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Role of the outer membrane of gram-negativebacteria in the intrinsic drug resistance

革兰氏阴性菌外膜在内在耐药性中的作用

基本信息

批准号：
62570197
负责人：
NAKAE Taiji
金额：
$ 1.34万
依托单位：
Tokai University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

The patients having cancer, leukemia, cystic fibrosis etc or the recipients of the organ or cell transplantation are normally under the low immune activity. Such immuncompromised patients often infect to the low virulent gram-negative bacteria such as Pseudomonas aeruginosa. Such opportunistic pathogens generally persist under the extensive antibiotic treatments. Aim of this study is to elucidate the role of the outer membrane barrier to the anibiotic of P. aeruginosa and related gram-negative bacteria.In the study of the outer membrane permeability, the following results were obtained. (i) When the diffusion rates of the uncharged saccharides of different M_r were determined using the liposome membranes containing the purified outer membrane of P. aeruginosa, the proteoliposome only allowed the free diffusion of pentose (M_r, 150) and hexose (M_r, 180). The diffusion rates of methlhexoses and N-acetylglucosamine were about 5% of that of pentose. The diffusion rates of saccharides larg … More er than disaccharides were undetectably low. This permeability of P. aeruginosa outer membrane is substantially lower than that of Escherichia coli. (ii) Since the investigators in other laboratories identified the protein F as the polysaccharide-permeable porin in the P. aeruginosa outer membrane and we were unable to confirm this result, the permeability of the outer membrane of the protein F-deficient mutants was compared with that of the wild type strain. The results showed that the diffusion rates of the permeable solutes as well as the apparent exclusion limits were fully comparable. Both outer membranes showed the presence of only monosaccharide-permeable pores. Determination of the diffusion rates and the MICs of -lactam antibiotics were again identical. The results ruled out the presence of large pore in the outer membrane of P. aeruginosa. (iii) To identify the pore-forming protein(s) in the outer membrane of P. aeruginosa, 7 outer membrane proteins were purified to an apparent homogeneity and their permeability properties were tested using the reconstituted proteoliposomes. Among them, proteins C,D,and E were identified to be the porin that form the identical permeability to the intact and the isolated outer membrane. (iv) Similar studies were carried out in other opportunistic gram-negative pathogens. (a) Alcaligenes faecalis have the small diffusion pores. (b) The porin of the outer membrane of A. faecalis is M_r, 43,000 protein. (c) Unlike P. aeruginosa, A. faecalis produces abundant porin. An anaerobic gram-negative opportunistic pathogen, Bacteroides fragilis was also shown to have smalll diffusion pores. These results contribute to the understanding of the antibiotic resistance of these gram-negative opportunistic pathogens. Less

癌症、白血病、囊性纤维化等患者或器官或细胞移植的接受者通常处于低免疫活性状态。这种免疫功能低下的患者经常感染低致病力的革兰氏阴性菌，如铜绿假单胞菌。这种机会致病菌通常在广泛的抗生素治疗下持续存在。本研究旨在阐明铜绿假单胞菌及其相关革兰阴性菌的外膜屏障作用。(i)用含有纯化的铜绿假单胞菌外膜的脂质体膜测定不同分子量的不带电糖的扩散速率时，脂蛋白体仅允许戊糖（M_r，150）和己糖（M_r，180）自由扩散。甲基己糖和N-乙酰氨基葡萄糖的扩散速率约为戊糖的5%。扩散速率 ...更多信息比二糖低得多。铜绿假单胞菌外膜的这种渗透性显著低于大肠杆菌的渗透性。(ii)由于其他实验室的研究人员鉴定出蛋白F是铜绿假单胞菌外膜中的多糖可渗透孔蛋白，我们无法证实这一结果，因此将蛋白F缺陷型突变体的外膜渗透性与野生型菌株的外膜渗透性进行了比较。结果表明，渗透性溶质的扩散速率以及表观排阻限是完全可比的。两种外膜均显示仅存在单糖可渗透孔。- 内酰胺抗生素的扩散速率和MIC的测定再次相同。结果排除了铜绿假单胞菌外膜中大孔的存在。(iii)为了鉴定铜绿假单胞菌外膜中的孔形成蛋白，将7种外膜蛋白纯化至表观均一性，并使用重构的脂蛋白体测试它们的渗透性。其中，蛋白质C、D和E被鉴定为对完整和分离的外膜形成相同渗透性的孔蛋白。(iv)在其他革兰氏阴性机会致病菌中也进行了类似的研究。(a)粪产碱菌具有较小的扩散孔。(b)A. faecalis为M_r，43，000蛋白。(c)与铜绿假单胞菌不同，A.粪菌产生丰富的孔蛋白。一种厌氧革兰氏阴性条件致病菌，脆弱拟杆菌也显示出具有小的扩散孔。这些结果有助于了解这些革兰阴性机会致病菌的抗生素耐药性。少