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標題: | 運用系統生物學研究細菌抗生素抗性及感受性之機制 Systems biology approaches to reveal the mechanism of antibiotics resistance and susceptibility |
作者: | Yi-Pin Lin 林宜屏 |
出版年 : | 2003 |
學位: | 碩士 |
摘要: | 枯草桿菌素是藉著和細菌胞壁合成必需的八異戊二烯焦磷酸相結合以抑 制細菌生長的常用抗生素。先前已發現八異戊二烯醇激?可造成細菌對枯 草桿菌素的抗藥性。在此論文我們使用系統生物學方法(包括跳躍子基因 破壞及蛋白質體學)來研究細菌對抗生素抗性及感受性之機制。跳躍子基 因破壞可用來篩選對枯草桿菌素具抗性及感受性之變種株。發現包括去掉 yadL ( putative fimbrial protein ) , artP ( ATPase of arginine transporter ) , evgS ( two-component system ) ,b145l ( membrane receptor for ion transport ) , b1121 ( virulence factor) , b1809 ,b1810,ybiW(putative formate acetyltransferase)及yehB ( Pilus usher protein )之變種株對枯草桿菌素 有較高桿受性。而去掉fimA ( major type subunit fimbrin ) ,pqqL(putative zinc peptidase ) ,xylF ( xylose binding protein transport system )及 hsdM ( a DNA methylase)導致較高抗性。這些結果顯示 XylFGH及ArtPQIM 分別可將枯草桿菌素吸入及排出。而 transporter assay 的確證明如此。 YehB是細菌表面纖毛疏密的決定因數,可決定biofilm的形成及對抗生 素的抗藥性。更進一步我們也用蛋白質體學研究得到 biofilm 的形成及 藥性基因活化的關聯。YehB 高度表現的菌種可形成 biofilm ,並?生 較多的 AadAI ( aminoglycoside adenylyltransferase ) , mdh ( malate dehyrogenase ) , ydjA , yehZ( putative transport system permease protein ) 和 wbdC ( mannosyl transferase )及較少的 YbfL(putative receptor ) , YgfD( putative nucleotide-binding protein )和 YgfP。如預期,此株對 kanamycin 呈現較高的抗性乃因其可用 AadA1 修飾此抗生素。 對付細菌細胞壁及細胞膜的抗生素可造成 biofilm 形成。而在枯草桿菌素 處理過的大腸桿菌可分泌費洛蒙到胞外造成 biofilm 形成。根據質譜儀、 核磁共振光譜儀及氣相層析質譜儀得知,此物質為具備豆蔻酸(14 :0)、 棕櫚酸(16 : 0)、棕櫚烯酸(16 : 1)、正十七烷酸(17 : l)及油酸(18 : 1) 及脂肪酸鏈的分子,其分子量為 1221.8 Da 。此大腸桿菌DH5分泌而導 致 biofilm 形成之物質完全不同於以往所發現autoinducers 。在培養基加 入 acetate 可增加此費洛蒙的表現量且可使細菌對抗枯草桿菌素的能力增 強說明 acetate 可能是其生合成之先驅。 為了瞭解細菌對外來抗生素的反應,大腸桿菌被枯草桿菌素處理過及未處 理過的膜蛋白或細胞質蛋白分別跑二維膠。在胞膜上有一未知蛋白 B2636 大量表達。其他蛋白包括 GlnA ,TerZ , Bla LAT-4 , EvgA , ManB , B2339 , DsbA , B2636 , BglG, LysR , YcgE , YihW, HisA , CoaA, YfbU , Cof,YqcD and YihS 在被抗生素處理的細胞表現較多。相反的 BtuE , FliY , FepC , ManA, FfoA , YghO , Fba , Gnd , and DapB 表現較少。 Bacitracin is a commonly used antibiotic that inhibits the bacterial cell wall biosynthesis by binding with undecaprenyl pyrophosphate (UPP) and blocking its dephosphorylation into a lipid carrier. Previous study had demonstrated that undecaprenol kinase confers resistant to bacitracin by supplementing the undecaprenyl phosphate. In the present study, the methods of system biologys (transposon gene knockout and 2-D proteomics technicals) were employed to identify other factors, which affect the toxicity of bacitracin as well as other antibiotics to E. coli. The mutant E. coli strains with a single gene disrupted by transposon were selected for increased bacitracin susceptibility and resistance. The removal of the proteins including yadL (putative fimbrial protein), artP (ATPase of arginine transporter), EvgS (two-component system), b1451 (membrane receptor for ion transport), bl121 (virulence factor), b1809, b1810, ybiW(putative formate acetyltransferase), and yehB (pilus usher protein) was found to cause higher sensitivity to bacitracin. On the other hand, disruption of fimA (major type 1 subunit fimbriae), pqqL (putative zinc peptidase), xylF (xylose binding protein transport system), and hsdM (a DNA methylase) resulted in more resistant strains. The ABC-transporters XyIFGH and ArtPQIM may be involved in the influx and efflux of bacitracin and other antibiotics, respectively. The transporter assay indeed confirmed this conclusion. YehB, the usher protein for pilus assembly, is essential to determine bioflim formation and thus antibiotic resistance. Furthermore, evidences linking bioflim formation and overexpression of antibiotic resistance genes were obtained from 2-D gel proteomic studies. The YehB overexpressed strain with more biofilm formation produced higher level of AadA2 (aminoglycoside adenylyltransferase), Mdh (malate dehydrogenase), YdjA, YehZ (putative transport system permease protein) and WbdC (mannosyl transferase) as well as lower level of YbfL (putative receptor), YgfD (putative nucleotide-binding protein), and YgfP. As expected, this strain showed much higher resistance to kanamycin which can be modified by AdaA2. Furthermore, the antibiotics targeting bacterial cell wall are more likely to induce bioflim formation. Also, a chemical (possibly a pheromone) secreted by the E. coli cells was found to cause formation of bioflim upon bacitracin treatment. Reverse phase silica gel and HPLC purification of the extract from the medium yielded a compound with a molecule mass of 1221.8Da according to mass analysis. Moreover, This compound contains five fatty acid chains, myristic acid, palmitic acid, Palmitoleic acid, heptadecenoic acid and Oleic acid, according to TOF-MS/MS, NMR and GC-MS analysis. This pheromone is structurally different from those autoinducers previously discovered from bacteria. To investigate the bacterial response under stress (antibiotic treatment), the E. coil cells treated with bacitracin at a concentration slightly lower than MIC were collected and the proteins with higher or lower expression level in comparison with the sample from untreated cells were analyzed by using 2-D gel electrophoresis. A putative transcription factor B2636 in the membrane of the treated cells was highly overexpressed. Other proteins including G1nA, TerZ, BlaLAT-4, EvgA, ManB, B2339, DsbA, B2636, Bg1G LysR, YcgE, YihW, HisA, CoaA, YfbU, Cof, YqcD and YihS were expressed at higher quantity and BtuE, FliY, FepC, ManA, FfoA, YgbO, Fba, Gnd, and DapB at lower amount. Their function in response to bacitracin will be further examined. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75442 |
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