分枝杆菌wecA基因的功能研究
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摘要
结核病是严重危害人类健康的最严重的疾病之一,自20世纪80年代起,已经受到控制的结核病疫情再次抬头。结核病的致病菌是结核分枝杆菌(Mycobacterium tuberculosis, Tb),该细菌具有特殊细胞壁结构,对其生存和繁殖极为重要。其核心结构由肽聚糖、聚阿拉伯半乳糖和分枝菌酸组成。其中聚阿拉伯半乳糖与肽聚糖之间靠L-鼠李糖-D-N-乙酰葡糖胺双糖衔接分子连接。衔接双糖是结核分枝杆菌细胞壁极其重要的结构,破坏这一结构,细胞壁的完整性将遭到破坏从而结核分枝杆菌不能生存。
L-鼠李糖-D-N-乙酰葡糖胺双糖衔接分子的合成第一步骤是在一个N-乙酰葡糖胺-1-磷酸转移酶的催化作用下,将GlcNAc-1-P (N-乙酰葡糖胺-1-磷酸)基团转移到C50-P (Decaprenyl phosphate)上形成C50-P-P-GlcNAc。显而易见,在以上的酶促反应中,这个N-乙酰葡糖胺-1-磷酸转移酶起到了非常关键的衔接作用,如果该酶被破坏掉,双糖衔接分子将不能合成。
在大肠杆菌(E. coli)中,WecA酶的功能是作为N-乙酰葡糖胺-1-磷酸转移酶而参与脂多糖(Lipopolysaccharide, LPS)的重要组成成分O-抗原多糖(O-antigenic polysaccharide, O-PS)的合成。BLAST分析表明,E. coli的WecA氨基酸序列与M. tuberculosis WecA氨基酸序列有27%的相似性,提示M. tuberculosis的WecA很可能也具有N-乙酰葡糖胺-1-磷酸转移酶的活性,可以将GlcNAc-P基团转移到C50-P上,再接受鼠李糖基完成双糖衔接分子的合成。
本文实验的目的是：(1)通过在wecA基因缺陷菌株E. coli MV501导入结核分枝杆菌Rv1302以及耻垢分枝杆菌MSMEG_4947基因,验证两个基因编码酶的功能以及利用高效液相和质谱进行酶功能的鉴定。(2)确定wecA基因为结核杆菌生长必需基因。用同源重组方法建立mc。155wecA基因敲除菌株。通过测定wecA基因敲除菌株的生长曲线,研究wecA基因与分枝杆菌生长的关系。利用温度转换实验,造成mc2155wecA基因敲除菌株缺乏WecA,通过比对扫描电镜(SEM)和透射电镜(TEM)结果来研究WecA缺乏对该突变株细菌的形态学以及结构的影响。在含有营救质粒的mc2155 wecA基因敲除菌液中加入不同浓度的衣霉素,通过绘制细菌生长曲线来观察衣霉素对WecA酶功能的抑制作用。(3)WecA酶在不同大肠杆菌宿主中的表达以及酶活性检测。利用表达载体pET16b携带Tb wecA分别转化到不同的大肠杆菌BL21(DE3),C41(DE3)以及ER2566中。表达WecA蛋白并提取上述各转化菌的膜蛋白,进行点杂交以及Western blot分析,并在适合条件下与底物一起进行酶促反应,通过高效液相色谱来检测底物UDP-GlcNAc浓度的变化,从而判断WecA是否在不同大肠杆菌宿主中得到表达。
本文获得以下结果：
1.对wecA基因缺陷菌株E.coli MV501中LPS合成缺陷的补偿以及WecA酶功能的鉴定
将pMD18-Sm wecA质粒(pYJ-1)和pMD18-Tb wecA质粒(pYJ-2)电转化到wecA基因缺陷菌株E.coli MV501中,提取LPS并且进行电泳。以未转化任何质粒的MV501以及转化了pUC 18质粒的MV501作为阴性对照。根据电泳结果,可以看到在转化了pYJ-1和pYJ-2的MV501中出现了清晰的LPS条带,而阴性对照细菌中提取的LPS则没有相应的条带。
提取上述三种质粒转化的E.coli MV501细菌的膜蛋白。将膜蛋白与底物C50-P以及UDP.GlcNAc一起进行孵育,反应上清中UDP.GlcNAc含量的变化通过高效液相以及高效液相-质谱联用仪来检测。检测的结果表明,转化了pYJ-1质粒和pYJ-2质粒的细胞膜蛋白反应体系中,底物UDP.GlcNAc的含量与阴性对照相比有所减少。
2.wecA基因是结核杆菌生长必需基因以及衣霉素对WecA蛋白的抑制作用
(1)wecA基因缺陷菌株mc2155 YJ-2的构建
酶切pUC4K质粒获得Kan抗性基因(KanR)并克隆到pYJ-1质粒中,产生Sm wecA::KanR突变基因。再将突变基因克隆到pPR27-xylE质粒,构建出条件性复制质粒pPR27-xylE-Sm wecA::KanR(pYJ-4).
酶切质粒pYJ-2,获得Tb wecA基因,克隆到pET23b-Phsp60质粒的相应位点,构建出pET23b-Phsp60-Tb wecA(pYJ-5)质粒。酶切pYJ-5,将酶切后的片断Phsp60-Tb wecA克隆到pCG76质粒,构建出pCG76-Tb wecA营救质粒(pYJ-6)。
将pYJ-4电转化到mc2155感受态细菌中,42℃条件下SmwecA::KanR与mc2 155基因组中的Sm wecA发生同源重组。Southern杂交分析显示发生第一次同源重组的mc2155 YJ-1突变菌株有两种同源重组方式,#2,7,8菌株以第一种方式发生同源重组,#3,4,13,1 6菌株以第二种方式发生同源重组。
将pYJ-6营救质粒电转化到mc2155 YJ-1感受态细菌中,用含10%蔗糖的选择性LB琼脂培养基,这种选择压力下,mc2155 YJ-1基因组中的Sm wecA::KanR与Sm wecA发生第二次同源重组。用Southern印迹方法筛选出mc2155 YJ-2突变菌株,即Sm wecA基因敲除菌株。
(2)mc2155 YJ-2生长曲线的测定
测定mc2155 YJ-2突变菌株在30℃和42℃的生长曲线,结果发现该突变菌株在30℃可以生长,但在42℃不生长,说明wecA基因是分枝杆菌生长必需基因。
在随后的温度转换实验中,让mc2155 YJ-2突变菌株在营救质粒可以复制、表达的30℃生长20小时,使之表达一定量Tb WecA蛋白后,提高温度至42℃,使pYJ-6不能复制,影响Tb WecA蛋白的表达。在Tb WecA逐渐缺乏的情况下,测定mc2155 YJ-2突变菌株的生长曲线。结果显示mc2155 YJ-2突变菌株可以在420C继续增殖1-2天,第三天以后细菌的生长受到抑制,菌量开始减少,这进一步说明wecA基因是分枝杆菌生长必需基因。
(3) WecA缺乏对突变菌株mc2155 YJ-2细菌形态的影响
在温度转换实验中,取mc2155 YJ-2突变菌株细菌用扫描电镜观察其细菌形态。结果显示在30℃条件下,该mc2155 YJ-2突变菌株均呈短棒状、轮廓饱满、表面光滑的形态。与野生型对照无明显差异。但在42℃条件下,营救质粒Tb wecA表达受影响致使WecA蛋白缺乏时,一些细菌表面出现明显易见的皱褶、凹陷,细菌之间有相互粘连以及融合,许多细菌的一端开始出现肿胀。生长144小时的细菌轮廓极不规则,细菌肿胀更加厉害,很多细菌表面有大量破损。
透射电镜结果显示,30℃培养生长72小时以及144小时的细菌结构正常,可见完整的细胞外膜,细菌形状规则。在42℃培养72小时后,细胞外膜出现皱褶,细胞膜有脱落。在42℃培养144小时后,细菌形状明显不规则,有胀大的头部和细长的尾部出现,细菌内部溶解并有大量空泡,部分细菌中部出现横隔。
扫描电镜以及透射电镜结果说明WecA蛋白质的缺乏影响细胞壁的合成,使细菌形态和结构发生改变,最终导致细菌死亡。
(4)衣霉素对Tb WecA酶功能的抑制
含有营救质粒的mc2155 YJ-2基因敲除菌株在30℃进行培养24小时,在菌液中加入不同浓度的衣霉素继续培养,每隔24小时进行一次菌液吸光度的测定,培养120小时之后绘制细菌生长曲线,根据细菌生长情况判断衣霉素对WecA蛋白功能的影响。生长曲线表明加入衣霉素的细菌生长受到一定程度的抑制,其抑制程度与衣霉素的浓度呈正相关。
3. Tb WecA酶在不同宿主大肠杆菌中的表达以及酶活性测定
将Tb wecA基因与pET16b载体相连后,质粒转化到不同的大肠杆菌宿主BL21 (DE3), C41 (DE3)以及ER2566菌株中,提取膜蛋白进行SDS-PAGE以及Western blot检测,未见到有明显杂交条带出现,但是点杂交结果显示为阳性。将上述所有膜蛋白与底物C50-P以及UDP-GlcNAc一起进行孵育,上清中UDP-GlcNAc的含量通过高效液相色谱来检测。检测结果表明,反应体系中的底物UDP-GlcNAc与对照相比都有不同程度的减少。
结论：
1.在E. coli wecA基因缺陷菌株MV501中导入携带Sm wecA和TbwecA的质粒,质粒可以表达并补偿E. coli MV501 WecA蛋白缺陷,补偿性合成LPS。初步判断在分枝杆菌中WecA具有N-乙酰葡糖胺-1-磷酸转移酶功能。
2.运用HPLC方法以及LC/MS/MS方法分别检测了在E. coli wecA基因缺陷菌株MV501中表达的Sm WecA和Tb WecA酶活性,并对其酶促反应条件进行了优化。证明了Sm wecA和Tb wecA基因编码的WecA蛋白具有N-乙酰葡糖胺-1-磷酸转移酶功能。
3.以耻垢分枝杆菌mc2155菌株为实验模型,构建了mc2155 wecA敲除突变菌株mc2155 YJ-2。通过研究mc2155 YJ-2在30℃和42℃生长情况,确定Tb wecA基因为分枝杆菌生长必需基因。
4.通过对mc2155 YJ-2在温度转换(从30℃到42℃)实验中生长的研究,进一步证实了wecA基因为结核分枝杆菌生长必需基因。
5.通过扫描电镜和透射电镜观察了mc2155 YJ-2在温度转换实验中细菌形态及结构的变化,证明了WecA缺乏会引起一系列细菌形态变化及结构的改变,最终可能引起细菌死亡。
6.在mc2155 YJ-2细菌中分别加入不同浓度衣霉素进行生长曲线的测定,结果表明衣霉素抑制Tb WecA酶的活性,并且其作用具有浓度依赖性。
7.本研究所获得wecA基因敲除菌株mc2155 YJ-2可作为筛选WecA抑制剂的细菌模型来筛选先导化合物,以便发现特异性高且对人体无毒副作用的抗结核新药。
8.初步尝试在不同大肠杆菌中表达膜蛋白WecA,并运用点杂交方法加以印证。通过HPLC检测酶促反应中底物的减少,再次印证Tb WecA具有N-乙酰葡糖胺-1-磷酸转移酶功能。
本研究结果确定了Tb wecA作为结核杆菌生长必需基因决定着细菌的存亡,并验证了Tb WecA的酶功能,分别用不同方法证实了WecA是N-乙酰葡糖胺-1-磷酸转移酶,参与细胞壁重要结构双糖衔接分子的合成,其酶活性可以被衣霉素所抑制。
未来研究方向：
1.选择合适的载体以及表达菌株,优化WecA的表达条件,实现膜蛋白WecA的高表达以及纯化。
2.在底物存在条件下体外模拟WecA酶促反应过程,用放射自显影或者TLC方法进一步确定WecA酶活性。
3.研究wecA基因敲除菌株的蛋白质组学。从在42℃生长的wecA基因敲除细菌中提取胞浆蛋白进行双向电泳,重复和优化双向电泳条件,并根据双向电泳结果,对感兴趣的蛋白质差异点用胰蛋白酶进行酶解,然后用质谱方法获得肽质谱并与数据库比对,以发现更多的靶点。然后进一步采用分子生物学的方法,克隆这些差异蛋白的基因,鉴定其功能,以期发现wecA基因与其它基因之间是否存在相互调控相互影响的关系。
4.分别提取30℃和42℃生长的wecA基因敲除细菌的细胞壁,用HPLC分析两者的细胞壁糖组分变化。
5.用TLC方法进一步筛选对WecA酶有抑制活性的先导化合物,以期寻找到新型抗结核药物。在此基础上,用本文构建的wecA基因敲除细菌模型进一步鉴定该化合物。
Tuberculosis (TB) is one of the most serious infective desease which threats the worldwide health. Mycobacterium tuberculosis, a species of the genus Mycobacteria is the pathogen of tuberculosis. TB has a certain cell wall structure, which is essential for its existence and reproduction. The mybacterial cell wall core consists of mycolic acids, arabinogalacan (AG) and peptidoglycan (PG). AG is attached to the PG via a linker disaccharide, the disaccharide linker plays so important role through the synthesis of mycobacterial cell wall that the integrality of cell wall will be destructed and the cell can not exist if the disaccharide linker destroyed.
The synthesis of the disaccharide linker is depended on two enzymatic reactions. In the first reaction, C50-P is added to N-acetylglucosamine-P group to form Cso-P-P-GlcNAc by a transferase, then the product accepts a Rha group under the catalysis of WbbL and converts to the final product disaccharide linker D-N-acetylglucosamine-L-rhamnose. Obviously, the enzyme in the first reaction is the key point of disaccharide linker synthesis. But in mycobacteria, this enzyme is not found till now and the gene which encodes this enzyme is uncertain.
In E. coli, the function of WecA is acting as a GlcNAc-1-P transferase to synthesis one of the most important components in LPS:O-PS. The BLAST analysis between WecA of M. tuberculosis and E. coli is showed that the two enzymes have 27% similarity. Based on this BLAST result, WecA of M. tuberculosis is probably has transferase enzyme activity as its homolog WecA of E. coli has. In M. tuberculosis, the function of WecA enzyme has not been clearly identified, but it may possibly have the same catalytic activity as a GlcNAc-1-P transferase and play a necessary role to help the synthesis of disaccharide linker.
The objective of our study are:(1) To introduce firstly pMD18-SmwecA plasmid containing Mycobacterium smegmatis (Sm) wecA gene and pMD18-Tb wecA plasmid containing Mycobacterium tuberculosis (Tb) wecA gene into wecA gene defective strain E. coli MV501 to investigate if WecA in mycobacteria have the similar enzyme activity as E. coli WecA has. Mycobacterial WecA protein function will be detected by HPLC and LC/MS/MS. (2) To generate Mycobacterium smegmatis mc2155 wecA gene knockout strain by homologous recombination strategy and test the essentiality of wecA gene for mycobacterial growth, and then to observe the cellular morphological and structural changes when mc2155 wecA gene knockout strain lacked Tb WecA by scanning electron microscope (SEM) and transmission electron microscope (TEM). Inhibitory function of Tunicamycin at different concentrations to Tb WecA protein in mc2155 YJ-2 (wecA gene knocked out strain) will be tested. (3) To overexpress Tb WecA protein in three different E. coli strains and detect WecA enzyme activity.
Followings are results we got in this study:
1. LPS synthesis of E. coli wecA gene defective strain MV501 was complemented with Sm wecA and Tb wecA gene respectively.
pMD18-Sm wecA (pYJ-1) and pMD18-Tb wecA (pYJ-2) was electroporated into wecA gene defective strain E. coli MV501, then LPS was extracted and electrophoresised. E. coli MV501 electroporated with pUC18 plasmid was used as a control. Only LPS from MV501 (pYJ-1) and MV501 (pYJ-2) cells showed a ladder like pattern characteristic of O side chain material.
The membrane protein of MV501 (pYJ-1) and MV501 (pYJ-2) was extracted and incubated with substrates of C50-P and UDP-GlcNAc, after the reactions have completed, supernatant was separated and detected with HPLC and LC/LC/MS. The analyzing results of HPLC and LC/LC/MS suggested that the substrate of UDP-GlcNAc was decreased in above two reactions compared to the membrane from MV501 (pUC18).
2. Mycobacterium smegmatis wecA gene knock out strain was constructed and essentiality of wecA gene was tested. The inhibitory activity of tunicamycin to Tb WecA enzyme function was detected.
(1) Constrction of mc2155 YJ-2 mutants (wecA gene knockout strains)
pUC4K was digested by BamHI to obtain the kanamycin resistance cassette (KanR). KanR was cloned to Sm wecA in pYJ-1 then subcloned to pPR27-xylE to construct pPR27-xlyE-Sm wecA::KanR (pYJ-4).
Tb wecA gene was cloned to pET23b-Phsp60 to generate pET23b-Phsp60-Tb wecA (pYJ-5), and Phsp60-Tb wecA fragment was cloned to pCG76 yielding pCG76-Tb wecA (pYJ-6). The expression of Tb WecA was controlled by the promoter of heat shock protein from M. bovis BCG. The plasmid pCG76 carries the same temperature-sensitive mycobacterial replication origin as pPR27-xylE.
pYJ-4 was electroporated into wild type Mycobacterium smegmatis mc2155 at 42℃, the single crossover event between Sm wecA-KanR in pYJ-4 and Sm wecA gene in the mc2155 genome occurred and resulted in mc2155 YJ-1 mutant, which carried both Sm wecA and Sm wecA::KanR. There were two homologous recombination pathways identified by Southern hybridization.
Rescue plasmids pCG76-Tb wecA were electroporated into mc2155 YJ-1 and the transformed mc2155 YJ-1 cells were incubated at 30℃on LB agar containing 10% sucrose and appropriate antibiotics. Since sacB gene product (levansucrase) was lethal to mc2155 YJ-1 for the existence of sucrose, the double crossover event between Sm wecA::KanR and Sm wecA of mc2155 YJ-1 genome occurred, and the Sm wecA gene was replaced by Sm wecA::KanR in the presence of pCG76-Tb wecA, resulting in mc2155 YJ-2 mutant.
(2) Growth of mc2155 YJ-2 mutants
The growth curve of mc2155 YJ-2 was detected at 30℃and 42℃. mc2155 YJ-2 grew only at 30℃, but not at 42℃. The results confirmed that Tb wecA gene was essential for mycobacterial growth.
In the following temperature shift experiment, mc2155 YJ-2 were grown at 30℃for 20 h to produce Tb WecA enzyme and then the cells were shifted to 42℃. Growth curves were measured in this Tb WecA lacking condition.
(3) Morphological and structural changes of mc2155 YJ-2 mutants
In temperature shift experiment, certain amount of mc2155 YJ-2 cells was acquired. The Scanning electron micrographs showed irregular surface with a crumpled appearance at 72 h and cell lysis appeared at 144 h, most cells began to swell, as contrasted with the control samples which were cultivated at 30℃.
TEM analysis showed that mc2155 YJ-2 grown at 42℃for 144 h became bigger (in diameter) and exhibited pear shape, compared to mc2155 YJ-2 cells grown at 30℃. The vacuoles also were observed in mc2155 YJ-2 cells grown at 42℃for 144 h.
(4) Inhibitory activity of tunicamycin to Tb WecA enzyme
Tunicamycin with different concentrations was added to mc2155 YJ-2 strain which had been cultured of 24 h under 30℃and absorbance was measured every 24 h until 120 h. From the growth curves we concluded that tunicamycin had inhibitory effects to Tb WecA enzyme.
3. pET16b-Tb wecA (pJY-7) was constructed and expression of Tb WecA in different E.coli strains was investigated.
pET16b-Tb wecA (pJY-7) was constructed and transformed into three E. coli expression strains BL21(DE3), C41(DE3) and ER2566, respectively. The membrane protein was extracted and analyzed by dot blot, SDS-PAGE and Western blot. The expressed Tb WecA protein was detected in three E. coli strains by dot blot. However, the expressed Tb WecA protein could not detected by SDS-PAGE and Western blot. The membrane protein from three strains was prepared and incubated with substrates of C50-P and UDP-GlcNAc. As soon as the reactions completed, the supernatant was separated and detected with HPLC. The analyzing results suggested that the substrate of UDP-GlcNAc was decreased from above reaction compared to the control reaction.
Summary:
1. LPS synthesis of E. coli wecA gene defective strain MV501 was complemented with Sm wecA and Tb wecA gene respectively.
2. The membrane protein of MV501 (pYJ-1) and MV501 (pYJ-2) contain GlcNAc-1-P transferase activity. Therefore, Sm wecA and Tb wecA was the gene to encode GlcNAc-1-P transferase.
3. Mycobacterium smegmatis mc2155 wecA gene knock out strain, YJ-2, was constructed to clarify the essentility of Tb wecA for mycobacterial growth.
4. The growth curves of me2155 YJ-2 after temperature shift further proved that wecA is essential to mycobacterial growth.
5. The cell morphorlogical and stuctural changes of mc2155 YJ-2 were observed by SEM and TEM when lacking Tb WecA protein. The SEM and TEM results showed that lacking WecA caused a serious of changes in cells and made cell death in the end.
6. Tunicamycin with different concentrations could inhibit the growth of mc2155 YJ-2. Therefore, tunicamycin can affect the function of Tb WecA.
7. mc2155 YJ-2 strain can be used a cell model to establish a fast method to screen lead compounds that are potential anti-tuberculosis drugs for further effective tuberculosis therapeutics.
8. The expressed Tb WecA membrane protein in different E. coli strains was detected by dot blot. WecA assay was performed using Tb WecA membrane protein and GlcNAc-1-P transferase activity was detected by HPLC method.
Further studies:
1. To select suitable vector and expression strains and optimize the expression conditions to realize overexpression of Tb WecA membrane protein.
2. To develop a simple and effective TLC method to determine WecA activity.
3. To study proteomics of mc2155 YJ-2 by using two-dimensional electrophoresis. Select differently expressed protein dots of interests according to the results of 2-D electrophoresis. Peptide mass fingerprinting was obtained by mass spectrometry for identifying these differently expressed proteins. The genes of interest were obtained by molecular cloning in order to find out the relationship between wecA gene and other genes.
4. To analyze the structural differences of cell wall between mc2155 YJ-2 and wild type mc2155 using HPLC.
5. To establish a fast and accurate Tb WecA enzyme assay to screen lead compounds that are potential anti-tuberculosis drugs for further effective tuberculosis therapeutics. At the same time, these compounds may be further confirmed by the wecA gene knockout strain as a cell model.
L-鼠李糖-D-N-乙酰葡糖胺双糖衔接分子的合成第一步骤是在一个N-乙酰葡糖胺-1-磷酸转移酶的催化作用下,将GlcNAc-1-P (N-乙酰葡糖胺-1-磷酸)基团转移到C50-P (Decaprenyl phosphate)上形成C50-P-P-GlcNAc。显而易见,在以上的酶促反应中,这个N-乙酰葡糖胺-1-磷酸转移酶起到了非常关键的衔接作用,如果该酶被破坏掉,双糖衔接分子将不能合成。
在大肠杆菌(E. coli)中,WecA酶的功能是作为N-乙酰葡糖胺-1-磷酸转移酶而参与脂多糖(Lipopolysaccharide, LPS)的重要组成成分O-抗原多糖(O-antigenic polysaccharide, O-PS)的合成。BLAST分析表明,E. coli的WecA氨基酸序列与M. tuberculosis WecA氨基酸序列有27%的相似性,提示M. tuberculosis的WecA很可能也具有N-乙酰葡糖胺-1-磷酸转移酶的活性,可以将GlcNAc-P基团转移到C50-P上,再接受鼠李糖基完成双糖衔接分子的合成。
本文实验的目的是：(1)通过在wecA基因缺陷菌株E. coli MV501导入结核分枝杆菌Rv1302以及耻垢分枝杆菌MSMEG_4947基因,验证两个基因编码酶的功能以及利用高效液相和质谱进行酶功能的鉴定。(2)确定wecA基因为结核杆菌生长必需基因。用同源重组方法建立mc。155wecA基因敲除菌株。通过测定wecA基因敲除菌株的生长曲线,研究wecA基因与分枝杆菌生长的关系。利用温度转换实验,造成mc2155wecA基因敲除菌株缺乏WecA,通过比对扫描电镜(SEM)和透射电镜(TEM)结果来研究WecA缺乏对该突变株细菌的形态学以及结构的影响。在含有营救质粒的mc2155 wecA基因敲除菌液中加入不同浓度的衣霉素,通过绘制细菌生长曲线来观察衣霉素对WecA酶功能的抑制作用。(3)WecA酶在不同大肠杆菌宿主中的表达以及酶活性检测。利用表达载体pET16b携带Tb wecA分别转化到不同的大肠杆菌BL21(DE3),C41(DE3)以及ER2566中。表达WecA蛋白并提取上述各转化菌的膜蛋白,进行点杂交以及Western blot分析,并在适合条件下与底物一起进行酶促反应,通过高效液相色谱来检测底物UDP-GlcNAc浓度的变化,从而判断WecA是否在不同大肠杆菌宿主中得到表达。
本文获得以下结果：
1.对wecA基因缺陷菌株E.coli MV501中LPS合成缺陷的补偿以及WecA酶功能的鉴定
将pMD18-Sm wecA质粒(pYJ-1)和pMD18-Tb wecA质粒(pYJ-2)电转化到wecA基因缺陷菌株E.coli MV501中,提取LPS并且进行电泳。以未转化任何质粒的MV501以及转化了pUC 18质粒的MV501作为阴性对照。根据电泳结果,可以看到在转化了pYJ-1和pYJ-2的MV501中出现了清晰的LPS条带,而阴性对照细菌中提取的LPS则没有相应的条带。
提取上述三种质粒转化的E.coli MV501细菌的膜蛋白。将膜蛋白与底物C50-P以及UDP.GlcNAc一起进行孵育,反应上清中UDP.GlcNAc含量的变化通过高效液相以及高效液相-质谱联用仪来检测。检测的结果表明,转化了pYJ-1质粒和pYJ-2质粒的细胞膜蛋白反应体系中,底物UDP.GlcNAc的含量与阴性对照相比有所减少。
2.wecA基因是结核杆菌生长必需基因以及衣霉素对WecA蛋白的抑制作用
(1)wecA基因缺陷菌株mc2155 YJ-2的构建
酶切pUC4K质粒获得Kan抗性基因(KanR)并克隆到pYJ-1质粒中,产生Sm wecA::KanR突变基因。再将突变基因克隆到pPR27-xylE质粒,构建出条件性复制质粒pPR27-xylE-Sm wecA::KanR(pYJ-4).
酶切质粒pYJ-2,获得Tb wecA基因,克隆到pET23b-Phsp60质粒的相应位点,构建出pET23b-Phsp60-Tb wecA(pYJ-5)质粒。酶切pYJ-5,将酶切后的片断Phsp60-Tb wecA克隆到pCG76质粒,构建出pCG76-Tb wecA营救质粒(pYJ-6)。
将pYJ-4电转化到mc2155感受态细菌中,42℃条件下SmwecA::KanR与mc2 155基因组中的Sm wecA发生同源重组。Southern杂交分析显示发生第一次同源重组的mc2155 YJ-1突变菌株有两种同源重组方式,#2,7,8菌株以第一种方式发生同源重组,#3,4,13,1 6菌株以第二种方式发生同源重组。
将pYJ-6营救质粒电转化到mc2155 YJ-1感受态细菌中,用含10%蔗糖的选择性LB琼脂培养基,这种选择压力下,mc2155 YJ-1基因组中的Sm wecA::KanR与Sm wecA发生第二次同源重组。用Southern印迹方法筛选出mc2155 YJ-2突变菌株,即Sm wecA基因敲除菌株。
(2)mc2155 YJ-2生长曲线的测定
测定mc2155 YJ-2突变菌株在30℃和42℃的生长曲线,结果发现该突变菌株在30℃可以生长,但在42℃不生长,说明wecA基因是分枝杆菌生长必需基因。
在随后的温度转换实验中,让mc2155 YJ-2突变菌株在营救质粒可以复制、表达的30℃生长20小时,使之表达一定量Tb WecA蛋白后,提高温度至42℃,使pYJ-6不能复制,影响Tb WecA蛋白的表达。在Tb WecA逐渐缺乏的情况下,测定mc2155 YJ-2突变菌株的生长曲线。结果显示mc2155 YJ-2突变菌株可以在420C继续增殖1-2天,第三天以后细菌的生长受到抑制,菌量开始减少,这进一步说明wecA基因是分枝杆菌生长必需基因。
(3) WecA缺乏对突变菌株mc2155 YJ-2细菌形态的影响
在温度转换实验中,取mc2155 YJ-2突变菌株细菌用扫描电镜观察其细菌形态。结果显示在30℃条件下,该mc2155 YJ-2突变菌株均呈短棒状、轮廓饱满、表面光滑的形态。与野生型对照无明显差异。但在42℃条件下,营救质粒Tb wecA表达受影响致使WecA蛋白缺乏时,一些细菌表面出现明显易见的皱褶、凹陷,细菌之间有相互粘连以及融合,许多细菌的一端开始出现肿胀。生长144小时的细菌轮廓极不规则,细菌肿胀更加厉害,很多细菌表面有大量破损。
透射电镜结果显示,30℃培养生长72小时以及144小时的细菌结构正常,可见完整的细胞外膜,细菌形状规则。在42℃培养72小时后,细胞外膜出现皱褶,细胞膜有脱落。在42℃培养144小时后,细菌形状明显不规则,有胀大的头部和细长的尾部出现,细菌内部溶解并有大量空泡,部分细菌中部出现横隔。
扫描电镜以及透射电镜结果说明WecA蛋白质的缺乏影响细胞壁的合成,使细菌形态和结构发生改变,最终导致细菌死亡。
(4)衣霉素对Tb WecA酶功能的抑制
含有营救质粒的mc2155 YJ-2基因敲除菌株在30℃进行培养24小时,在菌液中加入不同浓度的衣霉素继续培养,每隔24小时进行一次菌液吸光度的测定,培养120小时之后绘制细菌生长曲线,根据细菌生长情况判断衣霉素对WecA蛋白功能的影响。生长曲线表明加入衣霉素的细菌生长受到一定程度的抑制,其抑制程度与衣霉素的浓度呈正相关。
3. Tb WecA酶在不同宿主大肠杆菌中的表达以及酶活性测定
将Tb wecA基因与pET16b载体相连后,质粒转化到不同的大肠杆菌宿主BL21 (DE3), C41 (DE3)以及ER2566菌株中,提取膜蛋白进行SDS-PAGE以及Western blot检测,未见到有明显杂交条带出现,但是点杂交结果显示为阳性。将上述所有膜蛋白与底物C50-P以及UDP-GlcNAc一起进行孵育,上清中UDP-GlcNAc的含量通过高效液相色谱来检测。检测结果表明,反应体系中的底物UDP-GlcNAc与对照相比都有不同程度的减少。
结论：
1.在E. coli wecA基因缺陷菌株MV501中导入携带Sm wecA和TbwecA的质粒,质粒可以表达并补偿E. coli MV501 WecA蛋白缺陷,补偿性合成LPS。初步判断在分枝杆菌中WecA具有N-乙酰葡糖胺-1-磷酸转移酶功能。
2.运用HPLC方法以及LC/MS/MS方法分别检测了在E. coli wecA基因缺陷菌株MV501中表达的Sm WecA和Tb WecA酶活性,并对其酶促反应条件进行了优化。证明了Sm wecA和Tb wecA基因编码的WecA蛋白具有N-乙酰葡糖胺-1-磷酸转移酶功能。
3.以耻垢分枝杆菌mc2155菌株为实验模型,构建了mc2155 wecA敲除突变菌株mc2155 YJ-2。通过研究mc2155 YJ-2在30℃和42℃生长情况,确定Tb wecA基因为分枝杆菌生长必需基因。
4.通过对mc2155 YJ-2在温度转换(从30℃到42℃)实验中生长的研究,进一步证实了wecA基因为结核分枝杆菌生长必需基因。
5.通过扫描电镜和透射电镜观察了mc2155 YJ-2在温度转换实验中细菌形态及结构的变化,证明了WecA缺乏会引起一系列细菌形态变化及结构的改变,最终可能引起细菌死亡。
6.在mc2155 YJ-2细菌中分别加入不同浓度衣霉素进行生长曲线的测定,结果表明衣霉素抑制Tb WecA酶的活性,并且其作用具有浓度依赖性。
7.本研究所获得wecA基因敲除菌株mc2155 YJ-2可作为筛选WecA抑制剂的细菌模型来筛选先导化合物,以便发现特异性高且对人体无毒副作用的抗结核新药。
8.初步尝试在不同大肠杆菌中表达膜蛋白WecA,并运用点杂交方法加以印证。通过HPLC检测酶促反应中底物的减少,再次印证Tb WecA具有N-乙酰葡糖胺-1-磷酸转移酶功能。
本研究结果确定了Tb wecA作为结核杆菌生长必需基因决定着细菌的存亡,并验证了Tb WecA的酶功能,分别用不同方法证实了WecA是N-乙酰葡糖胺-1-磷酸转移酶,参与细胞壁重要结构双糖衔接分子的合成,其酶活性可以被衣霉素所抑制。
未来研究方向：
1.选择合适的载体以及表达菌株,优化WecA的表达条件,实现膜蛋白WecA的高表达以及纯化。
2.在底物存在条件下体外模拟WecA酶促反应过程,用放射自显影或者TLC方法进一步确定WecA酶活性。
3.研究wecA基因敲除菌株的蛋白质组学。从在42℃生长的wecA基因敲除细菌中提取胞浆蛋白进行双向电泳,重复和优化双向电泳条件,并根据双向电泳结果,对感兴趣的蛋白质差异点用胰蛋白酶进行酶解,然后用质谱方法获得肽质谱并与数据库比对,以发现更多的靶点。然后进一步采用分子生物学的方法,克隆这些差异蛋白的基因,鉴定其功能,以期发现wecA基因与其它基因之间是否存在相互调控相互影响的关系。
4.分别提取30℃和42℃生长的wecA基因敲除细菌的细胞壁,用HPLC分析两者的细胞壁糖组分变化。
5.用TLC方法进一步筛选对WecA酶有抑制活性的先导化合物,以期寻找到新型抗结核药物。在此基础上,用本文构建的wecA基因敲除细菌模型进一步鉴定该化合物。
Tuberculosis (TB) is one of the most serious infective desease which threats the worldwide health. Mycobacterium tuberculosis, a species of the genus Mycobacteria is the pathogen of tuberculosis. TB has a certain cell wall structure, which is essential for its existence and reproduction. The mybacterial cell wall core consists of mycolic acids, arabinogalacan (AG) and peptidoglycan (PG). AG is attached to the PG via a linker disaccharide, the disaccharide linker plays so important role through the synthesis of mycobacterial cell wall that the integrality of cell wall will be destructed and the cell can not exist if the disaccharide linker destroyed.
The synthesis of the disaccharide linker is depended on two enzymatic reactions. In the first reaction, C50-P is added to N-acetylglucosamine-P group to form Cso-P-P-GlcNAc by a transferase, then the product accepts a Rha group under the catalysis of WbbL and converts to the final product disaccharide linker D-N-acetylglucosamine-L-rhamnose. Obviously, the enzyme in the first reaction is the key point of disaccharide linker synthesis. But in mycobacteria, this enzyme is not found till now and the gene which encodes this enzyme is uncertain.
In E. coli, the function of WecA is acting as a GlcNAc-1-P transferase to synthesis one of the most important components in LPS:O-PS. The BLAST analysis between WecA of M. tuberculosis and E. coli is showed that the two enzymes have 27% similarity. Based on this BLAST result, WecA of M. tuberculosis is probably has transferase enzyme activity as its homolog WecA of E. coli has. In M. tuberculosis, the function of WecA enzyme has not been clearly identified, but it may possibly have the same catalytic activity as a GlcNAc-1-P transferase and play a necessary role to help the synthesis of disaccharide linker.
The objective of our study are:(1) To introduce firstly pMD18-SmwecA plasmid containing Mycobacterium smegmatis (Sm) wecA gene and pMD18-Tb wecA plasmid containing Mycobacterium tuberculosis (Tb) wecA gene into wecA gene defective strain E. coli MV501 to investigate if WecA in mycobacteria have the similar enzyme activity as E. coli WecA has. Mycobacterial WecA protein function will be detected by HPLC and LC/MS/MS. (2) To generate Mycobacterium smegmatis mc2155 wecA gene knockout strain by homologous recombination strategy and test the essentiality of wecA gene for mycobacterial growth, and then to observe the cellular morphological and structural changes when mc2155 wecA gene knockout strain lacked Tb WecA by scanning electron microscope (SEM) and transmission electron microscope (TEM). Inhibitory function of Tunicamycin at different concentrations to Tb WecA protein in mc2155 YJ-2 (wecA gene knocked out strain) will be tested. (3) To overexpress Tb WecA protein in three different E. coli strains and detect WecA enzyme activity.
Followings are results we got in this study:
1. LPS synthesis of E. coli wecA gene defective strain MV501 was complemented with Sm wecA and Tb wecA gene respectively.
pMD18-Sm wecA (pYJ-1) and pMD18-Tb wecA (pYJ-2) was electroporated into wecA gene defective strain E. coli MV501, then LPS was extracted and electrophoresised. E. coli MV501 electroporated with pUC18 plasmid was used as a control. Only LPS from MV501 (pYJ-1) and MV501 (pYJ-2) cells showed a ladder like pattern characteristic of O side chain material.
The membrane protein of MV501 (pYJ-1) and MV501 (pYJ-2) was extracted and incubated with substrates of C50-P and UDP-GlcNAc, after the reactions have completed, supernatant was separated and detected with HPLC and LC/LC/MS. The analyzing results of HPLC and LC/LC/MS suggested that the substrate of UDP-GlcNAc was decreased in above two reactions compared to the membrane from MV501 (pUC18).
2. Mycobacterium smegmatis wecA gene knock out strain was constructed and essentiality of wecA gene was tested. The inhibitory activity of tunicamycin to Tb WecA enzyme function was detected.
(1) Constrction of mc2155 YJ-2 mutants (wecA gene knockout strains)
pUC4K was digested by BamHI to obtain the kanamycin resistance cassette (KanR). KanR was cloned to Sm wecA in pYJ-1 then subcloned to pPR27-xylE to construct pPR27-xlyE-Sm wecA::KanR (pYJ-4).
Tb wecA gene was cloned to pET23b-Phsp60 to generate pET23b-Phsp60-Tb wecA (pYJ-5), and Phsp60-Tb wecA fragment was cloned to pCG76 yielding pCG76-Tb wecA (pYJ-6). The expression of Tb WecA was controlled by the promoter of heat shock protein from M. bovis BCG. The plasmid pCG76 carries the same temperature-sensitive mycobacterial replication origin as pPR27-xylE.
pYJ-4 was electroporated into wild type Mycobacterium smegmatis mc2155 at 42℃, the single crossover event between Sm wecA-KanR in pYJ-4 and Sm wecA gene in the mc2155 genome occurred and resulted in mc2155 YJ-1 mutant, which carried both Sm wecA and Sm wecA::KanR. There were two homologous recombination pathways identified by Southern hybridization.
Rescue plasmids pCG76-Tb wecA were electroporated into mc2155 YJ-1 and the transformed mc2155 YJ-1 cells were incubated at 30℃on LB agar containing 10% sucrose and appropriate antibiotics. Since sacB gene product (levansucrase) was lethal to mc2155 YJ-1 for the existence of sucrose, the double crossover event between Sm wecA::KanR and Sm wecA of mc2155 YJ-1 genome occurred, and the Sm wecA gene was replaced by Sm wecA::KanR in the presence of pCG76-Tb wecA, resulting in mc2155 YJ-2 mutant.
(2) Growth of mc2155 YJ-2 mutants
The growth curve of mc2155 YJ-2 was detected at 30℃and 42℃. mc2155 YJ-2 grew only at 30℃, but not at 42℃. The results confirmed that Tb wecA gene was essential for mycobacterial growth.
In the following temperature shift experiment, mc2155 YJ-2 were grown at 30℃for 20 h to produce Tb WecA enzyme and then the cells were shifted to 42℃. Growth curves were measured in this Tb WecA lacking condition.
(3) Morphological and structural changes of mc2155 YJ-2 mutants
In temperature shift experiment, certain amount of mc2155 YJ-2 cells was acquired. The Scanning electron micrographs showed irregular surface with a crumpled appearance at 72 h and cell lysis appeared at 144 h, most cells began to swell, as contrasted with the control samples which were cultivated at 30℃.
TEM analysis showed that mc2155 YJ-2 grown at 42℃for 144 h became bigger (in diameter) and exhibited pear shape, compared to mc2155 YJ-2 cells grown at 30℃. The vacuoles also were observed in mc2155 YJ-2 cells grown at 42℃for 144 h.
(4) Inhibitory activity of tunicamycin to Tb WecA enzyme
Tunicamycin with different concentrations was added to mc2155 YJ-2 strain which had been cultured of 24 h under 30℃and absorbance was measured every 24 h until 120 h. From the growth curves we concluded that tunicamycin had inhibitory effects to Tb WecA enzyme.
3. pET16b-Tb wecA (pJY-7) was constructed and expression of Tb WecA in different E.coli strains was investigated.
pET16b-Tb wecA (pJY-7) was constructed and transformed into three E. coli expression strains BL21(DE3), C41(DE3) and ER2566, respectively. The membrane protein was extracted and analyzed by dot blot, SDS-PAGE and Western blot. The expressed Tb WecA protein was detected in three E. coli strains by dot blot. However, the expressed Tb WecA protein could not detected by SDS-PAGE and Western blot. The membrane protein from three strains was prepared and incubated with substrates of C50-P and UDP-GlcNAc. As soon as the reactions completed, the supernatant was separated and detected with HPLC. The analyzing results suggested that the substrate of UDP-GlcNAc was decreased from above reaction compared to the control reaction.
Summary:
1. LPS synthesis of E. coli wecA gene defective strain MV501 was complemented with Sm wecA and Tb wecA gene respectively.
2. The membrane protein of MV501 (pYJ-1) and MV501 (pYJ-2) contain GlcNAc-1-P transferase activity. Therefore, Sm wecA and Tb wecA was the gene to encode GlcNAc-1-P transferase.
3. Mycobacterium smegmatis mc2155 wecA gene knock out strain, YJ-2, was constructed to clarify the essentility of Tb wecA for mycobacterial growth.
4. The growth curves of me2155 YJ-2 after temperature shift further proved that wecA is essential to mycobacterial growth.
5. The cell morphorlogical and stuctural changes of mc2155 YJ-2 were observed by SEM and TEM when lacking Tb WecA protein. The SEM and TEM results showed that lacking WecA caused a serious of changes in cells and made cell death in the end.
6. Tunicamycin with different concentrations could inhibit the growth of mc2155 YJ-2. Therefore, tunicamycin can affect the function of Tb WecA.
7. mc2155 YJ-2 strain can be used a cell model to establish a fast method to screen lead compounds that are potential anti-tuberculosis drugs for further effective tuberculosis therapeutics.
8. The expressed Tb WecA membrane protein in different E. coli strains was detected by dot blot. WecA assay was performed using Tb WecA membrane protein and GlcNAc-1-P transferase activity was detected by HPLC method.
Further studies:
1. To select suitable vector and expression strains and optimize the expression conditions to realize overexpression of Tb WecA membrane protein.
2. To develop a simple and effective TLC method to determine WecA activity.
3. To study proteomics of mc2155 YJ-2 by using two-dimensional electrophoresis. Select differently expressed protein dots of interests according to the results of 2-D electrophoresis. Peptide mass fingerprinting was obtained by mass spectrometry for identifying these differently expressed proteins. The genes of interest were obtained by molecular cloning in order to find out the relationship between wecA gene and other genes.
4. To analyze the structural differences of cell wall between mc2155 YJ-2 and wild type mc2155 using HPLC.
5. To establish a fast and accurate Tb WecA enzyme assay to screen lead compounds that are potential anti-tuberculosis drugs for further effective tuberculosis therapeutics. At the same time, these compounds may be further confirmed by the wecA gene knockout strain as a cell model.
引文
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1. Bayan AD, Dominique ML, Ahmed B. Purification and characterization of the bacterial UDP-GlcNAc:undecaprenyl-phosphate GlcNAc-1-phosphate transferase WecA. J Bacteriol.2008,190:7141-6.
2. Lehrer J, Vigeant KA, Tatar LD, Valvano MA. Functional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide. J Bacteriol.2007,189:2618-28.
3. Amal O. Amer, Miguel A. Valvano. Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine. Microbiology. 2001,147(pt 11):3015-25
4. Amal O. Amer, Miguel A. Valvano. The N-Terminal Region of the Escherichia coli WecA (Rfe) Protein, Containing Three Predicted Transmembrane Helices, Is Required for Function but Not for Membrane Insertion. J Bacteriol.2000, 182(2):498-503
2. Beveridge T J; Davies JA J Bacteriol. Cellular responses of Bacillus subtilis and Escherichia coli to the Gram strain. J Bacterio,1983; 156(2):846-58.
3. Funahara Y, Nikaido H. Asymmetric localization of lipopolysaccharides on the outer membrane of Salmonella typhimurium. J Bacteriol,1980; 141 (3):1463-65.
4. Galloway SM, Raetz CR. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. J Biol Chem,1990; 265(11):6394-402.
5. Imoto M, Yoshimura H, Sakaguchi N, Kusumoto S. Total synthesis of Escherichia coli lipid A. Tetrahedron Lett,1985; 26:1545-1548.
6. Liideritz O, Tanamoto K, Galanos C, McKenzie GR, Brade H, Zahringer U, Rietschel ET, Kusumoto S, Shiba T. Lipopolysaccharides:structural principles and biologic activities. Rev Infect Dis,1984; 6(4):428-31.
7. Kotani S, Takada H, Tsujimoto M, Ogawa T, Takahashi I, Ikeda T, Otsuka K, Shimauchi H, Kasai N, Mashimo J, et al. Synthetic lipid A with endotoxic and related biological activities comparable to those of a natural lipid A from an Escherichia coli re-mutant. J Infect. Immun,1985; 49(1):225-37.
8. Galanos C, Luderitz O, Rietschel ET, Westphal O, Brade H, Brade L, et al. Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur J Biochem,1985; 148(1):1-5.
9. Kotani S, Takada H, Tsujimoto M, Ogawa T, Harada K, Mori Y, Kawasaki A, Tanaka A, Nagao S, Tanaka S. Immunobiologically active lipid A analogs synthesized according to a revised structural model of natural lipid A. Infect Immun,1984; 45(1):293-6.
10. Takada H, Kotani S, Tanaka S, Ogawa T, Takahashi I, Tsujimoto M, Komuro T, Shiba T, Kusumoto S, Kusunose. Structural requirements of lipid A species in activation of clotting enzymes from the horseshoe crab, and the human complement cascade. Eur J Biochem,1988; 175(3):573-80.
11. Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zahringer U, Seydel U, Di Padova F. Bacterial endotoxin:molecular relationships of structure to activity and function. FASEB J,1994; 8:217-25.
12. Rietschel ET, Brade L, Schade U, Seydel U, Zahringer U, Brandenburg K, Helander I, Holst O, Kondo S, Kuhn H.-M, et al. Bacterial lipopolysaccharides: relationship of structure and conformation to endotoxic activity, serological specificity and biological function. Adv Exp Med Biol,1990; 256:81-99.
13. Raetz CRH. Biochemistry of endotoxins. Annu Rev Biochem,1990; 59:129-70.
14. Raetz, CRH. Escherichia coli and Salmonella. In:Niedhardt, FC., editor. Cellular and Molecular Biology. American Society for Microbiology; Washington, D.C.,1996; p.1035-63.
15. Raetz CRH. Bacterial endotoxins:extraordinary lipids that activate eucaryotic signal transduction. J Bacteriol,1993; 175:5745-53.
16. Wyckoff TJO, Raetz CRH, Jackman JE. Antibacterial and anti-inflammatory agents that target endotoxin. Trends Microbiol,1998; 6:154-9.
17. Anderson MS, Raetz CRH. Biosynthesis of lipid A precursors in Escherichia coli. A cytoplasmic acyltransferase that converts UDP-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. J Biol Chem,1987; 262:5159-69.
18. Anderson MS, Bull HS, Galloway SM, Kelly TM, Mohan S, et al. UDP-N-acetylglucosamine acyltransferase of Escherichia coli. The first step of endotoxin biosynthesis is thermodynamically unfavorable. J Biol Chem,1993; 268:19858-65.
19. Wyckoff TJ, Lin S, Cotter RJ, Dotson GD, Raetz CR. Hydrocarbon Rulers in UDP-N-acetylglucosamine Acyltransferases. J Biol Chem,1998; 273:32369-72.
20. Wyckoff TJ, Raetz CRH. The Active Site of Escherichia coli UDP-N-acetylglucosamine Acyltransferase. CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS. J Biol Chem,1999; 274:27047-55.
21. Kelly TM, Stachula SA, Raetz CRH, Anderson MS. The firA gene of Escherichia coli encodes UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. The third step of endotoxin biosynthesis. J Biol Chem,1993; 268:19866-74.
22. Lien E, Means TK, Heine H, Yoshimura A, Kusumoto S, et al. Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide. J Clin Invest,2000; 105:497-504.
23. Poltorak A, Ricciardi-Castagnoli P, Citterio S, Beutler B. Physical contact between lipopolysaccharide and toll-like receptor 4 revealed by genetic complementation. Proc Natl Acad Sci U S A,2000; 97:2163-7.
24. Schnaitman CA, Klena JD. Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol Rev,1993; 57:655-82.
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