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  • 學位論文

外膜蛋白OprI/OprF調控綠膿桿菌對陽離子胜肽(R3S1)3之感受性

Outer membrane protein, OprI/OprF, modulates the susceptibility of Pseudomonas aeruginosa to cationic peptide, (R3S1)3.

指導教授 : 陳垣崇
共同指導教授 : 廖有地

摘要


抗菌胜肽廣泛存在於植物、昆蟲、兩棲類和哺乳類,在先天性免疫之防禦機制上扮演重要的角色。在本論文中,我們合成一段含正電荷之胜肽-(R3S1)3,對綠膿桿菌有高度專一性的抗菌能力。我們發現綠膿桿菌之外膜蛋白OprI和OprF可和此胜肽結合,若加入過量之重組蛋白rOprI、rOprF、anti-OprI或anti-OprF專一性抗體,則可抑制胜肽之抗菌活性,因此推測外膜蛋白OprI和OprF可以調控綠膿桿菌對抗菌胜肽(R3S1)3之感受性。另外,以螢光和電子顯微鏡觀察顯示,綠膿桿菌在經過胜肽處理之後,細菌細胞膜之通透性顯著增強,外膜上會凸起產生水泡,內外膜分離,細胞質內物質有高度濃縮現象,最後會排出菌體外,而使細胞形成空洞狀。由免疫組織染色法得知,(R3S1)3穿透細菌細胞膜,累積在細胞質內高度濃縮區域,由於(R3S1)3可以和去氧核醣核酸(DNA)或核醣核酸(RNA)結合,因此我們推測此胜肽可與細胞內核酸結合,進而影響細菌之生理功能。 此胜肽(R3S1)3 除了具有殺菌特性外,也能穿透真核細胞HaCaT和HeLa之細胞膜及核膜,並聚集在細胞核某特定區域,但不造成細胞毒性,因此它可能為一具細胞穿透性之胜肽(Cell-penetrating peptide)。本論文有關(R3S1)3透過外膜蛋白OprI及OprF進入細菌,破壞及干擾菌體結構及成份之機制,提供了發展抗菌藥物之重要線索。

並列摘要


Short cationic antimicrobial peptides are widely present in living organisms for innate defense against invading microorganisms. They are generally amphipathic, small and cationic with at least two positive residues. We demonstrated a synthetic cationic peptide, (R3S1)3, possessing antimicrobial activity, especially against Pseudomonas aeruginosa. Our results showed that the outer membrane proteins of P. aeruginosa, OprI and OprF, interact with the peptide. The antimicrobial activity of (R3S1)3 were repressed by excess amounts of rOprI, rOprF, anti-OprI or anti-OprF. The bacterial membrane became permeable, the chromatin condensed in cytosol and blebs formed on outer membrane of bacteria after (R3S1)3 treatment analyzed by TEM. In addition, the (R3S1)3 translocated across the cytoplasmic membrane, localized in the cytosol of P. aeruginosa and bound to intracellular target, like nucleic acids, analyzed by immunohistochemistry. The (R3S1)3 exerted not only antimicrobial activity but also penetrated eukaryotic cell and nuclear membranes. These action mechanism of antimicrobial peptide (R3S1)3 against P. aeruginosa through the bacterial outer membrane proteins, OprI/OprF, provide important clues for development of new antimicrobial agents.

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