缺乏D-葡萄糖-1-磷酸胸苷转移酶对分枝杆菌生长和细胞形态的影响
|
摘要
结核病是严重危害人类健康的疾病之一,其致病菌是结核分枝杆菌(Mycobacterium tuberculosis, Tb)。由于耐多药(multi drug resistant, MDR)甚至是广泛耐药(extensive drug resistant, XDR)结核杆菌的出现,许多一线甚至一些二线抗结核药物都无法有效治疗结核病,因此寻找新的抗结核药物已迫在眉睫。分枝杆菌的特殊细胞壁结构,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。该双糖衔接分子中的L-鼠李糖是细菌特有的,人体内不存在,可作为理想且安全的新型抗结核药的靶标。dTDP-鼠李糖为鼠李糖残基供体, RmlA-D四种酶参与dTDP-鼠李糖的生物合成过程,其中rmlA基因编码的D-葡萄糖-1-磷酸胸苷转移酶(RmlA)催化第一步反应,即葡萄糖-1-磷酸和dTTP在RmlA的催化下生成dTDP-葡萄糖。结核分枝杆菌的rmlA-D基因存在于基因组的不同位置,其中rmlA基因单独存在, rmlB和rmlC基因位于一个操纵子内, rmlD基因与编码鼠李糖基转移酶(WbbL)的wbbL基因及manB基因组成一个操纵子。在本课题组以前的工作中,我们研究并证明了rmlB,C,D基因是分枝杆菌生长必需基因,还建立了结核分枝杆菌RmlB-D的酶活性检测方法,这些研究结果为筛选上述酶的抑制剂,从而发现新的抗结核药物提供了基础。
本论文以耻垢分枝杆菌(Mycobacterium smegmatis,Sm) mc2155菌株作为实验模式菌,用同源重组方法建立mc2155 rmlA基因敲除菌株。通过测定rmlA基因敲除菌株的生长曲线,确定rmlA基因是否为分枝杆菌生长必需基因。在此基础上,研究RmlA酶缺乏时rmlA基因敲除菌株细胞形态的改变,和在缺乏RmlA酶的情况下rmlA基因敲除菌株蛋白质谱的变化。研究结果将为参与dTDP-鼠李糖合成的D-葡萄糖-1-磷酸胸苷转移酶作为研发抗结核新药的靶标提供更有力的证据。
本文获得以下结果:
1.条件性复制质粒pPR27-xylE-Sm rmlA::KanR的构建:
根据Tb RmlA的氨基酸序列,对TIGR的耻垢分枝杆菌mc2155菌株基因组数据库进行BLAST分析,获得mc2155的rmlA和启动子(上游约500 bp)序列。从mc2155基因组中PCR扩增该基因及其启动子序列,克隆到pMD18-T克隆质粒,用BcaBEST Primer M13-47、RV-M对质粒上携带的Sm rmlA基因及其启动子序列进行DNA测序,并与已知的Sm rmlA基因及其启动子序列进行比对,结果表明PCR扩增的是正确的Sm rmlA基因。
用BamHI酶切pUC4K质粒获得Kan抗性基因(KanR) ,用Klenow补平KanR基因的两端并克隆到pMD18-Sm rmlA质粒中Sm rmlA上的StuI位点,产生Sm rmlA::KanR突变基因。再将Sm rmlA::KanR突变基因克隆到pPR27-xylE质粒,构建出条件性复制质粒pPR27-xylE-Sm rmlA::KanR。pPR27-xylE携带温度敏感型复制子,使质粒在允许温度(30°C)条件下可以复制,在非允许温度(42°C)条件下不能复制。pPR27-xylE还携带xylE基因和负选择标记sacB基因。
2.营救质粒pCG76-Tb rmlA的构建:
从结核分枝杆菌H37Rv基因组中PCR扩增Tb rmlA基因,克隆到pMD18-T克隆质粒,测序与比对结果表明PCR扩增的是正确的Tb rmlA基因。用NdeI和XhoI酶切质粒pMD18-Tb rmlA,获得Tb rmlA基因,克隆到pET23b-Phsp60质粒的相应位点,构建出pET23b-Phsp60-Tb rmlA质粒。再经XhoI酶切、Klenow补平和XbaI酶切获得Phsp60-Tb rmlA片段。其中的Phsp60是来源于分枝杆菌BCG热休克蛋白60的启动子。将Phsp60-Tb rmlA克隆到pCG76质粒,构建出pCG76-Tb rmlA营救质粒。由于pCG76不携带用于目的基因表达的启动子,目的基因Tb rmlA的表达由Phsp60启动子所控制。此外,pCG76质粒也携带温度敏感型复制子,使质粒在30°C条件下可以复制,在42°C条件下不能复制。因此,可以通过调节温度使pCG76-Tb rmlA营救质补偿或不补偿mc2155 Sm rmlA基因敲除菌株中Sm rmlA::KanR突变基因的功能。
3.第一次同源重组突变菌株mc~2155 M-1的筛选:
将条件复制性质粒pPR27-xylE-Sm rmlA::KanR电转化到mc2155感受态细胞中,由于pPR27-xylE-Sm rmlA::KanR在42oC不能复制,Sm rmlA::KanR与mc2155基因组中的Sm rmlA发生同源重组,使Sm rmlA::KanR以及sacB基因和xylE基因整合到mc2155基因组中。用Sm rmlA探针对SmaI酶切的7个黄色菌落基因组DNA进行Southern杂交分析,结果显示发生第一次同源重组的mc2155 M-1突变菌株具有预期的杂交条带(3.24 kb,7.72 kb和8.07 kb)。
4.第二次同源重组突变菌株mc2155 M-2 (Sm rmlA基因敲除)的筛选:
将pCG76-Tb rmlA营救质粒电转化到mc2155 M-1感受态细胞中,用含10%蔗糖的选择性LB琼脂培养基,在30°C培养转化的mc2155 M-1,由于条件复制质粒的sacB基因和xylE基因也被整合到mc2155 M-1基因组中,sacB基因的表达产物Levansucrase水解蔗糖,产生有害细菌生长的物质,引起mc2155 M-1死亡,所以在这种选择压力下,mc2155 M-1基因组中的Sm rmlA::KanR与Sm rmlA发生第二次同源重组,将Sm rmlA基因、scaB基因以及xylE基因从mc2155 M-1基因组中删除并被核酸酶水解,基因组中只存在Sm rmlA::KanR。如果Sm rmlA为分枝杆菌生长必需基因,营救质粒上的Tb rmlA基因必须在mc2155 M-2突变菌株中表达出Tb RmlA蛋白质以补偿Sm rmlA::KanR的功能。用Southern印迹方法筛选出mc2155 M-2突变菌株,即Sm rmlA基因敲除菌株。
5.生长曲线的测定
测定mc2155 M-2突变菌株在30°C和42°C的生长曲线,结果发现该突变菌株在30°C可以生长,但在42°C不生长,说明rmlA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 M-2突变菌株在营救质粒容许的30°C生长20小时,使之表达一定量Tb RmlA蛋白后,提高温度至42°C,使pCG76-Tb rmlA不能复制,影响Tb RmlA蛋白的表达。在Tb RmlA逐渐缺乏的情况下,测定mc2155 M-2突变菌株的生长曲线。结果显示mc2155 M-2突变菌株可以在42°C继续增殖1-2天,第三天以后逐渐有细菌死亡,菌量减少。进一步说明了rmlA基因是分枝杆菌生长必需基因。
6. RmlA缺乏对突变菌株mc2155 M-2细胞形态的影响在温度转换实验中,取mc2155 M-2突变菌株细胞用扫描电镜观察其细胞形态。结果显示在30°C条件下,该mc~2155 M-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°C条件下,营救质粒Tb rmlA表达受影响致使RmlA蛋白缺乏时,第三天菌体表面出现明显的塌陷和皱褶,第六天甚至出现大量破碎样物质,菌体轮廓亦不清晰,似乎都粘连在一起,部分细菌发生裂解。说明RmlA蛋白缺乏对细胞壁完整性有影响,可导致细胞裂解。
7. RmlA缺乏对突变菌株mc~2155 M-2蛋白质谱的影响
在温度转换以后,收获在42°C生长5天的mc2155 M-2细菌细胞提取胞浆蛋白,进行双向凝胶电泳,初步发现mc2155 M-2的蛋白质谱有所变化。与对照相比,rmlA基因敲除以后蛋白质点变少,由于RmlA的缺乏可能导致许多与胞壁代谢有关蛋白的降低,需要进一步确认这些点。
结论:
本研究以耻垢分枝杆菌mc~2155菌株作为实验模式菌证明rmlA基因与分枝杆菌生长的相关性。我们构建了携带Sm rmlA::KanR突变基因的条件复制质粒并转化mc~2155,用Southern印记方法筛选出发生第一次同源重组的mc~2155 M-1突变菌株。又构建了携带Tb rmlA基因的营救质粒pCG76-Tb rmlA,并转化mc~2155 M-1,当Tb rmlA基因表达时, mc~2155 M-1基因组中的Sm rmlA::KanR突变基因与Sm rmlA基因发生第二次同源重组,用Southern印记方法筛选出发生第二次同源重组的mc~2155 M-2突变菌株,即Sm rmlA基因敲除菌株。根据Sm rmlA基因敲除菌株在30°C和42°C条件下的生长曲线,以及在缺乏Tb RmlA蛋白条件下Sm rmlA基因敲除菌株细胞形态的改变和蛋白质谱的变化,我们证实了编码结核分枝杆菌D-葡萄糖-1-磷酸胸苷转移酶(RmlA)的rmlA基因是分枝杆菌生长所必需的基因。缺乏RmlA蛋白使细菌胞壁成分合成受阻,导致细菌裂解死亡。在缺乏RmlA蛋白时,细菌蛋白质谱也发生了改变。我们将进一步对这些蛋白质进行鉴定,以发现更多的药物作用靶点。
本研究结果为参与dTDP-鼠李糖合成的D-葡萄糖-1-磷酸胸苷转移酶作为研发抗结核新药的理想靶标提供更有力的证据,以便用D-葡萄糖-1-磷酸胸苷转移酶促反应筛选酶抑制剂,发现先导化合物。
未来研究方向:
1.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rmlA基因与其它基因之间是否存在相互调控相互影响的关系。
2.用高效液相色谱(HPLC)方法分析Sm rmlA基因敲除菌株与野生型mc2155菌株之间细胞壁聚糖(聚阿拉伯半乳糖)结构的差异;用气相色谱(GC)分析二者糖组成的差别;
3.通过RmlA酶活性检测方法的建立,筛选和优化有该酶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rmlA基因敲除细胞模型进一步鉴定该化合物。
Tuberculosis (TB) is today amongst the worldwide health threats. Mycobacterium tuberculosis, a species of the genus Mycobacteria is the pathogen of tuberculosis. The emergence of multi-drug and extra-drug resistant strains makes invalid of many anti- TB drugs. Thus, more efficient anti-TB drugs are desperately needed.
The cell wall is necessary for mycobacterial viability. The mybacterial cell wall core consists of mycolic acids, arabinogalacan (AG), and peptidoglycan. AG is attached to the peptidoglycan via a linker disaccharide,α-L-rhamnosyl-(1→3)-α-D-N acetyl- glucosaminosyl- 1-posphate. L-rhamnose in the linker disaccharide is special for the bacteria and not available in human beings. So, it is strongly suggests that the linker disaccharide is an ideal and safe target for new anti-TB drugs.
The L-rhamnosyl residue of linker disaccharide is provided with a sugar donor, dTDP-rhamnose. Four enzymes (RmlA-D) participate the dTDP-rhamnose biosynthesis. RmlA encoded by rmlA gene catalyzes the first reaction of the biosynthesis process: converting glucose-1-phosphate and dTTP to dTDP-glucose. The rmlA-D genes for the four enzymes are not located in a locus in the genome of M. tuberculosis H37Rv. The rmlA gene is isolated from any other rhamnosyl formation enzymes, the rmlB and rmlC genes are together in an operon, and rmlD gene is found in an operon with wbbL and manB.
In our previous works, we studied and verified the essentiality of rmlB, rmlC and rmlD genes for mycobacterial growth. We also established M. tuberculosis RmlB-D enzyme assays to screen inhibitors for developingnew tuberculosis therapeutics.
In this study, we generated Mycobacterium smegmatis mc2155 rmlA gene knockout strain by homologous recombination strategy and tested the essentiality of rmlA gene for mycobacterial growth. We then observed the cellular morphology changes when mc2155 rmlA gene knockout strain lacked RmlA by scanning electron microscope (SEM). We also studied the changes in protein profile when mc2155 rmlA gene knockout strain lacked RmlA by two-dimensional electrophoresis.
Followings are results we got in this study:
1. Construction of conditional replication plasmid pPR27-xylE-Sm rmlA::KanR
According to the amino acid sequences of Tb RmlA gained from M. tuberculosis genome, BLAST search was run against the genome of M. smegmatis mc2155. Sm rmlA and its promoter sequence (about 500bp upstream of rmlA gene) was obtained. Sm rmA and its upstream sequence were amplified from mc2155 genomic DNA. The PCR products was purified and cloned into pMD18-T clone vector and sequenced to be right. pUC4K was digested by BamHI to obtain the kanamycin resistance cassette (KanR). KanR was then cloned to the StuI site of Sm rmlA in pMD18-Sm rmlA to create Sm rmlA::KanR mutated gene. The Sm rmlA::KanR fragment was then subcloned to pPR27-xylE to construct pPR27-xlyE-Sm rmlA::KanR. pPR27-xylE carries the temperature-sensitive mycobacterial replication origin and thus can replicate at 30°C but is lost at 42°C and it also harbors xylE gene and a counter-selectable marker sacB.
2. Construction of rescue plasmids pCG76-Tb rmlA Tb rmlA gene was cloned to pET23b-Phsp60 to generate pET23b-Phsp60-Tb rmlA, and Phsp60-Tb rmlA fragment was cloned to pCG7 yielding pCG76-Tb rmlA. The expression of Tb RmlA 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.
3. Screening of mc2155 M-1 mutants
pPR27-xylE-Sm rmlA::KanR was electroporated to wild type mc2155 and transformed mc2155 cells were plated out on LB agar containing Kanat 42°C. Since the temperature-sensitive plasmid was able to replicate at 30°C but not at 42°C, the kanamycin resistant colonies that appear on LB agar containing Kan have necessarily integrated the KanR gene into their genome. Therefore, the single crossover event between Sm rmlA-KanR in pPR27-xylE-Sm rmlA::KanR and Sm rmlA gene in the mc~2155 genome occurred and Sm rmlA::KanR, sacB gene and xylE gene were integrated into the mc~2155 genome resulting in mc~2155 M-1 mutant, which carried both Sm rmlA and Sm rmlA::KanR. SmaI-digested genomic DNA from 7 yellow colonies grown at 42°C was analyzed by Southern hybridization of Sm rmlA probe and was proved to be mc~2155 M-1 strain, having the expected bands of 3.24 kb, 7.72 kb and 8.07 kb.
4. Screening of mc~2155 M-2 mutants (rmlA gene knockout strains)
Rescue plasmids pCG76-Tb rmlA were electroporated to mc~2155 M-1 and transformed mc~2155 M-1 cells were incubated at 30°C on LB agar containing 10% sucrose and appropriate antibiotics. Since sacB gene product (levansucrase) was lethal to mc~2155 M-1, the double crossover event between Sm rmlA::KanR and Sm rmlA of mc~2155 M-1 genome occurred, and the Sm rmlA gene was replaced by Sm rmlA::KanR in the presence of pCG76-Tb rmlA, resulting in mc~2155 M-2 mutant, that is Sm rmlA gene knock out strain. SmaI-digested genomic DNA from white colonies was analyzed by Southern hybridization, and all colonies were mc~2155 M-2 mutant (rmlA gene knockout strains).
5. Growth of mc~2155 M-2 mutants
The growth curve of mc~2155 M-2 was detected at 30°C and 42°C. mc~2155 M-2 grew only at 30°C, but not at 42°C. The results confirmed that Tb rmlA gene was essential for mycobacterial growth.
In the following temperature shift experiment, mc~2155 M-2 were grown at 30°C for 20 h to produce Tb RmlA enzyme and then the cells were grown at 42°C. Growth curves were detected in this Tb RmlA lacking condition. The results showed that mc~2155 M-2 grew for about 1-2 days before its dying.
6. Morphology of mc~2155 M-2 mutants
In temperature shift experiment, certain amount of mc~2155 M-2 cells was acquired. The Scanning electron micrographs showed many seriouscollapse and reductus formed the third day and cell lysis appeared the sixth day, as contrasted with the control samples which were cultivated at 30°C.
7. Effect of lacking RmlA on protein profile of mc2155 M-2 mutants
After temperature shift, mc2155 M-2 cells growing at 42°C for five days were collected and cytoplasmic protein were obtained. Changes in protein profile were tested by two-dimensional electrophoresis. The results showed that there were some changes in protein profile. The numbers of protein spots were less than those of control samples (mc2155 carrying pCG76). Because lacking RmlA may decrease many proteins relevant to cell wall metabolism, these protein spots need further confirmation by mass spectrometry.
Summary:
M. smegmatis mc2155 was used as model to clarify the essentility of Tb rmlA for the growth of mycobacterium tuberculosis. Conditional replication plasmid pPR27-xylE-Sm rmlA::KanR was constructed and electroporated to mc2155. The mc2155 M-1 mutants were screened by Southern blot analysis. Rescue plasmid pCG76-Tb rmlA was constructed and electroporated to mc2155 M-1 mutant, whose genomic Sm rmlA::KanR and Sm rmlA underwent second homologous recombination under selecting pressure and Sm rmlA was deleted. The mc2155 M-2 mutants (rmlA gene knockout strains) were screened by Southern blot analysis. We tested the growth of mc2155 M-2 at 30°C and 42°C. The result indicates rmlA gene is essential for mycobacterial growth. We tested the cell morphorlogical change by SEM when lacking RmlA protein. The SEM result shows that lacking RmlA causes cell lysis. We also checked proteomic profile of mc2155 M-2 when lacking RmlA. The result shows that the protein profile of mc2155 M-2 was changed compared to mc2155 cells. Therefore, Tb rmlA gene involved in dTDP-rhamnose synthesis in M. tuberculosis is a valid target for developing new anti-tuberculosis drugs.
Further studies:
1. To repeat and optimize the conditions for two-dimensional electrophoresis. Select differently expressed protein dots of interests according to the results of 2-D electrophoresis. Peptide mass fingerprinting were obtained by mass spectrometry for identifying these differentlyexpressed proteins. Genes of significance were obtained by molecular cloning in order to find out the relationship between rmlA gene and other genes.
2. To analyze the structural differences of cell wall glycan (arabinogalacan) between mc2155 M-2 and wild type mc2155 using HPLC. To detect the differences of cell wall sugar composition of cell wall between mc2155 M-2 and wild type mc2155 by using GC.
3. To establish a fast and accurate M. tuberculosis RmlA enzyme assay to screen and optimize lead compounds that are inhibitors of RmlA for further developing new tuberculosis therapeutics. At the same time, these compounds may be further confirmed by the rmlA gene knockout strain as a cell model.
本论文以耻垢分枝杆菌(Mycobacterium smegmatis,Sm) mc2155菌株作为实验模式菌,用同源重组方法建立mc2155 rmlA基因敲除菌株。通过测定rmlA基因敲除菌株的生长曲线,确定rmlA基因是否为分枝杆菌生长必需基因。在此基础上,研究RmlA酶缺乏时rmlA基因敲除菌株细胞形态的改变,和在缺乏RmlA酶的情况下rmlA基因敲除菌株蛋白质谱的变化。研究结果将为参与dTDP-鼠李糖合成的D-葡萄糖-1-磷酸胸苷转移酶作为研发抗结核新药的靶标提供更有力的证据。
本文获得以下结果:
1.条件性复制质粒pPR27-xylE-Sm rmlA::KanR的构建:
根据Tb RmlA的氨基酸序列,对TIGR的耻垢分枝杆菌mc2155菌株基因组数据库进行BLAST分析,获得mc2155的rmlA和启动子(上游约500 bp)序列。从mc2155基因组中PCR扩增该基因及其启动子序列,克隆到pMD18-T克隆质粒,用BcaBEST Primer M13-47、RV-M对质粒上携带的Sm rmlA基因及其启动子序列进行DNA测序,并与已知的Sm rmlA基因及其启动子序列进行比对,结果表明PCR扩增的是正确的Sm rmlA基因。
用BamHI酶切pUC4K质粒获得Kan抗性基因(KanR) ,用Klenow补平KanR基因的两端并克隆到pMD18-Sm rmlA质粒中Sm rmlA上的StuI位点,产生Sm rmlA::KanR突变基因。再将Sm rmlA::KanR突变基因克隆到pPR27-xylE质粒,构建出条件性复制质粒pPR27-xylE-Sm rmlA::KanR。pPR27-xylE携带温度敏感型复制子,使质粒在允许温度(30°C)条件下可以复制,在非允许温度(42°C)条件下不能复制。pPR27-xylE还携带xylE基因和负选择标记sacB基因。
2.营救质粒pCG76-Tb rmlA的构建:
从结核分枝杆菌H37Rv基因组中PCR扩增Tb rmlA基因,克隆到pMD18-T克隆质粒,测序与比对结果表明PCR扩增的是正确的Tb rmlA基因。用NdeI和XhoI酶切质粒pMD18-Tb rmlA,获得Tb rmlA基因,克隆到pET23b-Phsp60质粒的相应位点,构建出pET23b-Phsp60-Tb rmlA质粒。再经XhoI酶切、Klenow补平和XbaI酶切获得Phsp60-Tb rmlA片段。其中的Phsp60是来源于分枝杆菌BCG热休克蛋白60的启动子。将Phsp60-Tb rmlA克隆到pCG76质粒,构建出pCG76-Tb rmlA营救质粒。由于pCG76不携带用于目的基因表达的启动子,目的基因Tb rmlA的表达由Phsp60启动子所控制。此外,pCG76质粒也携带温度敏感型复制子,使质粒在30°C条件下可以复制,在42°C条件下不能复制。因此,可以通过调节温度使pCG76-Tb rmlA营救质补偿或不补偿mc2155 Sm rmlA基因敲除菌株中Sm rmlA::KanR突变基因的功能。
3.第一次同源重组突变菌株mc~2155 M-1的筛选:
将条件复制性质粒pPR27-xylE-Sm rmlA::KanR电转化到mc2155感受态细胞中,由于pPR27-xylE-Sm rmlA::KanR在42oC不能复制,Sm rmlA::KanR与mc2155基因组中的Sm rmlA发生同源重组,使Sm rmlA::KanR以及sacB基因和xylE基因整合到mc2155基因组中。用Sm rmlA探针对SmaI酶切的7个黄色菌落基因组DNA进行Southern杂交分析,结果显示发生第一次同源重组的mc2155 M-1突变菌株具有预期的杂交条带(3.24 kb,7.72 kb和8.07 kb)。
4.第二次同源重组突变菌株mc2155 M-2 (Sm rmlA基因敲除)的筛选:
将pCG76-Tb rmlA营救质粒电转化到mc2155 M-1感受态细胞中,用含10%蔗糖的选择性LB琼脂培养基,在30°C培养转化的mc2155 M-1,由于条件复制质粒的sacB基因和xylE基因也被整合到mc2155 M-1基因组中,sacB基因的表达产物Levansucrase水解蔗糖,产生有害细菌生长的物质,引起mc2155 M-1死亡,所以在这种选择压力下,mc2155 M-1基因组中的Sm rmlA::KanR与Sm rmlA发生第二次同源重组,将Sm rmlA基因、scaB基因以及xylE基因从mc2155 M-1基因组中删除并被核酸酶水解,基因组中只存在Sm rmlA::KanR。如果Sm rmlA为分枝杆菌生长必需基因,营救质粒上的Tb rmlA基因必须在mc2155 M-2突变菌株中表达出Tb RmlA蛋白质以补偿Sm rmlA::KanR的功能。用Southern印迹方法筛选出mc2155 M-2突变菌株,即Sm rmlA基因敲除菌株。
5.生长曲线的测定
测定mc2155 M-2突变菌株在30°C和42°C的生长曲线,结果发现该突变菌株在30°C可以生长,但在42°C不生长,说明rmlA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 M-2突变菌株在营救质粒容许的30°C生长20小时,使之表达一定量Tb RmlA蛋白后,提高温度至42°C,使pCG76-Tb rmlA不能复制,影响Tb RmlA蛋白的表达。在Tb RmlA逐渐缺乏的情况下,测定mc2155 M-2突变菌株的生长曲线。结果显示mc2155 M-2突变菌株可以在42°C继续增殖1-2天,第三天以后逐渐有细菌死亡,菌量减少。进一步说明了rmlA基因是分枝杆菌生长必需基因。
6. RmlA缺乏对突变菌株mc2155 M-2细胞形态的影响在温度转换实验中,取mc2155 M-2突变菌株细胞用扫描电镜观察其细胞形态。结果显示在30°C条件下,该mc~2155 M-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°C条件下,营救质粒Tb rmlA表达受影响致使RmlA蛋白缺乏时,第三天菌体表面出现明显的塌陷和皱褶,第六天甚至出现大量破碎样物质,菌体轮廓亦不清晰,似乎都粘连在一起,部分细菌发生裂解。说明RmlA蛋白缺乏对细胞壁完整性有影响,可导致细胞裂解。
7. RmlA缺乏对突变菌株mc~2155 M-2蛋白质谱的影响
在温度转换以后,收获在42°C生长5天的mc2155 M-2细菌细胞提取胞浆蛋白,进行双向凝胶电泳,初步发现mc2155 M-2的蛋白质谱有所变化。与对照相比,rmlA基因敲除以后蛋白质点变少,由于RmlA的缺乏可能导致许多与胞壁代谢有关蛋白的降低,需要进一步确认这些点。
结论:
本研究以耻垢分枝杆菌mc~2155菌株作为实验模式菌证明rmlA基因与分枝杆菌生长的相关性。我们构建了携带Sm rmlA::KanR突变基因的条件复制质粒并转化mc~2155,用Southern印记方法筛选出发生第一次同源重组的mc~2155 M-1突变菌株。又构建了携带Tb rmlA基因的营救质粒pCG76-Tb rmlA,并转化mc~2155 M-1,当Tb rmlA基因表达时, mc~2155 M-1基因组中的Sm rmlA::KanR突变基因与Sm rmlA基因发生第二次同源重组,用Southern印记方法筛选出发生第二次同源重组的mc~2155 M-2突变菌株,即Sm rmlA基因敲除菌株。根据Sm rmlA基因敲除菌株在30°C和42°C条件下的生长曲线,以及在缺乏Tb RmlA蛋白条件下Sm rmlA基因敲除菌株细胞形态的改变和蛋白质谱的变化,我们证实了编码结核分枝杆菌D-葡萄糖-1-磷酸胸苷转移酶(RmlA)的rmlA基因是分枝杆菌生长所必需的基因。缺乏RmlA蛋白使细菌胞壁成分合成受阻,导致细菌裂解死亡。在缺乏RmlA蛋白时,细菌蛋白质谱也发生了改变。我们将进一步对这些蛋白质进行鉴定,以发现更多的药物作用靶点。
本研究结果为参与dTDP-鼠李糖合成的D-葡萄糖-1-磷酸胸苷转移酶作为研发抗结核新药的理想靶标提供更有力的证据,以便用D-葡萄糖-1-磷酸胸苷转移酶促反应筛选酶抑制剂,发现先导化合物。
未来研究方向:
1.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rmlA基因与其它基因之间是否存在相互调控相互影响的关系。
2.用高效液相色谱(HPLC)方法分析Sm rmlA基因敲除菌株与野生型mc2155菌株之间细胞壁聚糖(聚阿拉伯半乳糖)结构的差异;用气相色谱(GC)分析二者糖组成的差别;
3.通过RmlA酶活性检测方法的建立,筛选和优化有该酶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rmlA基因敲除细胞模型进一步鉴定该化合物。
Tuberculosis (TB) is today amongst the worldwide health threats. Mycobacterium tuberculosis, a species of the genus Mycobacteria is the pathogen of tuberculosis. The emergence of multi-drug and extra-drug resistant strains makes invalid of many anti- TB drugs. Thus, more efficient anti-TB drugs are desperately needed.
The cell wall is necessary for mycobacterial viability. The mybacterial cell wall core consists of mycolic acids, arabinogalacan (AG), and peptidoglycan. AG is attached to the peptidoglycan via a linker disaccharide,α-L-rhamnosyl-(1→3)-α-D-N acetyl- glucosaminosyl- 1-posphate. L-rhamnose in the linker disaccharide is special for the bacteria and not available in human beings. So, it is strongly suggests that the linker disaccharide is an ideal and safe target for new anti-TB drugs.
The L-rhamnosyl residue of linker disaccharide is provided with a sugar donor, dTDP-rhamnose. Four enzymes (RmlA-D) participate the dTDP-rhamnose biosynthesis. RmlA encoded by rmlA gene catalyzes the first reaction of the biosynthesis process: converting glucose-1-phosphate and dTTP to dTDP-glucose. The rmlA-D genes for the four enzymes are not located in a locus in the genome of M. tuberculosis H37Rv. The rmlA gene is isolated from any other rhamnosyl formation enzymes, the rmlB and rmlC genes are together in an operon, and rmlD gene is found in an operon with wbbL and manB.
In our previous works, we studied and verified the essentiality of rmlB, rmlC and rmlD genes for mycobacterial growth. We also established M. tuberculosis RmlB-D enzyme assays to screen inhibitors for developingnew tuberculosis therapeutics.
In this study, we generated Mycobacterium smegmatis mc2155 rmlA gene knockout strain by homologous recombination strategy and tested the essentiality of rmlA gene for mycobacterial growth. We then observed the cellular morphology changes when mc2155 rmlA gene knockout strain lacked RmlA by scanning electron microscope (SEM). We also studied the changes in protein profile when mc2155 rmlA gene knockout strain lacked RmlA by two-dimensional electrophoresis.
Followings are results we got in this study:
1. Construction of conditional replication plasmid pPR27-xylE-Sm rmlA::KanR
According to the amino acid sequences of Tb RmlA gained from M. tuberculosis genome, BLAST search was run against the genome of M. smegmatis mc2155. Sm rmlA and its promoter sequence (about 500bp upstream of rmlA gene) was obtained. Sm rmA and its upstream sequence were amplified from mc2155 genomic DNA. The PCR products was purified and cloned into pMD18-T clone vector and sequenced to be right. pUC4K was digested by BamHI to obtain the kanamycin resistance cassette (KanR). KanR was then cloned to the StuI site of Sm rmlA in pMD18-Sm rmlA to create Sm rmlA::KanR mutated gene. The Sm rmlA::KanR fragment was then subcloned to pPR27-xylE to construct pPR27-xlyE-Sm rmlA::KanR. pPR27-xylE carries the temperature-sensitive mycobacterial replication origin and thus can replicate at 30°C but is lost at 42°C and it also harbors xylE gene and a counter-selectable marker sacB.
2. Construction of rescue plasmids pCG76-Tb rmlA Tb rmlA gene was cloned to pET23b-Phsp60 to generate pET23b-Phsp60-Tb rmlA, and Phsp60-Tb rmlA fragment was cloned to pCG7 yielding pCG76-Tb rmlA. The expression of Tb RmlA 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.
3. Screening of mc2155 M-1 mutants
pPR27-xylE-Sm rmlA::KanR was electroporated to wild type mc2155 and transformed mc2155 cells were plated out on LB agar containing Kanat 42°C. Since the temperature-sensitive plasmid was able to replicate at 30°C but not at 42°C, the kanamycin resistant colonies that appear on LB agar containing Kan have necessarily integrated the KanR gene into their genome. Therefore, the single crossover event between Sm rmlA-KanR in pPR27-xylE-Sm rmlA::KanR and Sm rmlA gene in the mc~2155 genome occurred and Sm rmlA::KanR, sacB gene and xylE gene were integrated into the mc~2155 genome resulting in mc~2155 M-1 mutant, which carried both Sm rmlA and Sm rmlA::KanR. SmaI-digested genomic DNA from 7 yellow colonies grown at 42°C was analyzed by Southern hybridization of Sm rmlA probe and was proved to be mc~2155 M-1 strain, having the expected bands of 3.24 kb, 7.72 kb and 8.07 kb.
4. Screening of mc~2155 M-2 mutants (rmlA gene knockout strains)
Rescue plasmids pCG76-Tb rmlA were electroporated to mc~2155 M-1 and transformed mc~2155 M-1 cells were incubated at 30°C on LB agar containing 10% sucrose and appropriate antibiotics. Since sacB gene product (levansucrase) was lethal to mc~2155 M-1, the double crossover event between Sm rmlA::KanR and Sm rmlA of mc~2155 M-1 genome occurred, and the Sm rmlA gene was replaced by Sm rmlA::KanR in the presence of pCG76-Tb rmlA, resulting in mc~2155 M-2 mutant, that is Sm rmlA gene knock out strain. SmaI-digested genomic DNA from white colonies was analyzed by Southern hybridization, and all colonies were mc~2155 M-2 mutant (rmlA gene knockout strains).
5. Growth of mc~2155 M-2 mutants
The growth curve of mc~2155 M-2 was detected at 30°C and 42°C. mc~2155 M-2 grew only at 30°C, but not at 42°C. The results confirmed that Tb rmlA gene was essential for mycobacterial growth.
In the following temperature shift experiment, mc~2155 M-2 were grown at 30°C for 20 h to produce Tb RmlA enzyme and then the cells were grown at 42°C. Growth curves were detected in this Tb RmlA lacking condition. The results showed that mc~2155 M-2 grew for about 1-2 days before its dying.
6. Morphology of mc~2155 M-2 mutants
In temperature shift experiment, certain amount of mc~2155 M-2 cells was acquired. The Scanning electron micrographs showed many seriouscollapse and reductus formed the third day and cell lysis appeared the sixth day, as contrasted with the control samples which were cultivated at 30°C.
7. Effect of lacking RmlA on protein profile of mc2155 M-2 mutants
After temperature shift, mc2155 M-2 cells growing at 42°C for five days were collected and cytoplasmic protein were obtained. Changes in protein profile were tested by two-dimensional electrophoresis. The results showed that there were some changes in protein profile. The numbers of protein spots were less than those of control samples (mc2155 carrying pCG76). Because lacking RmlA may decrease many proteins relevant to cell wall metabolism, these protein spots need further confirmation by mass spectrometry.
Summary:
M. smegmatis mc2155 was used as model to clarify the essentility of Tb rmlA for the growth of mycobacterium tuberculosis. Conditional replication plasmid pPR27-xylE-Sm rmlA::KanR was constructed and electroporated to mc2155. The mc2155 M-1 mutants were screened by Southern blot analysis. Rescue plasmid pCG76-Tb rmlA was constructed and electroporated to mc2155 M-1 mutant, whose genomic Sm rmlA::KanR and Sm rmlA underwent second homologous recombination under selecting pressure and Sm rmlA was deleted. The mc2155 M-2 mutants (rmlA gene knockout strains) were screened by Southern blot analysis. We tested the growth of mc2155 M-2 at 30°C and 42°C. The result indicates rmlA gene is essential for mycobacterial growth. We tested the cell morphorlogical change by SEM when lacking RmlA protein. The SEM result shows that lacking RmlA causes cell lysis. We also checked proteomic profile of mc2155 M-2 when lacking RmlA. The result shows that the protein profile of mc2155 M-2 was changed compared to mc2155 cells. Therefore, Tb rmlA gene involved in dTDP-rhamnose synthesis in M. tuberculosis is a valid target for developing new anti-tuberculosis drugs.
Further studies:
1. To repeat and optimize the conditions for two-dimensional electrophoresis. Select differently expressed protein dots of interests according to the results of 2-D electrophoresis. Peptide mass fingerprinting were obtained by mass spectrometry for identifying these differentlyexpressed proteins. Genes of significance were obtained by molecular cloning in order to find out the relationship between rmlA gene and other genes.
2. To analyze the structural differences of cell wall glycan (arabinogalacan) between mc2155 M-2 and wild type mc2155 using HPLC. To detect the differences of cell wall sugar composition of cell wall between mc2155 M-2 and wild type mc2155 by using GC.
3. To establish a fast and accurate M. tuberculosis RmlA enzyme assay to screen and optimize lead compounds that are inhibitors of RmlA for further developing new tuberculosis therapeutics. At the same time, these compounds may be further confirmed by the rmlA gene knockout strain as a cell model.
引文
1. World Health Organization. Global tuberculosis control: surveillance, planning, financing. WHO report 2007, Geneva, (WHO/HTM/TB/2007.376).
2. Susan ED, Richard EC. From magic bullets back to the Magic Mountain: the rise of extensively drug-resistant tuberculosis. Nature Medicine 2007,13(3): 295-8.
3. Raviglione MC and Smith IM. XDR Tuberculosis — Implications for Global Public Health. N. Engl. J. Med. 2007, 356: 656-659.
4. Wise J. Southern Africa is moving swiftly to combat the threat of XDR-TB. Bull World Health Organ. 2006, 84(12): 924-5.
5. Brennan PJ and Besra GS. Structure, function and biogenesis of the mycob- acterial cell wall. Biochemical Society Transactions 1997, 25(1): 188-94.
6. Khasnobis S, Escuyer VE and Chatterjee D. Emerging therapeutic targets in tuberculosis: post-genomic era. Expert Opinion on Therapeutic Targets 2002, 6(1): 21-40.
7. Brennan PJ and Nikaido H. The envelope of mycobacteria. Annual Reviews of Biochemistry 1995, 64: 29-63.
8. Crick DC, Brennan PJ and McNeil MR. The cell wall of Mycobacterium tuberculosis. In Tuberculosis (2 nd Edition), 2004,115-134. Edited by Rom WN and Garay SM, Lippincott Williams & Wilkins, Philadelphia.
9. Stevenson G, Neal B, Liu D, et al. Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster. Journal of Bacteriology 1994, 176: 4144-4156.
10. Jiang XM, Neal B, Santiago F, et al. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mole- cular Microbiology 1991, 5(3): 695-713.
11. Koplin R, Wang G, Hotte B, et al. A 3.9-kb DNA Region of Xanthomonas campestris pv. campestris that is necessary for lipopolysaccharide production encodes a set of enzymes involved in the synthesis of dTDP-Rhamnose. Journal of Bacteriology 1993, 175(24): 7786-7792.
12. Tsukioka Y, Yamashita Y, Oho T, et al. Biological function of the dTDP- Rhamnose synthesis pathway in Streptococcus mutans. Journal of Bacteriology 1997, 179(4): 1126-1134.
13. Ma Y, Mills J, Belisle JT, et al. Determination of the pathway for rhmnnose biosynthesis in mycobacteria: cloning, sequencing and expression of the M. tuberculosis gene encoding α-D-glucose-1-phosphate thymidylyltransferase. Microbiology 1997, 143: 937-945.
14. Boels IC, Beerthuyzen MM, Kosters MHW, et al. Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP- Rhamnose biosynthesis. Journal of Bacteriology 2004, 186(5): 1239-1248.
15. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393: 537-544.
16. Ma Y, Pan F and McNeil M. The formation of dTDP-rhamnose is essential for growth of mycobacteria. Journal of Bacteriology 2002, 184(12): 3392-5.
17. Li, W., Xin, Y., McNeil, M.R., Ma, Y. rmlB and rmlC genes are essential for growth of mycobacteria, Biochem. Biophys. Res. Comm. 2006, 342: 170-178.
18. Guilhot C, Otal I, Rompaey IV, et al. Efficienttransposition in Mycobacteria: Construction of Mycobacterium smegmatis insertional mutant libraries. Journal of Bacteriology 1994, 176(2): 535-539.
19. Pelicic, V., Reyrat, J.M., Gicquel, B. Generation of unmarked directed mutat- ions in mycobacteria using sucrose counter-selectable suicide vectors. Mol. Microbiol. 1996, 20: 919-925.
20. Pelicic V, and Gicquel B. Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on Mycobacteria. Journal of Bacteriology 1996, 178(4): 1197-1199.
21. Reyrat JM, Pelicic V, Gicquel B, et al. Counterselectable markers: Untapped tools for bacterial genetics and pathogenesis. Infection and Immunity 1998, 66(9): 4011-4017.
22. Mars AE, Kingma J, Kaschabek SR, et al.Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31. Journal of Bacteriology 1999, 181(4): 1309-1318.
23. Wulf B, Miryam A, Joseph S L. The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA). The EMBO journal. 2000, 19(24): 6652-63.
24. Daffe M, McNeil M, Brennan PJ. Major structural features of the cell wall arabinogalactans of Mycobacterium, Rhodococcus, and Nocardia spp. Carbo-hydr. Res. 1993,249: 383-398.
25. Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 2003,48: 77-84.
26. Apoorva B, Laurent K, Annie ZD. Conditional Depletion of KasA, a Key Enzyme of Mycolic Acid. Journal of bacteriology. 2005,187(22): 7596–7606.
27. Giraud MF, Naismith JH. The rhamnose pathway. Curr. Opin. in Structural Biol. 2000,10: 687-696.
28. Ma Y, Stern RJ, Scherman MS, et al., Drug targeting Mycobacterium tuberc- ulosis cell wall synthesis: Genetics of dTDP-Rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-Rhamnose. Antimicrobial Agents and Chemotherapy 2001, 45(5): 1407-1416.
29. Mills JA, Motichka K, Jucker M, et al. Inactivation of the mycobacterial rhamnosyltransferase, which is needed for the formation of the arabino- galactan-peptidoglycan linker, leads to irreversible loss of viability. J. Biol. Chem. 2004,279: 43540-43546.
30. Karlsson C, Jansson PE, Sorensen UB. The chemical structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A.. Eur. J. Biochem. 1998, 255(1): 296-302.
31. Schaffer C, Messner P. Surface-layer glycoproteins: an example for the diversity of bacterial glycosylation with promising impacts on nanobiote- chnology. Glycobiology 2004, 14: 31R-42R.
32. Mikusova K, Slayden RA, Besra GS, et al. Biogenesis of the mycobacterial cell wall and the site of action of ethambutol. Antimicrob. Agents Chemother. 1995,39: 2484-89.
33. Escuyer VE, Lety MA, Torrelles JB, et al. The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan. J Biol Chem. 2001, 276: 48854-62.
1. World Health Organization. Global tuberculosis control: surveillance, planning, financing. WHO report 2007. Geneva, (WHO/HTM/TB/2007.376).
2. World Health Organization. Tuberculosis. WHO fact sheet 104. 2007
3. World Health Organization. What is DOTS? A guide to understanding the WHO-recommended TB control strategy known as DOTS. Geneva, 1999 (document WHO/CDS/CPC/TB/99.270).
4. Susan ED, Richard EC. From magic bullets back to the Magic Mountain: the rise of extensively drug-resistant tuberculosis. Nature Medicine 2007,13(3): 295-8.
5. Centers for Disease Control and Prevention (CDC). Emergence of Mycoba- cterium tuberculosis with extensive resistance to second-line drugs-- worldwide, 2000-2004. MMWR Morb. Mortal. Wkly. Rep. 2006, 55(11): 301-305.
6. World Health Organization. The Stop TB Strategy. 2006, WHO/HTM/STB/ 2006.37.
7. World Health Organization. Treatment of tuberculosis: guidelines for national programmes, third edition. Geneva, 2003 (WHO/CDS/TB/2003.313).
8. Henry M, Michael K, Robert MJ. Treatment of TB and latent TB infection. JAMA, 2005, 293(22): 2776-85.
9. Banerjee A, Dubnau E, Quemard A, et al. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 1994, 263: 227–230.
10. Johnsson K, Froland WA, Schultz PG. Overexpression, purification, and characterization of the catalase-peroxidase KatG from Mycobacterium tuberculosis. J. Biol. Chem. 1997, 272: 2835-2840.
11. Lei B, Wei CJ, Tu SC. Action mechanism of antitubercular isoniazid. Activation by Mycobacterium tuberculosis KatG, isolation, and character- ization of inha inhibitor. J. Biol. Chem. 2000, 275: 2520–2526.
12. Ghiladi RA, Medzihradszky KF, Rusnak FM, et al. Correlation between isoniazid resistance and superoxide reactivity in mycobacterium tuberculosis KatG. J. Am. Chem. Soc. 2005, 127:13428–13442.
13. Rozwarski DA, Grant GA, Barton DH, et al. Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. Science 1998,279(5347): 98–102.
14. Ducasse-Cabanot S, Cohen-Gonsaud M, Marrakchi H, et al. In vitro inhibition of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein reductase MabA by isoniazid. Antimicrob. Agents Chemother. 2004, 48: 242–249.
15. Argyrou A, Vetting MW, Aladegbami B, et al. Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid. Nat. Struct. Mol. Biol. 2006, 13: 408–413.
16. Scior T, Meneses Morales I, Garces Eisele SJ, et al. Antitubercular isoniazid and drug resistance of Mycobacterium tuberculosis--a review. Arch. Pharm. 2002, 335: 511–525
17. Cohen T, Becerra MC, Murray MB. Isoniazid resistance and the future of drug-resistant tuberculosis. Microb. Drug Resist. 2004, 10: 280–285.
18. Salman S, Param T, Christian B, et al. Isoniazid induces its own resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother., 2007, 10:1128
19. Jarvis B, Harriet M. Rifapentine. Drugs. 1998, 56: 607–616.
20. Lilly KWY, David L, Peter JC. Bacteriological and molecular analysis of rifampin-resistant mycobacterium tuberculosis strains isolated in Australia. J Clin Microbiol. 1999, 37(12): 3844–3850.
21. Cynamon MH, Speirs RJ, Welch JT, In vitro antimycobacterial activity of 5-chloropyrazinamide. Antimicrob. Agents Chemother. 1998, 42: 462–463.
22. Heifets LB, Flory MA, Lindholm-Levy PJ. Does pyrazinoic acid as an active moiety of pyrazinamide have specific activity against Mycobacterium tuberculosis? Antimicrob. Agents Chemother. 1989, 33: 1252–1254.
23. Morlock GP, Crawford JT, Butler WR, et al. Phenotypic characterization of pncA mutants of Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2000, 44: 2291–2295.
24. Speirs RJ, Welch JT, Cynamon M. Activity of n-propyl pyrazinoate against pyrazinamide-resistant Mycobacterium tuberculosis: investigations into mechanism of action of and mechanism of resistance to pyrazinamide. Antim- icrob. Agents Chemother. 1995, 39: 1269–1271.
25. Zhang Y, Scorpio A, Nikaido H, et al. Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide. J. Bacteriol. 1999, 181: 2044–2049.
26. Mikusova K, Slayden RA, Besra GS, et al. Biogenesis of the mycobacterial cellwall and the site of action of ethambutol. Antimicrob. Agents Chemother. 1995,39: 2484-89.
27. Plinke C, Rusch-Gerdes S and Niemann S. Significance of mutations in embB codon 306 for prediction of ethambutol resistance in clinical Mycobacterium tuberculosis isolates. Antimicrob Agents Chemother. 2006, 50(5): 1900-2.
28. Carter AP, Clemons WM, Brodersen D,et al. Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 2000, 407: 340–348.
29. Lambert PA. Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria. J. Appl. Microbiol. 2002, 92: 46S–54S.
30. Mailaender C, Reiling N, Engelhardt H, et al. The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis. Microbiology 2004, 150: 853–864.
31. Stephan J, Mailaender C, Etienne G, et al. Multidrug resistance of a porin deletion mutant of Mycobacterium smegmatis. Antimicrob. Agents Chemother. 2004, 48: 4163–4170.
32. Honore N, Cole ST. Streptomycin resistance in mycobacteria. Antimicrob. Agents Chemother. 1994, 38: 238–242.
33. Bottger EC. Resistance to drugs targeting protein synthesis in mycobacteria. Trends Microbiol. 1994, 2: 416–421.
34. Levine C, Hiasa H, Marians KJ. DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities. Biochem. Biophys. Acta 1998, 1400: 29–43.
35. Champoux JJ. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 2001, 70: 369–413.
36. Corbett KD, Berger JM. Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu. Rev. Biophys. Biomol. Struct. 2004, 33: 95–118.
37. Aubry A, Pan XS, Fisher LM, et al. Mycobacterium tuberculosis DNA gyrase: interaction with quinolones and correlation with antimycobacterial drug activity. Antimicrob. Agents Chemother. 2004, 48: 1281–1288.
38. Aubry A, Fisher LM, Jarlier V, et al. First functional characterization of a singly expressed bacterial type II topoisomerase: the enzyme from Mycoba-cterium tuberculosis. Biochem. Biophys. Res. Commun. 2006, 348: 158–165.
39. Campbell EA, Korzheva N, Mustaev A, et al. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 2001, 104: 901–912.
40. Zenkin N, Kulbachinskiy A, Bass I, et al. Different rifampin sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA polymerases are not explained by the difference in the ?-subunit rifampin regions I and II. Antimicrobial Agents and Chemotherapy 2005, 49: 1587-1590.
41. Zhang Y, Permar S, Sun Z. Conditions that may affect the results of suscepti- bility testing of Mycobacterium tuberculosis to pyrazinamide. Journal of Medical Microbiology 2002, 51: 42–49.
42. Scorpio A, Zhang Y. Mutations in pncA, a gene encoding pyrazinamidase/ nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nature Medicine 1996, 2: 662–667.
43. Zhang Y, Scorpio A, Nikaido H, et al. Role of acid pH and deficient efflux of pyrazinoic acid in the unique susceptibility of Mycobacterium tuberculosis to pyrazinamide. Journal of Bacteriology 1999, 181: 2044–2049.
44. Boshoff HI, Mizrahi V, Barry CE 3rd. Effects of pyrazinamide on fatty acid synthesis by whole mycobacterial cells and purified fatty acid synthase I. Journal of Bacteriology 2002, 184: 2167–2172.
45. Zhang Y, Wade MM, Scorpio A, et al. Mode of action of pyrazinamide: disrupt- tion of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid. Journal of Antimicrobial Chemotherapy 2003, 52: 790–795.
46. Kaim G, Dimroth P. ATP synthesis by the F1Fo ATP synthase of Escherichia coli is obligatorily dependent on the electric potential. FEBS Letters 1998, 434: 57–60.
47. Telenti A, Philipp WJ, Sreevatsan S, et al. The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nature Med. 1997, 3: 567–570.
48. Alcaide F, Pfyffer GE, Telenti A. Role of embB in natural and acquired resistance to ethambutol in mycobacteria. Antimicrob. Agents Chemother. 1997,41: 2270–2273.
49. Escuyer VE, Lety MA, Torrelles JB, et al. The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan. J Biol Chem. 2001, 276: 48854-62.
50. Mailaender C, Reiling N, Engelhardt H, et al. The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis. Microbiology 2004, 150: 853-64.
2. Susan ED, Richard EC. From magic bullets back to the Magic Mountain: the rise of extensively drug-resistant tuberculosis. Nature Medicine 2007,13(3): 295-8.
3. Raviglione MC and Smith IM. XDR Tuberculosis — Implications for Global Public Health. N. Engl. J. Med. 2007, 356: 656-659.
4. Wise J. Southern Africa is moving swiftly to combat the threat of XDR-TB. Bull World Health Organ. 2006, 84(12): 924-5.
5. Brennan PJ and Besra GS. Structure, function and biogenesis of the mycob- acterial cell wall. Biochemical Society Transactions 1997, 25(1): 188-94.
6. Khasnobis S, Escuyer VE and Chatterjee D. Emerging therapeutic targets in tuberculosis: post-genomic era. Expert Opinion on Therapeutic Targets 2002, 6(1): 21-40.
7. Brennan PJ and Nikaido H. The envelope of mycobacteria. Annual Reviews of Biochemistry 1995, 64: 29-63.
8. Crick DC, Brennan PJ and McNeil MR. The cell wall of Mycobacterium tuberculosis. In Tuberculosis (2 nd Edition), 2004,115-134. Edited by Rom WN and Garay SM, Lippincott Williams & Wilkins, Philadelphia.
9. Stevenson G, Neal B, Liu D, et al. Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster. Journal of Bacteriology 1994, 176: 4144-4156.
10. Jiang XM, Neal B, Santiago F, et al. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mole- cular Microbiology 1991, 5(3): 695-713.
11. Koplin R, Wang G, Hotte B, et al. A 3.9-kb DNA Region of Xanthomonas campestris pv. campestris that is necessary for lipopolysaccharide production encodes a set of enzymes involved in the synthesis of dTDP-Rhamnose. Journal of Bacteriology 1993, 175(24): 7786-7792.
12. Tsukioka Y, Yamashita Y, Oho T, et al. Biological function of the dTDP- Rhamnose synthesis pathway in Streptococcus mutans. Journal of Bacteriology 1997, 179(4): 1126-1134.
13. Ma Y, Mills J, Belisle JT, et al. Determination of the pathway for rhmnnose biosynthesis in mycobacteria: cloning, sequencing and expression of the M. tuberculosis gene encoding α-D-glucose-1-phosphate thymidylyltransferase. Microbiology 1997, 143: 937-945.
14. Boels IC, Beerthuyzen MM, Kosters MHW, et al. Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP- Rhamnose biosynthesis. Journal of Bacteriology 2004, 186(5): 1239-1248.
15. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393: 537-544.
16. Ma Y, Pan F and McNeil M. The formation of dTDP-rhamnose is essential for growth of mycobacteria. Journal of Bacteriology 2002, 184(12): 3392-5.
17. Li, W., Xin, Y., McNeil, M.R., Ma, Y. rmlB and rmlC genes are essential for growth of mycobacteria, Biochem. Biophys. Res. Comm. 2006, 342: 170-178.
18. Guilhot C, Otal I, Rompaey IV, et al. Efficienttransposition in Mycobacteria: Construction of Mycobacterium smegmatis insertional mutant libraries. Journal of Bacteriology 1994, 176(2): 535-539.
19. Pelicic, V., Reyrat, J.M., Gicquel, B. Generation of unmarked directed mutat- ions in mycobacteria using sucrose counter-selectable suicide vectors. Mol. Microbiol. 1996, 20: 919-925.
20. Pelicic V, and Gicquel B. Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on Mycobacteria. Journal of Bacteriology 1996, 178(4): 1197-1199.
21. Reyrat JM, Pelicic V, Gicquel B, et al. Counterselectable markers: Untapped tools for bacterial genetics and pathogenesis. Infection and Immunity 1998, 66(9): 4011-4017.
22. Mars AE, Kingma J, Kaschabek SR, et al.Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31. Journal of Bacteriology 1999, 181(4): 1309-1318.
23. Wulf B, Miryam A, Joseph S L. The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA). The EMBO journal. 2000, 19(24): 6652-63.
24. Daffe M, McNeil M, Brennan PJ. Major structural features of the cell wall arabinogalactans of Mycobacterium, Rhodococcus, and Nocardia spp. Carbo-hydr. Res. 1993,249: 383-398.
25. Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 2003,48: 77-84.
26. Apoorva B, Laurent K, Annie ZD. Conditional Depletion of KasA, a Key Enzyme of Mycolic Acid. Journal of bacteriology. 2005,187(22): 7596–7606.
27. Giraud MF, Naismith JH. The rhamnose pathway. Curr. Opin. in Structural Biol. 2000,10: 687-696.
28. Ma Y, Stern RJ, Scherman MS, et al., Drug targeting Mycobacterium tuberc- ulosis cell wall synthesis: Genetics of dTDP-Rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-Rhamnose. Antimicrobial Agents and Chemotherapy 2001, 45(5): 1407-1416.
29. Mills JA, Motichka K, Jucker M, et al. Inactivation of the mycobacterial rhamnosyltransferase, which is needed for the formation of the arabino- galactan-peptidoglycan linker, leads to irreversible loss of viability. J. Biol. Chem. 2004,279: 43540-43546.
30. Karlsson C, Jansson PE, Sorensen UB. The chemical structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A.. Eur. J. Biochem. 1998, 255(1): 296-302.
31. Schaffer C, Messner P. Surface-layer glycoproteins: an example for the diversity of bacterial glycosylation with promising impacts on nanobiote- chnology. Glycobiology 2004, 14: 31R-42R.
32. Mikusova K, Slayden RA, Besra GS, et al. Biogenesis of the mycobacterial cell wall and the site of action of ethambutol. Antimicrob. Agents Chemother. 1995,39: 2484-89.
33. Escuyer VE, Lety MA, Torrelles JB, et al. The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan. J Biol Chem. 2001, 276: 48854-62.
1. World Health Organization. Global tuberculosis control: surveillance, planning, financing. WHO report 2007. Geneva, (WHO/HTM/TB/2007.376).
2. World Health Organization. Tuberculosis. WHO fact sheet 104. 2007
3. World Health Organization. What is DOTS? A guide to understanding the WHO-recommended TB control strategy known as DOTS. Geneva, 1999 (document WHO/CDS/CPC/TB/99.270).
4. Susan ED, Richard EC. From magic bullets back to the Magic Mountain: the rise of extensively drug-resistant tuberculosis. Nature Medicine 2007,13(3): 295-8.
5. Centers for Disease Control and Prevention (CDC). Emergence of Mycoba- cterium tuberculosis with extensive resistance to second-line drugs-- worldwide, 2000-2004. MMWR Morb. Mortal. Wkly. Rep. 2006, 55(11): 301-305.
6. World Health Organization. The Stop TB Strategy. 2006, WHO/HTM/STB/ 2006.37.
7. World Health Organization. Treatment of tuberculosis: guidelines for national programmes, third edition. Geneva, 2003 (WHO/CDS/TB/2003.313).
8. Henry M, Michael K, Robert MJ. Treatment of TB and latent TB infection. JAMA, 2005, 293(22): 2776-85.
9. Banerjee A, Dubnau E, Quemard A, et al. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 1994, 263: 227–230.
10. Johnsson K, Froland WA, Schultz PG. Overexpression, purification, and characterization of the catalase-peroxidase KatG from Mycobacterium tuberculosis. J. Biol. Chem. 1997, 272: 2835-2840.
11. Lei B, Wei CJ, Tu SC. Action mechanism of antitubercular isoniazid. Activation by Mycobacterium tuberculosis KatG, isolation, and character- ization of inha inhibitor. J. Biol. Chem. 2000, 275: 2520–2526.
12. Ghiladi RA, Medzihradszky KF, Rusnak FM, et al. Correlation between isoniazid resistance and superoxide reactivity in mycobacterium tuberculosis KatG. J. Am. Chem. Soc. 2005, 127:13428–13442.
13. Rozwarski DA, Grant GA, Barton DH, et al. Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. Science 1998,279(5347): 98–102.
14. Ducasse-Cabanot S, Cohen-Gonsaud M, Marrakchi H, et al. In vitro inhibition of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein reductase MabA by isoniazid. Antimicrob. Agents Chemother. 2004, 48: 242–249.
15. Argyrou A, Vetting MW, Aladegbami B, et al. Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid. Nat. Struct. Mol. Biol. 2006, 13: 408–413.
16. Scior T, Meneses Morales I, Garces Eisele SJ, et al. Antitubercular isoniazid and drug resistance of Mycobacterium tuberculosis--a review. Arch. Pharm. 2002, 335: 511–525
17. Cohen T, Becerra MC, Murray MB. Isoniazid resistance and the future of drug-resistant tuberculosis. Microb. Drug Resist. 2004, 10: 280–285.
18. Salman S, Param T, Christian B, et al. Isoniazid induces its own resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother., 2007, 10:1128
19. Jarvis B, Harriet M. Rifapentine. Drugs. 1998, 56: 607–616.
20. Lilly KWY, David L, Peter JC. Bacteriological and molecular analysis of rifampin-resistant mycobacterium tuberculosis strains isolated in Australia. J Clin Microbiol. 1999, 37(12): 3844–3850.
21. Cynamon MH, Speirs RJ, Welch JT, In vitro antimycobacterial activity of 5-chloropyrazinamide. Antimicrob. Agents Chemother. 1998, 42: 462–463.
22. Heifets LB, Flory MA, Lindholm-Levy PJ. Does pyrazinoic acid as an active moiety of pyrazinamide have specific activity against Mycobacterium tuberculosis? Antimicrob. Agents Chemother. 1989, 33: 1252–1254.
23. Morlock GP, Crawford JT, Butler WR, et al. Phenotypic characterization of pncA mutants of Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2000, 44: 2291–2295.
24. Speirs RJ, Welch JT, Cynamon M. Activity of n-propyl pyrazinoate against pyrazinamide-resistant Mycobacterium tuberculosis: investigations into mechanism of action of and mechanism of resistance to pyrazinamide. Antim- icrob. Agents Chemother. 1995, 39: 1269–1271.
25. Zhang Y, Scorpio A, Nikaido H, et al. Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide. J. Bacteriol. 1999, 181: 2044–2049.
26. Mikusova K, Slayden RA, Besra GS, et al. Biogenesis of the mycobacterial cellwall and the site of action of ethambutol. Antimicrob. Agents Chemother. 1995,39: 2484-89.
27. Plinke C, Rusch-Gerdes S and Niemann S. Significance of mutations in embB codon 306 for prediction of ethambutol resistance in clinical Mycobacterium tuberculosis isolates. Antimicrob Agents Chemother. 2006, 50(5): 1900-2.
28. Carter AP, Clemons WM, Brodersen D,et al. Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 2000, 407: 340–348.
29. Lambert PA. Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria. J. Appl. Microbiol. 2002, 92: 46S–54S.
30. Mailaender C, Reiling N, Engelhardt H, et al. The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis. Microbiology 2004, 150: 853–864.
31. Stephan J, Mailaender C, Etienne G, et al. Multidrug resistance of a porin deletion mutant of Mycobacterium smegmatis. Antimicrob. Agents Chemother. 2004, 48: 4163–4170.
32. Honore N, Cole ST. Streptomycin resistance in mycobacteria. Antimicrob. Agents Chemother. 1994, 38: 238–242.
33. Bottger EC. Resistance to drugs targeting protein synthesis in mycobacteria. Trends Microbiol. 1994, 2: 416–421.
34. Levine C, Hiasa H, Marians KJ. DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities. Biochem. Biophys. Acta 1998, 1400: 29–43.
35. Champoux JJ. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 2001, 70: 369–413.
36. Corbett KD, Berger JM. Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu. Rev. Biophys. Biomol. Struct. 2004, 33: 95–118.
37. Aubry A, Pan XS, Fisher LM, et al. Mycobacterium tuberculosis DNA gyrase: interaction with quinolones and correlation with antimycobacterial drug activity. Antimicrob. Agents Chemother. 2004, 48: 1281–1288.
38. Aubry A, Fisher LM, Jarlier V, et al. First functional characterization of a singly expressed bacterial type II topoisomerase: the enzyme from Mycoba-cterium tuberculosis. Biochem. Biophys. Res. Commun. 2006, 348: 158–165.
39. Campbell EA, Korzheva N, Mustaev A, et al. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 2001, 104: 901–912.
40. Zenkin N, Kulbachinskiy A, Bass I, et al. Different rifampin sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA polymerases are not explained by the difference in the ?-subunit rifampin regions I and II. Antimicrobial Agents and Chemotherapy 2005, 49: 1587-1590.
41. Zhang Y, Permar S, Sun Z. Conditions that may affect the results of suscepti- bility testing of Mycobacterium tuberculosis to pyrazinamide. Journal of Medical Microbiology 2002, 51: 42–49.
42. Scorpio A, Zhang Y. Mutations in pncA, a gene encoding pyrazinamidase/ nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nature Medicine 1996, 2: 662–667.
43. Zhang Y, Scorpio A, Nikaido H, et al. Role of acid pH and deficient efflux of pyrazinoic acid in the unique susceptibility of Mycobacterium tuberculosis to pyrazinamide. Journal of Bacteriology 1999, 181: 2044–2049.
44. Boshoff HI, Mizrahi V, Barry CE 3rd. Effects of pyrazinamide on fatty acid synthesis by whole mycobacterial cells and purified fatty acid synthase I. Journal of Bacteriology 2002, 184: 2167–2172.
45. Zhang Y, Wade MM, Scorpio A, et al. Mode of action of pyrazinamide: disrupt- tion of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid. Journal of Antimicrobial Chemotherapy 2003, 52: 790–795.
46. Kaim G, Dimroth P. ATP synthesis by the F1Fo ATP synthase of Escherichia coli is obligatorily dependent on the electric potential. FEBS Letters 1998, 434: 57–60.
47. Telenti A, Philipp WJ, Sreevatsan S, et al. The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nature Med. 1997, 3: 567–570.
48. Alcaide F, Pfyffer GE, Telenti A. Role of embB in natural and acquired resistance to ethambutol in mycobacteria. Antimicrob. Agents Chemother. 1997,41: 2270–2273.
49. Escuyer VE, Lety MA, Torrelles JB, et al. The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan. J Biol Chem. 2001, 276: 48854-62.
50. Mailaender C, Reiling N, Engelhardt H, et al. The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis. Microbiology 2004, 150: 853-64.