【作者】 陈安群；

【导师】 田德英；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 目的：探讨新型突变mucA56基因编码蛋白的亚细胞定位,并观察mucA56基因对生物被膜形成的影响,探讨mucA56基因影响生物被膜形成的机制。方法：采用Ncol和XbaI酶切位点的引物,从pMD18T-HR扩增mucA56基因的全长克隆到pMF54构建pCAQ56, XbaI单酶切pPH07载体中的碱性磷酸酶基因全长插入到pCAQ56的开放阅读框中,构建mucA56-phoA融合表达载体pCAQ57。对构建的pCAQ57载体进行PCR鉴定及序列测定,并使用碱性磷酸酶抗体验证融合蛋白的表达。将pCAQ57、pKMG170阴性对照质粒)、pKMG176(阳性对照质粒)分别转化大肠杆菌和铜绿假单胞菌,划线接种在含有碱性磷酸酶作用底物BCIP的LB琼脂平板上,观察各自菌落颜色以评估碱性磷酸酶活性;从pGFPuv质粒中扩增表达增强型绿色荧光蛋白的基因全长,克隆入大肠杆菌-铜绿假单胞菌穿梭质粒(pUCP20),构建一个稳定的铜绿假单胞菌绿色荧光蛋白表达载体(pUCP20/GFPuv),采用电转化的方式将pUCP20/GFPuv导入到铜绿假单胞菌PAOl、PDO300(含经典突变mucA22基因的粘液型PA01同源菌株)、PAOmucA56(含新型突变mucA56基因的粘液型PAO1同源菌株)中,采用改良的流动介质体外培养生物被膜,应用激光共聚焦显微镜观察生物被膜形成第l、3、5天的形态和结构；收集三者生物被膜形成第一、三、五天的生物被膜细菌,提取细菌全蛋白进行蛋白质二维电泳,找出PA01、PAOmucA56表达水平相同而与PDO300表达水平差异的蛋白点,采用基质辅助的激光解吸／电离飞行时间(MALDI-TOF MS)分析差异蛋白质。结果：对构建的融合表达载体pCAQ57进行PCR鉴定及序列测定,结果表明phoA基因正向插入到mucA56的开放阅码框中；Western Blot进一步证实了融合蛋白的表达；以上提示成功构建了mucA56-phoA融合蛋白表达载体pCAQ57。转化了pCAQ57的大肠杆菌和铜绿假单胞菌,以及转化了pKMG170的大肠杆菌和铜绿假单胞菌,在含有碱性磷酸酶作用底物BCIP的LB琼脂平板上,均表现为白色菌落,提示为无活性的碱性磷酸酶活动。而转化了pKMG176的大肠杆菌和铜绿假单胞菌,在含有碱性磷酸酶作用底物BCIP的LB琼脂平板上均表现为蓝色菌落,提示为有活性的碱性磷酸酶活动；激光共聚焦显微镜观察PAOmucA56、PA01、PD0300第1、3、5天生物被膜形态,结果显示：PAOmucA56、PAO1的形成方式为水平铺散,其成熟生物被膜形态为均一的薄膜状,基质覆盖完全,PAOmucA56戎熟生物被膜平均厚度约为30μm,PAO1成熟生物被膜平均厚度约为25μm；PDO300的形成方式为局部堆积,呈山丘状,成熟生物被膜具有很大的异质性,以低的基质覆盖和大的微生物菌落间隔为特点,生物被膜最高厚度约为36μm。生物被膜形成第一天、第三天、第五天的生物被膜细菌全蛋白二维电泳,PAO1和PAOmucA56表达相同而与PDO300差异表达的蛋白点分别有8个、6个、15个。29个蛋白的质谱分析结果表明,有12个蛋白的质谱分析结果可信度不高(MS<66)。筛选出来的17个候选蛋白中,DNA指导的RNA聚合酶p亚单位、谷氨酰胺合成酶、延长因子Tu、DNA指导的RNA聚合酶α亚单位、内肽酶Clp2、延长因子G1、热休克蛋白HtpG、ATP依赖的Clp蛋白ATP结合亚单位clpX、触发因子在PDO300中相对低表达,而30S核糖体蛋白S18、磷酸烯醇丙酮酸羧激酶、丙酮酸脱氢酶复合物二氢硫辛酰赖氨酸基乙酰转移酶、PasP、生物素羧化酶、精氨酸脱亚氨酸、鸟氨酸氨甲酰转移酶、丝氨酸羟甲基转移酶3在PD0300中相对高表达；其中clp蛋白酶家族(内肽酶Clp2和ATP依赖的Clp蛋白ATP结合亚单位clpX)分别在第三天、第五天生物被膜细菌中有PD0300的相对低表达。结论：mucA56基因编码的产物定位在细胞质；藻酸盐可以影响生物被膜结构,而mucA56基因存在藻酸盐以外的途径影响生物被膜的形成；mucA56基因是一个多效调节基因,其调节范围涉及细菌的压力应激、藻酸盐合成等很多方面。clp蛋白酶家族的差异表达可能是PAOmucA56生物被膜形态特殊的重要原因。更多还原

【Abstract】 Objective:To investigate the subcellular localization of the protein encoded by newly found mucA mutation gene (mucA56), observe the influnence of the mucA56 gene on biofilm formation and structure, and further explore the mechanism of the influnence.Methods:mucA56 gene is amlified from pMD18T-HR with Ncol and Xbal restrictive endonuclease site and cloned into pMF54 expression vector, resulting in pCAQ56; alkaline phosphatase gene fragment resulting from Xbal digesting of pPHO7 is fused to the open reading frame (ORF) of pCAQ56, resulting in the mucA56-phoA fusion expression vector pCAQ57; Gene fusion vector pCAQ57 is confirmed first by PCR and sequencing, then by western blot using anti-alkaline phosphatase antibody. pCAQ57, pKMG170 (negative control plasmid), pKMG176(positive control plasmid) are transformed into Ecoli. DH5a and Pseudomonas aeruginosa PAO1, then transformants are cultured on the LB agars containing alkaline phosphatase substrate BCIP, which could evaluate the activity of alkaline phosphatase; A enhanced green fluorescent protein gene is amplified from pGFPuv and cloned into E.coli-P.aeruginosa Shuttle vector pUCP20, resulting in a green fluorescent protein expression vector pUCP20/GFPuv which can label P.aeruginosa; pUCP20/GFPuv are transformed into P. aeruginosa PAO1, PDO300 (the mucoid PAO1 derivative with classic mucA22 mutation) and PAOmucA56 (the mucoid PAO1 derivative with new mucA56 mutation) by electrotransformation. Biofilms with 1-day-aged, three-day-aged and five-day-aged formed by GFP labeled P. aeruginosa are observed by laser scanning confocal microscope. The P.aeruginosa biofilm cells with 1-day-aged, three-day-aged and five-day-aged were collected, total bacterial protein extract of which are then gone through two-dimensional electrophoresis. The protein spots which represent the same expression level between PAO and PAOmucA56 but different from PDO300 are picked up for further identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF MS) analysis. Results:The constructed mucA56-phoA fusion expression vector pCAQ57 is confirmed right by PCR and sequencing,WB further confirmed the expression of hybrid proteins. The colony colour of E. coilDH5a(pCAQ57), PAO1(pCAQ57), E. coliDH5a(pKMG170) and PAO1(pKMG176) on LB plate containing BCIP are all white, indicating no activity for the alkaline phosphatase. Observation of the biofilms with 1-day-old, three-day-old and five-day-old of PAO1,PAOmucA56 and PDO300 by Laser scanning confocal microscope reveal that PAO1 and PAOmucA56 biofilm develope from a uniform monolayer of attached cells into a biofilm characterized by an almost complete substratum coverage and even biomass distribution, the average thickness of the PAO1 and PAOmucA56 biofilm is 30μm and 25μm respectively; PDO300 forms a significantly different biofilm architecture, with attached cells growing exclusively in discrete microcolonies resulting in a low substratum coverage and high structural heterogeneity, the maximum thickness of PDO300 is 36μm; Proteomes of total protein extract of the biofilm bacterial with 1-day-aged, three-day-aged and five-day-aged reveals that the number of the protein spots whose abundance are same between PAO1 and PAOmucA56 but different from PDO300, were 8,6 and 15, respectively. Mass spectrometry results show the reliability of twelve proteins are not high. Among the seventeen candidate proteins, DNA directed RNA polymeraseβsubunit, glutamine synthetase, endopeptidase Clp2, elongation factor Tu, DNA directed RNA polymerase a subunit, elongation factor G1, heat shock protein HtpG, ATP dependent Clp protein ATP binding subunit clpX, trigger factor are relatively down-regulated in PDO300; 30S ribosomal protein S18, Phosphoenolpyruvate carboxykinase, Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, PasP, Biotin carboxylase, Arginine deiminase,Ornithine carbamoyltransferase, Serine hydroxymethyltransferase 3 are relatively up-regulated in PDO300; Especially,one clp protease family (Endopeptidase Clp2 and ATP dependent Clp protein ATP binding subunit clpX)is relatively down-regulated in the third day and fifth day of biofilm bacteria in PDO300, respectively. Conclusion:The MucA56 protein encoded by mucA56 gene is localized in the cytoplasm; Alginate might affect the biofilm formation, however, mucA56 gene influence the biofilm formation in another pathway other than alginate; mucA56 gene is a pleiotropic regulatory gene,which regulate many genes including genes involved in bacterial stress response, alginate synthesis and so on.The differentially expression of clp protease family may be the reason of the special biofilm morphology of PAOmucA56.更多还原