结核分枝杆菌rmlB和rmlC基因是分枝杆菌生长相关基因
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摘要
结核分枝杆菌(M．tuberculosis)是引起结核病的病原体。由于耐多药菌株的出现和HIV的协同作用，使得一度被控制的结核病的发病率在全球范围内显著回升，因此，寻找新一代抗结核药物已迫在眉睫。细胞壁是分枝杆菌赖以生存的结构基础，其核心结构由分枝菌酸、聚阿拉伯糖半乳糖及肽聚糖三种组分组成。分枝菌酸和聚阿拉伯糖半乳糖通过衔接双糖(L-鼠李糖-D-N-乙酰葡糖胺)共价连接到肽聚糖大分子上，衔接双糖尤其是鼠李糖可作为研发新一代抗结核药物的理想靶标。衔接双糖中鼠李糖的糖基供体是dTDP-鼠李糖，四种酶(Rm1A-D)参与了由底物葡萄糖-1-磷酸合成dTDP-鼠李糖的过程，编码这四种酶的rm1A-D基因存在于结核分枝杆菌基因组中不同的位置，其中rm1B和rm1C同属于一个操纵子。尽管我们已发现在结核分枝杆菌中不存在dTDP-鼠李糖生物合成的补救代谢途径，但仍有必要从理论上证明编码结核分枝杆菌dTDP-鼠李糖合成酶系的rm1A-D基因为结核分枝杆菌的生长相关基因。
本论文的目的是用基因剔除方法证明结核分枝杆菌中编码Rm1B(dTDP-D-葡萄糖-4，6-脱氢酶)和Rm1C(dTDP-4-酮基-6-脱氧-D-葡萄糖3，5表异构酶)的rm1B和C基因是分枝杆菌生长过程中所必需的基因，这将为dTDP-鼠李糖作为研发抗结核新药的理想靶标提供更有力的证据。
利用与结核分枝杆菌有相同细胞壁结构，但生长快速且无致病性的耻垢分枝杆菌(Mycobacterium smegmatis，Sm)mc~2155菌株作为实验模型。用携带Sm rm1B：：Kan~R突变基因的条件复制性质粒转化mc~2155菌株，在42℃条件下筛选出mc~2155突变菌株M1(其基因组中同时存在Sm rm1B和Sm rm1B：：Kan~R)。用携带结核分枝杆菌M．tuberculosis，Tb)rm1B和rm1C基因的营救质粒转化mc~2155 M1突变菌株，Tb rm1B和Tb rm1C基因的表达产物可补偿mc~2155 M1突变菌株中突变的Sm rm1B以及突变的Sm rm1C基因的功能，在30℃条件下筛选出mc~2155 M2突变菌株(其基因组中只存在Sm rm1B：：Kan~R，即Sm rm1B基因剔除；同时Sm rm1B：：Kan~R下游的Sm rm1C基因发生框移突变)。分别在30℃和42℃条件下测定
Mycobacterium tuberculosis, a species of the genus Mycobacteria is the pathogen of tuberculosis. M. tuberculosis had once been controlled, but now it poses a major public health problem. The emergence of multi-drug resistant strains and co-infection of M. tuberculosis with HIV are the two greatest factors that have contributed to the global resurgence of TB. Thus, more efficient anti-TB drugs are desperately needed.
The cell wall is necessary for mycobacterial viability. The mybacterial cell wall core consists of three interconnected 'macromolecules': the outermost, mycolic acids are esterified to the middle component arabinogalacan (AG), and AG is attached to the peptidoglycan via a linker disaccharide, α-L-rhamnosyl-(1→3)-α-D-N acetyl-glucosaminosyl-1-posphate. The structure of the mycobacteria cell wall strongly suggests that the linker disaccharide is an important component. The L-rhamnosyl residue of linker disaccharide is provided with a sugar donor, dTDP-rhamnose. The biosynthetic pathway of dTDP-rhamnose consists of four-step reactions from α-D-glucose-1-phosphate and TTP to dTDP-rhamnose through three intermediates, and four reactions are catalyzed by four enzymes, α-D-glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-D-glucose-4, 6-dehydratase (RmlB), dTDP-4-keto-6-deoxy-glucose-3, 5-epimerase (RmlC), and dTDP-6-deoxy-L-lyxo-4-hexulose reductase (dTDP-4-keto-L-rhamnose reductase) (RmlD), respectively. The rmlA (Rv0334), rmlB (Rv3464), rmlC (Rv3465), and rmlD (Rv3266c) 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 is found in an operon with wbbL (Rv3265c) and manB (Rv3264c). The findings and the facts that there is no salvage pathway for the formation of dTDP-rhamnose and L-rhamnosyl residues are not found in humans support the enzymes involved in dTDP-rhamnose biosynthesis are important targets for
本论文的目的是用基因剔除方法证明结核分枝杆菌中编码Rm1B(dTDP-D-葡萄糖-4，6-脱氢酶)和Rm1C(dTDP-4-酮基-6-脱氧-D-葡萄糖3，5表异构酶)的rm1B和C基因是分枝杆菌生长过程中所必需的基因，这将为dTDP-鼠李糖作为研发抗结核新药的理想靶标提供更有力的证据。
利用与结核分枝杆菌有相同细胞壁结构，但生长快速且无致病性的耻垢分枝杆菌(Mycobacterium smegmatis，Sm)mc~2155菌株作为实验模型。用携带Sm rm1B：：Kan~R突变基因的条件复制性质粒转化mc~2155菌株，在42℃条件下筛选出mc~2155突变菌株M1(其基因组中同时存在Sm rm1B和Sm rm1B：：Kan~R)。用携带结核分枝杆菌M．tuberculosis，Tb)rm1B和rm1C基因的营救质粒转化mc~2155 M1突变菌株，Tb rm1B和Tb rm1C基因的表达产物可补偿mc~2155 M1突变菌株中突变的Sm rm1B以及突变的Sm rm1C基因的功能，在30℃条件下筛选出mc~2155 M2突变菌株(其基因组中只存在Sm rm1B：：Kan~R，即Sm rm1B基因剔除；同时Sm rm1B：：Kan~R下游的Sm rm1C基因发生框移突变)。分别在30℃和42℃条件下测定
Mycobacterium tuberculosis, a species of the genus Mycobacteria is the pathogen of tuberculosis. M. tuberculosis had once been controlled, but now it poses a major public health problem. The emergence of multi-drug resistant strains and co-infection of M. tuberculosis with HIV are the two greatest factors that have contributed to the global resurgence of TB. Thus, more efficient anti-TB drugs are desperately needed.
The cell wall is necessary for mycobacterial viability. The mybacterial cell wall core consists of three interconnected 'macromolecules': the outermost, mycolic acids are esterified to the middle component arabinogalacan (AG), and AG is attached to the peptidoglycan via a linker disaccharide, α-L-rhamnosyl-(1→3)-α-D-N acetyl-glucosaminosyl-1-posphate. The structure of the mycobacteria cell wall strongly suggests that the linker disaccharide is an important component. The L-rhamnosyl residue of linker disaccharide is provided with a sugar donor, dTDP-rhamnose. The biosynthetic pathway of dTDP-rhamnose consists of four-step reactions from α-D-glucose-1-phosphate and TTP to dTDP-rhamnose through three intermediates, and four reactions are catalyzed by four enzymes, α-D-glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-D-glucose-4, 6-dehydratase (RmlB), dTDP-4-keto-6-deoxy-glucose-3, 5-epimerase (RmlC), and dTDP-6-deoxy-L-lyxo-4-hexulose reductase (dTDP-4-keto-L-rhamnose reductase) (RmlD), respectively. The rmlA (Rv0334), rmlB (Rv3464), rmlC (Rv3465), and rmlD (Rv3266c) 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 is found in an operon with wbbL (Rv3265c) and manB (Rv3264c). The findings and the facts that there is no salvage pathway for the formation of dTDP-rhamnose and L-rhamnosyl residues are not found in humans support the enzymes involved in dTDP-rhamnose biosynthesis are important targets for
引文
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15. 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.
16. 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.
17. 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.
18. Boels 1C, 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.
19. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393: 537-544.
20. Ma Y, Stern RJ, Scherman MS, et al., Drug targeting Mycobacterium tuberculosis 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.
21. Weston ARJ, Stern RE, Lee PM, et al. Biosynthetic origin of mycobacterial cell wall galactofuranosyl residues. Tuber. Lung Dis. 1997, 78:123-131.
22. 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.
23. Pelicic V, Jackson M, Reyrat JM, et al. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 1997, 94:10955-10960.
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28. Guilhot C, Otal I, Rompaey IV, et al. Efficienttransposition in Mycobacteria: Construction of Mycobacterium smegmatis insertional mutant libraries. Journal of Bacteriology 1994, 176(2):535-539.29. Pan F, Jackson M, Ma Y and McNeil M. Determination that cell wall core galactofuran synthesis is essential for growth of mycobacteria. Journal of Bacteriology 2001, 183(13): 3991-8.
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2. Corbett EL, et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch. Intern. Med. 2003, 163:1009-1021.
3. Dye C, Scheele S, Dolin P, et al. Global burden of tuberculosis estimated incidence, prevalence and mortality by country. JAMA 1999, 282(7):677-686.
4. Murray CJL and Lopez AD. Global mortality, disability, and the contribution of risk factors: Global Burden of Disease Survey. Lancet 1997,349(9063): 1436-1442.
5. Bloom BR and Murrary CJL. Tuberculosis: commentary on a reemergent killer. Science 1992, 257:1055-1064.
6. O'Brien RJ. Tuberculosis. Scientific Blueprint for Tuberculosis Drug Development. Tuberculosis (Edinb) 2001,81 (S1): 1-52.
7. Barry III CE and Duncan K. Tuberculosis - strategies towards anti-infectives for a chronic disease. Drug Discovery Today: Therapeutic Strategies 2004, 1(4): 491-496.
8. Warner DF, and Mizrahi V.Mycobacterial genetics in target validation. Drug Discovery Today: Technologies 2004, l(2):93-98.
9. Brennan PJ and Besra GS. Structure, function and biogenesis of the mycobacterial cell wall. Biochemical Society Transactions 1997, 25(1):188-94.
10. 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.
11. Brennan PJ and Nikaido H. The envelope of mycobacteria. Annual Reviews of Biochemistry 1995,64:29-63.
12. 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.
13. 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.
14. Jiang XM, Neal B, Santiago F, et al. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Molecular Microbiology 1991, 5(3):695-713.
15. 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.
16. 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.
17. 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.
18. Boels 1C, 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.
19. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393: 537-544.
20. Ma Y, Stern RJ, Scherman MS, et al., Drug targeting Mycobacterium tuberculosis 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.
21. Weston ARJ, Stern RE, Lee PM, et al. Biosynthetic origin of mycobacterial cell wall galactofuranosyl residues. Tuber. Lung Dis. 1997, 78:123-131.
22. 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.
23. Pelicic V, Jackson M, Reyrat JM, et al. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 1997, 94:10955-10960.
24. 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.
25. 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.
26. Pelicic V, Reyrat JM and Gicquel B. Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors. Molecular Microbiology 1996, 20(5):919-925.
27. 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.
28. Guilhot C, Otal I, Rompaey IV, et al. Efficienttransposition in Mycobacteria: Construction of Mycobacterium smegmatis insertional mutant libraries. Journal of Bacteriology 1994, 176(2):535-539.29. Pan F, Jackson M, Ma Y and McNeil M. Determination that cell wall core galactofuran synthesis is essential for growth of mycobacteria. Journal of Bacteriology 2001, 183(13): 3991-8.
30. Cole ST, Eiglmeier K, Parkhill J, et al. Massive gene decay in the leprosy bacillus. Nature 2001,409:1007-1011.
31. The genome of Mycobacterium leprae: a minimal mycobacterialgene set. Genome Biology 2001, 2(8): reviews 1023.1-1023.8
32. Cole ST. Comparative mycobacterial genomics as a tool for drug target and antigen discovery. Eur Respir Journal 2002,20: Suppl. 36:78s-86s.
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