| Budget Amount *help |
¥8,400,000 (Direct Cost: ¥8,400,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1998: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥3,500,000 (Direct Cost: ¥3,500,000)
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| Research Abstract |
Pseudomonas aeruginosa is a major pathogen in the hospital that exhibits resistance to many structurally and functionally diverse antibiotics, This multidrug resistance is mainly attributable to a tight outer membrane permeability and expression of the xenobiotic efflux pumps. The MexAB-OprM efflux pump consists of three subunits such as inner membrane protein, MexB, inner membrane associated periplasmic protein, MexA and outer membrane protein, OprM, functioning as the energy dependent efflux pump, the membrane fusion protein and the xenobiotic exit channel, respectively, We studied structure and function of these pump subunit proteins. (I) To acess the role of the subunit proteins in the antibiotic export, we constructed by mean of gene replacement technique the mutant lacking either MexA, MexB, OprM or all combinations of these subunits. The antibiotic susceptibility test of these mutant revealed that the mutants lacking MexA or MexB are equally hypersusceptible to many antibiotics,
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whereas the mutants lacking the OprM subunit are more antibiotic hypersusceptible than the MexA or MixB mutants. (ii) To quantitate the pump function in real time, we developed the method to determine the fluorescent dye accumulation in stead of measuring the antibiotic susceptibility. The dyes accumulation in the cell was proportionally related with the antibiotic susceptibility of the cells. Thus, the measurement of fluorescent dye accumulation could be used as a pump function monitor. (iii) P. aeruginosa encodes MexCD-OprJ pump upon the regulator mutation, the nfxB gene. Since the amino acid sequence of the MexAB-OprM and MexCD-OprJ has high similarity, we tested a possible interchangeability of the pump subunits, expressing the hybrid pump. To do it, OprJ subunit, for instance, was expressed in the host lacking OprM subunit and producing the MexA and MexB subunits. Results of such study revealed that the outer membrane subunit could be substituted, but not the inner membrane subunit. Thus, it became clear that P. aeruginosa expressing more than two pump systems produces the hybrid pumps. (iv) The MexB subunit is assumed to be the energy dependent efflux pump crossing the cytoplasmic membrane several times. However, there is no experimental manifestation to this assumption. We studied the membrane topology of the MexB protein by the reporter gene (phoA ) fusion method to the various length of carboxyl terminal truncated mexB gene. Expression of such fusion genes showed that the alkaline phosphatase activity of the fusion protein, of which fusion-site is faced to the periplasm would be high. On one hand, the enzyme activity would be undetectably low in the hybrid protein, of which reporter gene is located in the cytoplasm. We constructed a total 28 hybrid proteins. The result showed that the MexB protein bears 12 transmembrane domains leaving the amino and carboxyl termini in the cytoplasm. Interestingly, the MexB protein had two large hydrophilic domains containing 311 and 314 amino acid residues. Thus, it was suggested that these two large domains may interact with the MexA and Oprm subunits. Less
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