结核分枝杆菌RmlA和Rv0228的药物靶标研究
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摘要
结核病(Tuberculosis, TB)是严重危害人类健康的传染病。近年来多耐药TB、广泛耐药TB的不断出现和TB/AIDS疫情的加重，使得新一代抗结核药物的开发势在必行。以特异靶标为基础的化合物筛选是药物开发的主要策略之一。我们可以针对已知的药物靶标，建立高通量筛选的方法从化合物库中筛选抑制剂；也可以在结核分枝杆菌(Mycobacterium tuberculosis)中探寻新的靶标，验证它们对细菌生长或感染的必需性，从而为开发具有全新作用机制的抗结核药物奠定基础。
α-D-葡萄糖-1-磷酸胸苷转移酶(RmlA)与其它三个酶(RmlB, RmlC和RmlD)共同催化了分枝杆菌细胞壁中鼠李糖的活性供体：dTDP-鼠李糖的合成。鼠李糖是细胞壁核心结构的重要组成成分之一，缺乏RmlA蛋白的分枝杆菌会因细胞壁合成受阻而无法生长。因此，RmlA是已被确定的、可用于抗结核药物开发的潜在靶标。
Rv0228是经计算机系统分析预测的、抗结核药物的候选靶点。Rv0228对于结核分枝杆菌生长的必需性尚未通过实验验证,其基因功能也尚不明确。
本论文的实验目的是：(1)为已确定的药物靶标RmlA建立快速有效的、可用于高通量筛选抑制剂的酶活性测定方法。利用此方法确定M. tuberculosis RmlA的酶促反应动力学特征和常数，包括初速度范围、最适温度、最适pH值、最适Mg~(2+)浓度以及Km和Vmax值。(2)验证候选的药物靶标Rv0228基因对分枝杆菌生长的必需性。利用Mycobacterium smegmatis mc~2155作为实验模式菌，以同源重组的方法敲除Rv0228的同源基因MSMEG_0319，建立MSMEG_0319基因敲除菌珠，作为Rv0228生长必需性研究的模型。通过研究MSMEG_0319基因敲除菌珠的生长情况，分析Rv0228基因对结核分枝杆菌生长的必需性。(3)初步探索候选的药物靶标Rv0228的基因功能。利用生物信息学方法对Rv0228序列和结构进行分析，预测Rv0228的功能范围。利用温度转换实验，获取一定量的MSMEG_0319基因敲除菌，作为Rv0228功能研究的模型。观察MSMEG_0319基因敲除菌在MSMEG_0319缺失时的形态变化和细胞壁糖组分变化，从而确定MSMEG_0319是否与分枝杆菌细胞壁的代谢相关。利用多种蛋白表达系统在大肠杆菌和耻垢分枝杆菌中表达Rv0228蛋白，筛选出高效表达Rv0228蛋白的菌珠，为获得一定量的Rv0228蛋白纯品和最终鉴定Rv0228的功能奠定基础。
本文获得如下结果：
1. RmlA酶活性方法的建立和酶促反应动力学分析
(1) RmlA酶蛋白的表达纯化：利用pET16b表达载体在大肠肝菌BL21(DE3)中表达了结核分枝杆菌RmlA蛋白，并利用His-Ni~(2+)亲和层析对RmlA蛋白进行了纯化。SDS-PAGE和Western blot检测结果显示：纯化的RmlA酶蛋白分子量大小正确，纯度良好。
(2) RmlA酶蛋白的活性测定：利用传统的高效液相层析(HPLC)方法对纯化的RmlA蛋白进行活性鉴定，结果显示纯化的RmlA酶蛋白具有α-D-葡萄糖-1-磷酸胸苷转移酶活性，催化底物dTTP和D-Glc-1-P生成了产物dTDP-D-Glc和PPi。
(3)高通量RmlA酶活性测定方法的建立：在RmlA酶促反应体系中偶联焦磷酸酶，将产物PPi分解为Pi，最终用孔雀石绿显色剂显色。结果显示，此方法可以有效检测RmlA酶蛋白活性。方法学评价结果表明，此方法样品/空白信号比大于2，Z’值为0.7，可用于RmlA酶蛋白抑制剂的高通量筛选。
(4)结核分枝杆菌RmlA的酶促反应动力学研究：利用上述建立的孔雀石绿方法测定了结核分枝杆菌RmlA酶促反应动力学特征和常数。结果显示：RmlA酶促反应的最适温度为37℃，最适pH值为7.5，最适Mg~(2+)浓度为5mM。RmlA对底物dTTP的Km值为0.020±0.004mM，Vmax值为0.003±0.0003mM/min； RmlA对底物D-Glc-1-P的Km值为0.069±0.0050mM，Vmax值为0.002±0.0001mM/min。
(5) RmlA抑制剂的筛选：利用孔雀石绿方法对23种天然产物进行了筛选。结果显示，23种天然产物对结核分枝杆菌RmlA无抑制作用。
2. Rv0228对分枝杆菌生长必需性的研究
(1) MSMEG_0319基因敲除菌珠的建立
将Kan抗性基因(kan~R)插入到MSMEG_0319基因中，产生突变基因MSMEG_0319::kan~R。再将此突变基因克隆到pPR27-xylE载体中，构建出温度敏感的条件复制质粒pPR27-xylE-MSMEG_0319::kan~R(pMS-3)。
将Rv0228基因克隆pET23b-Phsp60载体中，然后再将Phsp60-Rv0228片段克隆到pCG76载体中，构建出温度敏感的营救质粒pCG76-Phsp60-Rv0228(pMS-6)。
将pMS-3电转化至mc~2155感受态细胞中，在温度和抗生素的选择性压力下，pMS-3上的突变基因MSMEG_0319::kan~R与mc~2155基因组上的MSMEG_0319发生第一次同源重组，突变基因MSMEG_0319::kan~R整合至mc~2155的基因组中。Southern blot筛选出7个第一次同源重组正确的突变菌mc~2155MS-1。将营救质粒转化至mc~2155MS-1中，在蔗糖的选择性压力下，mc~2155MS-1基因组自身发生第二次同源重组，剔除正常的MSMEG_0319基因，只保留突变基因MSMEG_0319::kan~R。Southern blot筛选出7个第二次同源重组正确的突变菌mc~2155MS-2，即为MSMEG_0319基因敲除菌珠。
(2) MSMEG_0319基因敲除菌珠生长曲线的绘制
以MSMEG_0319基因敲除菌珠作为Rv228基因必需性研究的模型，测定MSMEG_0319基因敲除菌株在30C和42C培养时的生长曲线，结果显示MSMEG_0319基因敲除菌株在30C可以生长，在42C条件下不能生长；然而，对照菌(携带pCG76质粒的mc~2155)在30C和42C条件下均可正常生长。此结果表明Rv0228是分枝杆菌生长的必需基因。
3.初步探索Rv0228基因的功能
(1) Rv0228的生物信息学分析：利用基因信息的数据库查看Rv0228基因组上相邻基因的功能；利用序列比对分析搜寻Rv0228同源蛋白，查看它们的功能及种属来源。结果显示：Rv0228在基因组中存在于单独的操纵子中，相邻基因多与细胞壁代谢相关；Rv0228在结核分枝杆菌及放线菌门的某些菌属中具有同源基因，在人类细胞、其它原核生物以及放线菌门的链霉菌中没有同源基因。总结这些信息，推测Rv0228基因的功能可能与结核分枝杆菌细胞壁肽聚糖以外的结构代谢相关。
(2)获取MSMEG_0319基因敲除菌作为Rv0228功能研究的模型：利用温度转换实验，先将MSMEG_0319基因敲除菌置于30℃培养20小时，允许其中的营救质粒复制并表达Rv0228蛋白以补偿缺失的MSMEG_0319基因功能，然后将培养温度升高至42℃，此时营救质粒不再复制和表达Rv0228蛋白，随着培养时间的延长，敲除菌内的Rv0228蛋白逐渐消耗，整个细菌也逐渐表现出缺乏MSMEG_0319基因功能产生的形态学和细胞组分的变化。利用此方法我们收集到了一定量MSMEG_0319蛋白缺失的MSMEG_0319基因敲除菌敲除菌，作为Rv0228功能研究的模型。
(3) MSMEG_0319基因敲除菌形态的观察：利用扫描电镜观察温度转换实验获得的MSMEG_0319基因敲除菌，结果显示，敲除菌在转换至42C生长72小时后出现形态改变，局部膨大、大小不一、表面出现皱褶；144小时后，MSMEG_0319基因敲除菌细菌形态改变更加明显，细菌形态不规则，细胞表面出现大量刺状突起，极不光滑。同时，持续在30C生长72小时和144小时的MSMEG_0319基因敲除菌形态正常，与野生型mc~2155无明显差别。利用透射电镜观察温度转换实验获得的MSMEG_0319基因敲除菌，结果显示，敲除菌在转换至42C生长144小时时，细胞壁不再完整，出现明显破损，并可观察到胞质溢出。扫描电镜和透射电镜的结果表明，Rv0228基因对分枝杆菌的生长至关重要，其功能可能与细胞壁合成有关。
(4) MSMEG_0319基因敲除菌细胞壁糖组分含量分析：提取温度转换实验获得的MSMEG_0319基因敲除菌和野生型mc~2155的细胞壁核心结构mAGP，利用HPLC测定聚阿拉伯糖半乳糖层中半乳糖和肽聚糖层中胞壁酸的含量。结果显示，MSMEG_0319基因敲除菌中半乳糖与胞壁酸的比值明显低于野生型mc~2155。此结果说明，Rv0228的基因功能可能与分枝杆菌细胞壁聚半乳糖的形成直接或间接相关。
(5) R0228蛋白的表达：构建了pET29b-Rv0228, pET16b-Rv0228, pBAD-Rv0228-C和pBAD-Rv0228-N表达质粒，在大肠杆菌中诱导表达Rv0228蛋白；同时构建了pVV16-Rv0228表达质粒，在耻垢分枝杆菌中表达Rv0228蛋白。Dot blot检测结果显示，携带pET16b-Rv0228的E. coli C41(DE3)和E. coli ER2566菌珠以及携带pBAD-RV0228-N的E. coli TOP10菌珠可以高效表达结核分枝杆菌Rv0228蛋白。
结论：
1.利用pET16b表达载体在E. coli BL21(DE3)中高效表达了结核分枝杆菌RmlA酶蛋白，纯化后的RmlA酶蛋白具有α-D-葡萄糖-1-磷酸胸苷转移酶活性。
2.通过偶联焦磷酸酶和使用孔雀石绿显色，建立了快速有效且稳定可靠的、可用于高通量筛选RmlA抑制剂的RmlA酶活性检测方法(孔雀石绿法)。
3.利用孔雀石绿法确定了RmlA酶促反应动力学特征和常数，包括最适温度、最适pH、最适Mg~(~(~(2+)))浓度，以及RmlA酶对反应底物的Km和Vmax值。
4.以耻垢分枝杆菌mc~2155作为实验模式菌，利用同源重组的方法敲除了结核分枝杆菌候选靶标Rv0228的同源基因：MSMEG_0319，建立了MSMEG_0319基因敲除菌珠，作为Rv0228生长必需性研究的模型。通过观察MSMEG_0319基因敲除菌珠的生长情况，确定了Rv0228是分枝杆菌生长的必需基因。
5.通过生物信息学方法对Rv0228的功能进行分析预测，确定了Rv0228的功能可能与结核分枝杆菌细胞壁肽聚糖以外的结构代谢相关。
6.通过温度转换(从30℃到42℃)实验，获得了一定量的MSMEG_0319基因敲除菌，作为Rv0228基因功能研究的模型。利用扫描电镜和透射电镜观察了MSMEG_0319基因敲除菌在缺失MSMEG_0319蛋白功能时的形态变化，并通过HPLC分析了它们细胞壁中半乳糖和胞壁酸含量比值的变化，从而确定了Rv0228的功能可能与结核分枝杆菌细胞壁中聚半乳糖的形成直接或间接相关。
7.利用不同的表达载体在大肠杆菌和耻垢分枝杆菌中表达Rv02228蛋白，筛选得到了3个高效表达Rv0228蛋白的大肠杆菌菌珠：携带pET16b-Rv0228的E. coliC41(DE3)菌珠、携带pET16b-Rv0228的E. coli ER2566菌珠、以及携带pBAD-Rv0228-N的E. coli TOP10菌珠。
未来的研究方向：
1.对于已知的药物靶标RmlA可开展如下工作：
(1)利用孔雀石绿显色的方法，从小分子化合物库中筛选RmlA酶蛋白抑制剂。
(2)优化RmlA酶蛋白的表达条件，获得更大量的RmlA纯化蛋白进行RmlA的结构研究，根据RmlA酶蛋白的结构设计或挑选合适的小分子化合物进行RmlA抑制作用的测试。
2.对于候选靶标Rv0228可开展如下工作：
(1)利用MSMEG_0319基因敲除菌珠作为功能研究的模型，通过测定细胞壁中更多单糖成分如阿拉伯糖、鼠李糖、N-乙酰葡糖胺的含量变化，从而分析Rv0228的具体功能。
(2)利用Rv0228高效表达菌珠在大体积培养基中大量表达Rv0228蛋白，提取细菌膜组分并纯化Rv0228蛋白，同时建立酰基转移酶的酶活性测定方法，为Rv0228功能的最终确定奠定基础。
Tuberculosis (TB) remains one of the deadliest threats to public health. The casesof multi-drug resistant TB, extensive-drug resistant TB as well as co-infection of M.tuberculosis with HIV are increasing fast in recent years. There is an urgent need fornew TB drugs. Target-based approach is one of the main strategies in TB drugdevelopment. For the targets that have been identified by experiments, high-throughputscreening of inhibitors from compound libraries can be performed with a suitable assay.For target candidates, their essentiality for mycobacterial growth should be confirmedfirst of all.
D-glucose-1-phosphate thymidylyltransferase (RmlA) together with other threeenzymes, RmlB, RmlC and RmlD, catalyze the formation of dTDP-L-rhamnose, a sugardonor providing L-rhamnosyl residue in the synthesis of M. tuberculosis cell wall. Theprevious study on RmlA essentiality has proved RmlA as a potential target for TB drugdevelopment.
M. tuberculosis Rv0228is a target candidate for TB drug development predictedthrough computational methods. Its essentiality and function for mycobacterial growthare still unknown.
The objectives of this study are:(1) to develop a rapid enzyme assay forpotential drug target RmlA which is suitable for high-throughput screening of inhibitors,and using this assay, to determine the kinetic properties of M. tuberculosis RmlAincluding initial velocity, optimal temperature, optimal pH, the effect of Mg~(2+)andkinetic parameters;(2) to test the essentiality of target candidate Rv0228formycobacterial growth. Using M. smegmatis as a model organism, MSMEG_0319(theortholog of Rv0228) gene knockout strain will be constructed via homologousrecombination method. Rv0228’s essentiality for mycobacterial growth will beconfirmed through growing MSMEG_0319knockout strain at different temperatures;(3) to analyze the function of target candidate Rv0228primarily. The function of Rv0228will be first predicted through bioinformatic analysis and further studied usingMSMEG_0319knockout strain as a model strain. An amount of MSMEG_0319knockout cells will be acquired through temperature shift experiment, and whether thefunction of Rv0228is related with the cell wall metabolism will be confirmed bycellular morphology observation and cell wall sugar analysis of MSMEG_0319knockout cells. For further function study of Rv0228, the expression of Rv0228proteinin Escherichia coli and M. smegmatis with different expression systems will also betried in this study.
Followings are results we obtained in this study:
1. Development of a colorimetric assay and kinetic analysis for M. tuberculosisRmlA.
(1) Expression and purification of M. tuberculosis RmlA: RmlA protein with afused his-tag at N-terminus was expressed in E. coli BL21(DE3) cells and purified byNi~(2+)affinity chromatography. SDS-PAGE and Western blot analysis confirmed that thepurified RmlA with an expected molecular weight of33.67kD and a perfecthomogeneity.
(2) HPLC analysis of M. tuberculosis RmlA activity: The enzyme activity of thepurified RmlA was detected by HPLC analysis. The result confirmed that the purifiedRmlA had the D-glucose-1-phosphate thymidylyltransferase activity, which catalyzedsubstrate dTTP and Glc-1-P converting to dTDP-D-Glc and PPi.
(3) Development of a colorimetric RmlA assay for high-throughput screening ofM. tuberculosis RmlA inhibitors: In this assay, pyrophosphatase was coupled tocatalyze the product PPi of RmlA reaction to Pi, and Pi molecules were finally detectedby the malachite green reagent. The results showed RmlA catalytic reaction had anapparent color change from yellow-green to blue-green. The assay had adequatesensitivity and reproducibility with the ratio of signal to background (S/B) above2.0and the Z’ value about0.70statistically, which was suitable for high-throughputscreening of M. tuberculosis RmlA inhibitors.
(4) Kinetic study of M. tuberculosis RmlA: Using the colorimetric assay, thekinetic properties of M. tuberculosis RmlA was determined. The results showed M.tuberculosis RmlA had the relative maximal activity in the buffer of pH7.5containing5mM Mg~(2+)at the temperature of37C. The Km and Vmax against dTTP were0.020±0.004mM and0.003±0.0003mM/min respectively, and against D-Glc-1-P were0.069 ±0.0050mM and0.002±0.0001mM/min respectively.
(5) RmlA inhibitors screening: Twenty-three natural products were screenedby the colorimetric assay and none of them was found to inhibit the activity ofM. tuberculosis RmlA.
2. The essentiality study of M. tuberculosis Rv0228for mycobacterial growth
(1) Construction of MSMEG_0319gene knockout strain
The kanamycin resistant gene (kan~R) was inserted into M. smegmgtis MSMEG_0319gene, resulting in a mutant MSMEG_0319gene (MSMEG_0319::kan~R).
MSMEG_0319::kan~Rfragment was then cloned into pPR27-xylE plasmid, yielding a conditional replication plasmid pPR27-xylE-MSMEG_0319::kan~R(pMS-3), which is sensitive to temperature.
M. tuberculosis Rv0228gene was cloned into pET23b-Phsp60plasmid resultingin a plasmid pET23b-Phsp60-Rv0228. Phsp60-Rv0228was then cloned into pCG76plasmid, yielding a rescue plasmid pCG76-Phsp60-Rv0228(pMS-6), which is alsosensitive to temperature.
pMS-3plasmid was elctroporated to M. smegmatis mc~2155competent cells. In theselective pressure of temperature and antibiotics, the first homologous recombinationoccurred between MSMEG_0319::kan~Rfragment in pMS-3plasmid and MSMEG_0319gene in mc~2155genome, thus MSMEG_0319::kan~Rfragment was integrated intomc~2155genome. Seven mc~2155MS-1mutant strains with correct homologousrecombination were screened by Southern blot analysis, and one of them waselectroporated with pMS-6plasmid. In the selective pressure of sucrose, the secondhomologous recombination occurred within the genome of mc~2155MS-1. The normalMSMEG_0319gene was deleted from the mc~2155mutant genome and only the mutantMSMEG_0319(MSMEG_0319::kan~R) was reserved. Seven mc~2155MS-2mutantstrains, i.e. MSMEG_0319knockout strains, were confirmed by Southern blot analysis.
(2) Growth of MSMEG_0319knockout strains at different temperatures
As the model strain for studying the essentiality of Rv0228for mycobacterialgrowth, MSMEG_0319knockout strain was grown at both30℃and42℃. The growthcurve showed that they could grow at30℃but not at42℃, while the mc~2155carryingpCG76plasmid as a control could grow at both30℃and42℃. The result demonstratedthat Rv0228was essential for mycobacterial growth.
3. The functional analysis of M. tuberculosis Rv0228
(1) Bioinformatic analysis of Rv0228: The information of Rv0228and its neighboring genes in M. tuberculosis genome was understood in GenBank and TBdatabases, and the homologous proteins of Rv0228were confirmed through BLASTanalysis. The result showed Rv0228located in M. tuberculosis genome without formingan operon with other open reading frames, and its neighboring genes were mostinvolved in cell wall biosynthesis. The orthologs exist in mycobacteria and someactinobacterial bacteria, but not found in human and other prokaryotic organisms. It wasconcluded from these information that Rv0228was probably involved in cell wall(except for peptidoglycan) biosynthesis.
(2) Acquirement of MSMEG_0319gene knockout cells as a model for functionalstudy of Rv0228. In the temperature shift experiment, MSMEG_0319knockout cellswere grown at30℃for20h, then the cells were transferred to a42℃incubator andgrown at42℃. The Rv0228protein expressed at30℃allowed the cells to grow at42℃for a certain period, however, with the Rv0228protein reduced gradually, the effects oflack of MSMEG_0319protein on the cells emerged. With this method, a certain amountof MSMEG_0319knockout cells were acquired for further function study of Rv0228.
(3) Morphology of MSMEG_0319knockout cells: MSMEG_0319knockout cellsgrown at42℃for72h and144h in the temperature shift experiment were observed viaSEM. The cells significantly bulged at72h and some cells exhibited an irregularsurface and were even lysed at144h. TEM analysis of the MSMEG_0319knockoutcells grown at42℃for144h found that the cell wall ruptured and intracellular contentoverflowed. In contrast, the MSMEG_0319knockout cells grown at30℃exhibitednormal shape and integrated cell wall structure as wild type mc~2155both in SEM andTEM. These results again confirmed that Rv0228was essential for mycobacterialgrowth, and indicated that the function of Rv0228related to the cell wall biosynthesis.
(4) Sugar analysis of cell wall in MSMEG_0319knockout cells: Themycolyl-arabinogalactan-peptidoglycan (mAGP) complex, the core structure of cellwall, of the MSMEG_0319knockout cells in the temperature shift experiment wasprepared and the contents of galactose (Gal) and muramic acid (MuAc) were measuredby HPLC. The result showed that the ratio of Gal to MuAc decreased in MSMEG_0319knockout cells compared with the wild type mc~2155. The result indicated that thefunction of Rv0228was involved in the formation of galactan in mycobacterial cell walldirectly or indirectly.
(5) Expression of Rv0228protein: The expression plasmids pET29b-Rv0228,pET16b-Rv0228, pBAD-Rv0228-C and pBAD-Rv0228-N were constructed to express Rv0228protein in E. coli, and pVV16-Rv0228was constructed to express Rv0228protein in M. smegmatis. Dot blot analysis showed that pET16b-Rv0228/E. coliC41(DE3), pET16b-Rv0228/E. coli ER2566and pBAD-RV0228-N/E. coli TOP10canexpress Rv0228protein effectively.
Followings are conclusions we obtained from this study:
1. M. tuberculosis RmlA protein was expressed in E. coli BL21(DE3) cells andpurified with Ni~(2+)affinity chromatography.
2. A colorimetric assay, which was coupled with pyrophosphatase and detectedwith malachite green reagent, was established to detect RmlA activity rapidly andsteadily. It was suitable for high-throughput screening of M. tuberculosis RmlAinhibitors.
3. The kinetic properties of M. tuberculosis RmlA including optimal temperature,optimal pH, the effect of Mg~(2+)and kinetic parameters Km and Vmax were determinedwith the established colorimetric assay.
4. M. smegmatis MSMEG_0319gene knockout strain was constructed via homologous recombination strategy. It was a model strain for studying the essentiality of M. tuberculosis Rv0228gene (a target candidate) for mycobacterial growth. Through observing the growth of MSMEG_0319knockout strains at different
temperatures, Rv0228gene was confirmed essential for mycobacterial growth.
5. The function of Rv0228was predicted probably involved in the biosynthesis ofmycobacterial cell wall structure except for peptidoglycan through bioinformaticanalysis.
6. MSMEG_0319knockout cells were acquired via temperature shift experiment,which could be used for function study of Rv0228. The morphology changes ofMSMEG_0319knockout cells were observed via SEM and TEM, and the content ratiochange of Gal to MuAc in the cell wall of MSMEG_0319knockout cells was analyzedby HPLC. The result demonstrated that Rv0228was involved in the formation ofgalactan in mycobacterial cell wall directly or indirectly.
7. Though expressing Rv0228protein with different vectors in different host cells,three E. coli strains pET16b-Rv0228/E. coli C41(DE3), pET16b-Rv0228/E. coliER2566and pBAD-Rv0228-N/E. coli TOP10, which could express Rv0228proteineffectively were obtained.
Followings are further studies for the potential target RmlA:(1) Screening ofRmlA inhibitors from chemical compound libraries with the colorimetric assay established in this study.(2) Structural analysis of purified M. tuberculosis RmlAprotein for further structure-based screening of inhibitors or TB drug design.
Followings are further studies for target candidate Rv0228:(1) Identify thefunction of Rv0228using MSMEG_0319knockout strain as model strain. The contentof other monosaccharide such as D-arabinose, L-rhamnose or N-acetylglucosamine inthe cell wall of MSMEG_0319knockout cells can be detected to confirm the functionof Rv0228.(2) Express Rv0228protein in large volume and purify Rv0228proteinthrough Ni~(2+)affinity chromatography, and establish enzyme assay of acyltransferasemeanwhile. These works will also be helpful for identification of Rv0228’s functionfinally.
α-D-葡萄糖-1-磷酸胸苷转移酶(RmlA)与其它三个酶(RmlB, RmlC和RmlD)共同催化了分枝杆菌细胞壁中鼠李糖的活性供体：dTDP-鼠李糖的合成。鼠李糖是细胞壁核心结构的重要组成成分之一，缺乏RmlA蛋白的分枝杆菌会因细胞壁合成受阻而无法生长。因此，RmlA是已被确定的、可用于抗结核药物开发的潜在靶标。
Rv0228是经计算机系统分析预测的、抗结核药物的候选靶点。Rv0228对于结核分枝杆菌生长的必需性尚未通过实验验证,其基因功能也尚不明确。
本论文的实验目的是：(1)为已确定的药物靶标RmlA建立快速有效的、可用于高通量筛选抑制剂的酶活性测定方法。利用此方法确定M. tuberculosis RmlA的酶促反应动力学特征和常数，包括初速度范围、最适温度、最适pH值、最适Mg~(2+)浓度以及Km和Vmax值。(2)验证候选的药物靶标Rv0228基因对分枝杆菌生长的必需性。利用Mycobacterium smegmatis mc~2155作为实验模式菌，以同源重组的方法敲除Rv0228的同源基因MSMEG_0319，建立MSMEG_0319基因敲除菌珠，作为Rv0228生长必需性研究的模型。通过研究MSMEG_0319基因敲除菌珠的生长情况，分析Rv0228基因对结核分枝杆菌生长的必需性。(3)初步探索候选的药物靶标Rv0228的基因功能。利用生物信息学方法对Rv0228序列和结构进行分析，预测Rv0228的功能范围。利用温度转换实验，获取一定量的MSMEG_0319基因敲除菌，作为Rv0228功能研究的模型。观察MSMEG_0319基因敲除菌在MSMEG_0319缺失时的形态变化和细胞壁糖组分变化，从而确定MSMEG_0319是否与分枝杆菌细胞壁的代谢相关。利用多种蛋白表达系统在大肠杆菌和耻垢分枝杆菌中表达Rv0228蛋白，筛选出高效表达Rv0228蛋白的菌珠，为获得一定量的Rv0228蛋白纯品和最终鉴定Rv0228的功能奠定基础。
本文获得如下结果：
1. RmlA酶活性方法的建立和酶促反应动力学分析
(1) RmlA酶蛋白的表达纯化：利用pET16b表达载体在大肠肝菌BL21(DE3)中表达了结核分枝杆菌RmlA蛋白，并利用His-Ni~(2+)亲和层析对RmlA蛋白进行了纯化。SDS-PAGE和Western blot检测结果显示：纯化的RmlA酶蛋白分子量大小正确，纯度良好。
(2) RmlA酶蛋白的活性测定：利用传统的高效液相层析(HPLC)方法对纯化的RmlA蛋白进行活性鉴定，结果显示纯化的RmlA酶蛋白具有α-D-葡萄糖-1-磷酸胸苷转移酶活性，催化底物dTTP和D-Glc-1-P生成了产物dTDP-D-Glc和PPi。
(3)高通量RmlA酶活性测定方法的建立：在RmlA酶促反应体系中偶联焦磷酸酶，将产物PPi分解为Pi，最终用孔雀石绿显色剂显色。结果显示，此方法可以有效检测RmlA酶蛋白活性。方法学评价结果表明，此方法样品/空白信号比大于2，Z’值为0.7，可用于RmlA酶蛋白抑制剂的高通量筛选。
(4)结核分枝杆菌RmlA的酶促反应动力学研究：利用上述建立的孔雀石绿方法测定了结核分枝杆菌RmlA酶促反应动力学特征和常数。结果显示：RmlA酶促反应的最适温度为37℃，最适pH值为7.5，最适Mg~(2+)浓度为5mM。RmlA对底物dTTP的Km值为0.020±0.004mM，Vmax值为0.003±0.0003mM/min； RmlA对底物D-Glc-1-P的Km值为0.069±0.0050mM，Vmax值为0.002±0.0001mM/min。
(5) RmlA抑制剂的筛选：利用孔雀石绿方法对23种天然产物进行了筛选。结果显示，23种天然产物对结核分枝杆菌RmlA无抑制作用。
2. Rv0228对分枝杆菌生长必需性的研究
(1) MSMEG_0319基因敲除菌珠的建立
将Kan抗性基因(kan~R)插入到MSMEG_0319基因中，产生突变基因MSMEG_0319::kan~R。再将此突变基因克隆到pPR27-xylE载体中，构建出温度敏感的条件复制质粒pPR27-xylE-MSMEG_0319::kan~R(pMS-3)。
将Rv0228基因克隆pET23b-Phsp60载体中，然后再将Phsp60-Rv0228片段克隆到pCG76载体中，构建出温度敏感的营救质粒pCG76-Phsp60-Rv0228(pMS-6)。
将pMS-3电转化至mc~2155感受态细胞中，在温度和抗生素的选择性压力下，pMS-3上的突变基因MSMEG_0319::kan~R与mc~2155基因组上的MSMEG_0319发生第一次同源重组，突变基因MSMEG_0319::kan~R整合至mc~2155的基因组中。Southern blot筛选出7个第一次同源重组正确的突变菌mc~2155MS-1。将营救质粒转化至mc~2155MS-1中，在蔗糖的选择性压力下，mc~2155MS-1基因组自身发生第二次同源重组，剔除正常的MSMEG_0319基因，只保留突变基因MSMEG_0319::kan~R。Southern blot筛选出7个第二次同源重组正确的突变菌mc~2155MS-2，即为MSMEG_0319基因敲除菌珠。
(2) MSMEG_0319基因敲除菌珠生长曲线的绘制
以MSMEG_0319基因敲除菌珠作为Rv228基因必需性研究的模型，测定MSMEG_0319基因敲除菌株在30C和42C培养时的生长曲线，结果显示MSMEG_0319基因敲除菌株在30C可以生长，在42C条件下不能生长；然而，对照菌(携带pCG76质粒的mc~2155)在30C和42C条件下均可正常生长。此结果表明Rv0228是分枝杆菌生长的必需基因。
3.初步探索Rv0228基因的功能
(1) Rv0228的生物信息学分析：利用基因信息的数据库查看Rv0228基因组上相邻基因的功能；利用序列比对分析搜寻Rv0228同源蛋白，查看它们的功能及种属来源。结果显示：Rv0228在基因组中存在于单独的操纵子中，相邻基因多与细胞壁代谢相关；Rv0228在结核分枝杆菌及放线菌门的某些菌属中具有同源基因，在人类细胞、其它原核生物以及放线菌门的链霉菌中没有同源基因。总结这些信息，推测Rv0228基因的功能可能与结核分枝杆菌细胞壁肽聚糖以外的结构代谢相关。
(2)获取MSMEG_0319基因敲除菌作为Rv0228功能研究的模型：利用温度转换实验，先将MSMEG_0319基因敲除菌置于30℃培养20小时，允许其中的营救质粒复制并表达Rv0228蛋白以补偿缺失的MSMEG_0319基因功能，然后将培养温度升高至42℃，此时营救质粒不再复制和表达Rv0228蛋白，随着培养时间的延长，敲除菌内的Rv0228蛋白逐渐消耗，整个细菌也逐渐表现出缺乏MSMEG_0319基因功能产生的形态学和细胞组分的变化。利用此方法我们收集到了一定量MSMEG_0319蛋白缺失的MSMEG_0319基因敲除菌敲除菌，作为Rv0228功能研究的模型。
(3) MSMEG_0319基因敲除菌形态的观察：利用扫描电镜观察温度转换实验获得的MSMEG_0319基因敲除菌，结果显示，敲除菌在转换至42C生长72小时后出现形态改变，局部膨大、大小不一、表面出现皱褶；144小时后，MSMEG_0319基因敲除菌细菌形态改变更加明显，细菌形态不规则，细胞表面出现大量刺状突起，极不光滑。同时，持续在30C生长72小时和144小时的MSMEG_0319基因敲除菌形态正常，与野生型mc~2155无明显差别。利用透射电镜观察温度转换实验获得的MSMEG_0319基因敲除菌，结果显示，敲除菌在转换至42C生长144小时时，细胞壁不再完整，出现明显破损，并可观察到胞质溢出。扫描电镜和透射电镜的结果表明，Rv0228基因对分枝杆菌的生长至关重要，其功能可能与细胞壁合成有关。
(4) MSMEG_0319基因敲除菌细胞壁糖组分含量分析：提取温度转换实验获得的MSMEG_0319基因敲除菌和野生型mc~2155的细胞壁核心结构mAGP，利用HPLC测定聚阿拉伯糖半乳糖层中半乳糖和肽聚糖层中胞壁酸的含量。结果显示，MSMEG_0319基因敲除菌中半乳糖与胞壁酸的比值明显低于野生型mc~2155。此结果说明，Rv0228的基因功能可能与分枝杆菌细胞壁聚半乳糖的形成直接或间接相关。
(5) R0228蛋白的表达：构建了pET29b-Rv0228, pET16b-Rv0228, pBAD-Rv0228-C和pBAD-Rv0228-N表达质粒，在大肠杆菌中诱导表达Rv0228蛋白；同时构建了pVV16-Rv0228表达质粒，在耻垢分枝杆菌中表达Rv0228蛋白。Dot blot检测结果显示，携带pET16b-Rv0228的E. coli C41(DE3)和E. coli ER2566菌珠以及携带pBAD-RV0228-N的E. coli TOP10菌珠可以高效表达结核分枝杆菌Rv0228蛋白。
结论：
1.利用pET16b表达载体在E. coli BL21(DE3)中高效表达了结核分枝杆菌RmlA酶蛋白，纯化后的RmlA酶蛋白具有α-D-葡萄糖-1-磷酸胸苷转移酶活性。
2.通过偶联焦磷酸酶和使用孔雀石绿显色，建立了快速有效且稳定可靠的、可用于高通量筛选RmlA抑制剂的RmlA酶活性检测方法(孔雀石绿法)。
3.利用孔雀石绿法确定了RmlA酶促反应动力学特征和常数，包括最适温度、最适pH、最适Mg~(~(~(2+)))浓度，以及RmlA酶对反应底物的Km和Vmax值。
4.以耻垢分枝杆菌mc~2155作为实验模式菌，利用同源重组的方法敲除了结核分枝杆菌候选靶标Rv0228的同源基因：MSMEG_0319，建立了MSMEG_0319基因敲除菌珠，作为Rv0228生长必需性研究的模型。通过观察MSMEG_0319基因敲除菌珠的生长情况，确定了Rv0228是分枝杆菌生长的必需基因。
5.通过生物信息学方法对Rv0228的功能进行分析预测，确定了Rv0228的功能可能与结核分枝杆菌细胞壁肽聚糖以外的结构代谢相关。
6.通过温度转换(从30℃到42℃)实验，获得了一定量的MSMEG_0319基因敲除菌，作为Rv0228基因功能研究的模型。利用扫描电镜和透射电镜观察了MSMEG_0319基因敲除菌在缺失MSMEG_0319蛋白功能时的形态变化，并通过HPLC分析了它们细胞壁中半乳糖和胞壁酸含量比值的变化，从而确定了Rv0228的功能可能与结核分枝杆菌细胞壁中聚半乳糖的形成直接或间接相关。
7.利用不同的表达载体在大肠杆菌和耻垢分枝杆菌中表达Rv02228蛋白，筛选得到了3个高效表达Rv0228蛋白的大肠杆菌菌珠：携带pET16b-Rv0228的E. coliC41(DE3)菌珠、携带pET16b-Rv0228的E. coli ER2566菌珠、以及携带pBAD-Rv0228-N的E. coli TOP10菌珠。
未来的研究方向：
1.对于已知的药物靶标RmlA可开展如下工作：
(1)利用孔雀石绿显色的方法，从小分子化合物库中筛选RmlA酶蛋白抑制剂。
(2)优化RmlA酶蛋白的表达条件，获得更大量的RmlA纯化蛋白进行RmlA的结构研究，根据RmlA酶蛋白的结构设计或挑选合适的小分子化合物进行RmlA抑制作用的测试。
2.对于候选靶标Rv0228可开展如下工作：
(1)利用MSMEG_0319基因敲除菌珠作为功能研究的模型，通过测定细胞壁中更多单糖成分如阿拉伯糖、鼠李糖、N-乙酰葡糖胺的含量变化，从而分析Rv0228的具体功能。
(2)利用Rv0228高效表达菌珠在大体积培养基中大量表达Rv0228蛋白，提取细菌膜组分并纯化Rv0228蛋白，同时建立酰基转移酶的酶活性测定方法，为Rv0228功能的最终确定奠定基础。
Tuberculosis (TB) remains one of the deadliest threats to public health. The casesof multi-drug resistant TB, extensive-drug resistant TB as well as co-infection of M.tuberculosis with HIV are increasing fast in recent years. There is an urgent need fornew TB drugs. Target-based approach is one of the main strategies in TB drugdevelopment. For the targets that have been identified by experiments, high-throughputscreening of inhibitors from compound libraries can be performed with a suitable assay.For target candidates, their essentiality for mycobacterial growth should be confirmedfirst of all.
D-glucose-1-phosphate thymidylyltransferase (RmlA) together with other threeenzymes, RmlB, RmlC and RmlD, catalyze the formation of dTDP-L-rhamnose, a sugardonor providing L-rhamnosyl residue in the synthesis of M. tuberculosis cell wall. Theprevious study on RmlA essentiality has proved RmlA as a potential target for TB drugdevelopment.
M. tuberculosis Rv0228is a target candidate for TB drug development predictedthrough computational methods. Its essentiality and function for mycobacterial growthare still unknown.
The objectives of this study are:(1) to develop a rapid enzyme assay forpotential drug target RmlA which is suitable for high-throughput screening of inhibitors,and using this assay, to determine the kinetic properties of M. tuberculosis RmlAincluding initial velocity, optimal temperature, optimal pH, the effect of Mg~(2+)andkinetic parameters;(2) to test the essentiality of target candidate Rv0228formycobacterial growth. Using M. smegmatis as a model organism, MSMEG_0319(theortholog of Rv0228) gene knockout strain will be constructed via homologousrecombination method. Rv0228’s essentiality for mycobacterial growth will beconfirmed through growing MSMEG_0319knockout strain at different temperatures;(3) to analyze the function of target candidate Rv0228primarily. The function of Rv0228will be first predicted through bioinformatic analysis and further studied usingMSMEG_0319knockout strain as a model strain. An amount of MSMEG_0319knockout cells will be acquired through temperature shift experiment, and whether thefunction of Rv0228is related with the cell wall metabolism will be confirmed bycellular morphology observation and cell wall sugar analysis of MSMEG_0319knockout cells. For further function study of Rv0228, the expression of Rv0228proteinin Escherichia coli and M. smegmatis with different expression systems will also betried in this study.
Followings are results we obtained in this study:
1. Development of a colorimetric assay and kinetic analysis for M. tuberculosisRmlA.
(1) Expression and purification of M. tuberculosis RmlA: RmlA protein with afused his-tag at N-terminus was expressed in E. coli BL21(DE3) cells and purified byNi~(2+)affinity chromatography. SDS-PAGE and Western blot analysis confirmed that thepurified RmlA with an expected molecular weight of33.67kD and a perfecthomogeneity.
(2) HPLC analysis of M. tuberculosis RmlA activity: The enzyme activity of thepurified RmlA was detected by HPLC analysis. The result confirmed that the purifiedRmlA had the D-glucose-1-phosphate thymidylyltransferase activity, which catalyzedsubstrate dTTP and Glc-1-P converting to dTDP-D-Glc and PPi.
(3) Development of a colorimetric RmlA assay for high-throughput screening ofM. tuberculosis RmlA inhibitors: In this assay, pyrophosphatase was coupled tocatalyze the product PPi of RmlA reaction to Pi, and Pi molecules were finally detectedby the malachite green reagent. The results showed RmlA catalytic reaction had anapparent color change from yellow-green to blue-green. The assay had adequatesensitivity and reproducibility with the ratio of signal to background (S/B) above2.0and the Z’ value about0.70statistically, which was suitable for high-throughputscreening of M. tuberculosis RmlA inhibitors.
(4) Kinetic study of M. tuberculosis RmlA: Using the colorimetric assay, thekinetic properties of M. tuberculosis RmlA was determined. The results showed M.tuberculosis RmlA had the relative maximal activity in the buffer of pH7.5containing5mM Mg~(2+)at the temperature of37C. The Km and Vmax against dTTP were0.020±0.004mM and0.003±0.0003mM/min respectively, and against D-Glc-1-P were0.069 ±0.0050mM and0.002±0.0001mM/min respectively.
(5) RmlA inhibitors screening: Twenty-three natural products were screenedby the colorimetric assay and none of them was found to inhibit the activity ofM. tuberculosis RmlA.
2. The essentiality study of M. tuberculosis Rv0228for mycobacterial growth
(1) Construction of MSMEG_0319gene knockout strain
The kanamycin resistant gene (kan~R) was inserted into M. smegmgtis MSMEG_0319gene, resulting in a mutant MSMEG_0319gene (MSMEG_0319::kan~R).
MSMEG_0319::kan~Rfragment was then cloned into pPR27-xylE plasmid, yielding a conditional replication plasmid pPR27-xylE-MSMEG_0319::kan~R(pMS-3), which is sensitive to temperature.
M. tuberculosis Rv0228gene was cloned into pET23b-Phsp60plasmid resultingin a plasmid pET23b-Phsp60-Rv0228. Phsp60-Rv0228was then cloned into pCG76plasmid, yielding a rescue plasmid pCG76-Phsp60-Rv0228(pMS-6), which is alsosensitive to temperature.
pMS-3plasmid was elctroporated to M. smegmatis mc~2155competent cells. In theselective pressure of temperature and antibiotics, the first homologous recombinationoccurred between MSMEG_0319::kan~Rfragment in pMS-3plasmid and MSMEG_0319gene in mc~2155genome, thus MSMEG_0319::kan~Rfragment was integrated intomc~2155genome. Seven mc~2155MS-1mutant strains with correct homologousrecombination were screened by Southern blot analysis, and one of them waselectroporated with pMS-6plasmid. In the selective pressure of sucrose, the secondhomologous recombination occurred within the genome of mc~2155MS-1. The normalMSMEG_0319gene was deleted from the mc~2155mutant genome and only the mutantMSMEG_0319(MSMEG_0319::kan~R) was reserved. Seven mc~2155MS-2mutantstrains, i.e. MSMEG_0319knockout strains, were confirmed by Southern blot analysis.
(2) Growth of MSMEG_0319knockout strains at different temperatures
As the model strain for studying the essentiality of Rv0228for mycobacterialgrowth, MSMEG_0319knockout strain was grown at both30℃and42℃. The growthcurve showed that they could grow at30℃but not at42℃, while the mc~2155carryingpCG76plasmid as a control could grow at both30℃and42℃. The result demonstratedthat Rv0228was essential for mycobacterial growth.
3. The functional analysis of M. tuberculosis Rv0228
(1) Bioinformatic analysis of Rv0228: The information of Rv0228and its neighboring genes in M. tuberculosis genome was understood in GenBank and TBdatabases, and the homologous proteins of Rv0228were confirmed through BLASTanalysis. The result showed Rv0228located in M. tuberculosis genome without formingan operon with other open reading frames, and its neighboring genes were mostinvolved in cell wall biosynthesis. The orthologs exist in mycobacteria and someactinobacterial bacteria, but not found in human and other prokaryotic organisms. It wasconcluded from these information that Rv0228was probably involved in cell wall(except for peptidoglycan) biosynthesis.
(2) Acquirement of MSMEG_0319gene knockout cells as a model for functionalstudy of Rv0228. In the temperature shift experiment, MSMEG_0319knockout cellswere grown at30℃for20h, then the cells were transferred to a42℃incubator andgrown at42℃. The Rv0228protein expressed at30℃allowed the cells to grow at42℃for a certain period, however, with the Rv0228protein reduced gradually, the effects oflack of MSMEG_0319protein on the cells emerged. With this method, a certain amountof MSMEG_0319knockout cells were acquired for further function study of Rv0228.
(3) Morphology of MSMEG_0319knockout cells: MSMEG_0319knockout cellsgrown at42℃for72h and144h in the temperature shift experiment were observed viaSEM. The cells significantly bulged at72h and some cells exhibited an irregularsurface and were even lysed at144h. TEM analysis of the MSMEG_0319knockoutcells grown at42℃for144h found that the cell wall ruptured and intracellular contentoverflowed. In contrast, the MSMEG_0319knockout cells grown at30℃exhibitednormal shape and integrated cell wall structure as wild type mc~2155both in SEM andTEM. These results again confirmed that Rv0228was essential for mycobacterialgrowth, and indicated that the function of Rv0228related to the cell wall biosynthesis.
(4) Sugar analysis of cell wall in MSMEG_0319knockout cells: Themycolyl-arabinogalactan-peptidoglycan (mAGP) complex, the core structure of cellwall, of the MSMEG_0319knockout cells in the temperature shift experiment wasprepared and the contents of galactose (Gal) and muramic acid (MuAc) were measuredby HPLC. The result showed that the ratio of Gal to MuAc decreased in MSMEG_0319knockout cells compared with the wild type mc~2155. The result indicated that thefunction of Rv0228was involved in the formation of galactan in mycobacterial cell walldirectly or indirectly.
(5) Expression of Rv0228protein: The expression plasmids pET29b-Rv0228,pET16b-Rv0228, pBAD-Rv0228-C and pBAD-Rv0228-N were constructed to express Rv0228protein in E. coli, and pVV16-Rv0228was constructed to express Rv0228protein in M. smegmatis. Dot blot analysis showed that pET16b-Rv0228/E. coliC41(DE3), pET16b-Rv0228/E. coli ER2566and pBAD-RV0228-N/E. coli TOP10canexpress Rv0228protein effectively.
Followings are conclusions we obtained from this study:
1. M. tuberculosis RmlA protein was expressed in E. coli BL21(DE3) cells andpurified with Ni~(2+)affinity chromatography.
2. A colorimetric assay, which was coupled with pyrophosphatase and detectedwith malachite green reagent, was established to detect RmlA activity rapidly andsteadily. It was suitable for high-throughput screening of M. tuberculosis RmlAinhibitors.
3. The kinetic properties of M. tuberculosis RmlA including optimal temperature,optimal pH, the effect of Mg~(2+)and kinetic parameters Km and Vmax were determinedwith the established colorimetric assay.
4. M. smegmatis MSMEG_0319gene knockout strain was constructed via homologous recombination strategy. It was a model strain for studying the essentiality of M. tuberculosis Rv0228gene (a target candidate) for mycobacterial growth. Through observing the growth of MSMEG_0319knockout strains at different
temperatures, Rv0228gene was confirmed essential for mycobacterial growth.
5. The function of Rv0228was predicted probably involved in the biosynthesis ofmycobacterial cell wall structure except for peptidoglycan through bioinformaticanalysis.
6. MSMEG_0319knockout cells were acquired via temperature shift experiment,which could be used for function study of Rv0228. The morphology changes ofMSMEG_0319knockout cells were observed via SEM and TEM, and the content ratiochange of Gal to MuAc in the cell wall of MSMEG_0319knockout cells was analyzedby HPLC. The result demonstrated that Rv0228was involved in the formation ofgalactan in mycobacterial cell wall directly or indirectly.
7. Though expressing Rv0228protein with different vectors in different host cells,three E. coli strains pET16b-Rv0228/E. coli C41(DE3), pET16b-Rv0228/E. coliER2566and pBAD-Rv0228-N/E. coli TOP10, which could express Rv0228proteineffectively were obtained.
Followings are further studies for the potential target RmlA:(1) Screening ofRmlA inhibitors from chemical compound libraries with the colorimetric assay established in this study.(2) Structural analysis of purified M. tuberculosis RmlAprotein for further structure-based screening of inhibitors or TB drug design.
Followings are further studies for target candidate Rv0228:(1) Identify thefunction of Rv0228using MSMEG_0319knockout strain as model strain. The contentof other monosaccharide such as D-arabinose, L-rhamnose or N-acetylglucosamine inthe cell wall of MSMEG_0319knockout cells can be detected to confirm the functionof Rv0228.(2) Express Rv0228protein in large volume and purify Rv0228proteinthrough Ni~(2+)affinity chromatography, and establish enzyme assay of acyltransferasemeanwhile. These works will also be helpful for identification of Rv0228’s functionfinally.
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