| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1988: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1987: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
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
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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
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