resistance; bacteria; membrane; permeability; extrusion pump; active afflux; antibiotic target; beta-lactamase; 薬剤; 耐性; 透過性; 排出; 膜蛋白; 抗生物質; 消毒薬; 楽剤耐性; 細菌感染; 多剤耐性; 病院内感染; MRSA; 膜; 緑膿菌; beta-ラクタマーゼ; クローニング

Antibiotic susceptibility of bvacterial cells may be determined by several factors including the outer membrane permiability, active drug efflux, interplay of the antibiotic modifying enzyme or altered target sensitivity with the membrane permiability. This project team focused her studies on these problems.1. Outer membrane permeability and antibiotic susceptibility. (1) We studied role of the Pseudomonas porins on the antibiotic susceptiblity constructing chromosomal deletions of single or muliple porin gene (s). Susceptibility of porin-deletion mutants to most antibiotics tested was unchanged except that all the potein D-deletion mutant showed 8-fold elevated MIC.(2) It was found that high imipenem resistance could be achieved by the interplay of protein D deficiency and beta-lactamase production. In the beta-lactamase induction, it is likely that products of the penicillin inding protein (s) wrer involved.2. We discovered that active antibiotic extrusion from the cells are a major … More factor of antibiotic resistance. (1) We cloned, sequenced and identified genes coded for an antibiotic extrusion machinery (mex gene) in Pseudomonas aeruginosa. (2) Multiple antibiotic sresistance in P.aeruginosa was found to be attributable to high expression of the operon. (3) Amino acid residues responsible for the tetracycline extrusion in Escherichia coli were identified by the cite-directed mutagenesis and that was found to be acidic amino acid residues. (4) Interplay of the antibiotic extrusion system such as nfxB and nfxC with target alteration resulted in high quinolone resistance.3. Problems of antibiotics and disinfectant tesistance Staphylococcus were subject of present study. (1) We studied how the mec A gene was integrated into MRSA chromosome and found that mexA was intgrated at specific sites of the chromosome. For the expression of mec operon, it was discovered that two regulatory genes were involbed. (2) We have screened the genes coded for the disinfectant extrsion proteins and found that all Staphylococcus conerved the gene.Search of homologue gene coded for the disinfectnt extrusion protein was extended to P.aeruginosa and E.coli and found that these bacteria possess qac E hmologue. (3) Structures of membrane damaging pathogenic factors of MRSA,leucocidin and gamma-hemolysine were analyzed and found that these toxins shares one of the subunit proteins. Amino acid residues of this protein responsible for leucocidin and gamma-hemolysine function were determined.This project team studied role of membranes in the antibiotic resistance in bacteria and largly contribute to understanding of the mechaism of antibiotic rsistance in which the membrane function is involved. It was timely to organize this collaborative working team, since past few years were the time that impotance of membrane functions in antibioti resistance was rcognized world wide. Less

细菌细胞的抗生素敏感性可由几个因素决定，包括外膜渗透性、主动药物外排、抗生素修饰酶的相互作用或改变的靶敏感性与膜渗透性。这个项目小组把她的研究集中在这些问题上。外膜通透性和抗生素敏感性。(1)我们研究了假单胞菌孔蛋白对抗生素敏感性的作用，构建了单个或多个孔蛋白基因的染色体缺失。孔蛋白缺失突变体对大多数测试的抗生素的敏感性是不变的，除了所有的potein D-缺失突变体显示出8倍的MIC升高。(2)结果发现，高亚胺培南耐药可以通过蛋白D缺乏和β-内酰胺酶产生的相互作用来实现。在β-内酰胺酶的诱导过程中，可能涉及青霉素结合蛋白的产物。我们发现，从细胞中挤出活性抗生素是一个主要的 ...更多信息 抗生素耐药性因素。(1)我们克隆、测序并鉴定了铜绿假单胞菌抗生素挤出机制（mex基因）的编码基因。(2)铜绿假单胞菌对多种抗生素的耐药性与操纵子的高表达有关。(3)通过定点突变鉴定了大肠杆菌中四环素挤出的氨基酸残基，发现其为酸性氨基酸残基。(4)抗生素外排系统如nfxB和nfxC与靶点改变的相互作用导致高喹诺酮耐药.葡萄球菌对抗生素和消毒剂的耐药性问题是目前研究的主题。(1)我们研究了mec A基因是如何整合到MRSA染色体上的，发现mexA基因整合在染色体的特定位点上。对于mec操纵子的表达，发现涉及两个调控基因。(2)我们对编码消毒剂挤出蛋白的基因进行了筛选，发现所有葡萄球菌都含有该基因，并将编码消毒剂挤出蛋白的同源基因的搜索扩展到铜绿假单胞菌和大肠杆菌，发现这两种细菌都具有qac同源基因。(3)对MRSA膜损伤致病因子、杀白细胞素和γ-溶血素的结构进行了分析，发现它们具有相同的亚基蛋白。确定了该蛋白质中负责杀白细胞素和γ-溶血素功能的氨基酸残基。该项目组研究了膜在细菌抗生素耐药性中的作用，并为理解膜功能参与的抗生素耐药性机制做出了很大贡献。由于近几年来膜功能在抗生素耐药性中的重要性在世界范围内得到了广泛的认识，因此组建这个合作工作组是及时的。少

项目成果

Yamaguchi,A.,T.Samejima and T.Sawai.: "His-257 is a Uniquely important Histidine Residue for Tetracycline/H^+ Antiport Function but Not Mandatory for Full Activity of the Transposon Tn10-Encoded Metal-Tetracycline/H^+ Antiporter." Biochemistry. (in press)

Yamaguchi,A.,T.Samejima 和 T.Sawai.：“His-257 是对四环素/H^ 逆向转运功能特别重要的组氨酸残基，但对于转座子 Tn10 编码的金属四环素/H^ 反向转运蛋白的全部活性不是必需的。

Yoneyama,H.,Nakae,T.: "Mechanism of efficient elimination of protein D2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa." Antimicrob.Agents Chemother.37. 2385-2390 (1993)

Yoneyama, H., Nakae, T.：“有效消除亚胺培南耐药铜绿假单胞菌外膜中蛋白质 D2 的机制。”

Nakae,T.: "Role of membrane permeability in determining antibiotic resistance inPseudomonas aeruginosa." Microbiol.Immunol.39. 211-229 (1995)

Nakae,T.：“膜通透性在确定铜绿假单胞菌抗生素耐药性中的作用。”

Gotoh,N.,Tsujimoto,H.,Poole,K.,Yamagishi,J-I.,and Nishino,T.: "The outer membrane protein OprM of Pseudomonas aeruginosa is encoded by the oprK gene in the mexA-mexB-oprKmultidrug resistance operon." Antimicrob.Agents Chemother.39. 2567-2569 (1995)

Gotoh,N.、Tsujimoto,H.、Poole,K.、Yamagishi,J-I. 和 Nishino,T.：“铜绿假单胞菌的外膜蛋白 OprM 由 mexA-mexB-oprK 多药耐药操纵子中的 oprK 基因编码

Hayashi,T.,Matsumoto,H.,Ohnishi,M.,Yokota,S-I.,Shimoniya,T.,Kageyama,M.,and Terawaki,Y.: "Cytotoxin-converting phages,ΦCTX and PS21,are R pyocin-related phages." FEMS Microbiol.Lett.122. 239-244 (1994)

Hayashi, T.、Matsumoto, H.、Ohnishi, M.、Yokota, S-I.、Shimoniya, T.、Kageyama, M. 和 Terawaki, Y.：“细胞毒素转化噬菌体 ΦCTX 和 PS21 是 R pyocin-相关噬菌体。”FEMS Microbiol. Lett. 122. 239-244 (1994)