【作者】 吴腾飞；

【导师】 刘波； Robert. A. Nicholas；

【作者基本信息】 吉林大学 ， 基础兽医学， 2010， 博士

【摘要】 淋病是由淋球菌引起的泌尿生殖系统的化脓性感染,严重危害着人类的健康。临床上主要采用抗生素治疗,近年来因抗生素的滥用和乱用,耐药菌株大量出现,急需调整抗生素用药策略和研发新型的抗淋球菌药物和药物作用靶点。溶解性转糖酶C（LtgC）通过催化切割肽聚糖β-1,4糖苷键断裂,在淋球菌分裂和生长中发挥重要作用,可能是潜在的药物作用靶点。本实验采用分子克隆和同源重组技术研究溶解性转糖酶C的活性位点和特异的结构域三对淋球菌生长分裂的影响,阐述作用机制,为研发新型抗淋球菌药物提供一定的理论和实践依据。采用分子生物学方法获得淋球菌FA19ltgC敲除株（FA19△ltgC）、D393A突变株(FA19ltgC_D393A)、D405A突变株(FA19ltgC_D405A)和结构域三缺失株(FA19ltgC_△DM3)和结构域三表面七个氨基酸突变株(FA19ltgC_7M)。野生型淋球菌FA19菌落表面光滑且明亮、无褶皱、边缘完整;但FA19△ltgC的菌落表面出现褶皱和凸凹不平,菌体生长缓慢,四小时停止生长;且FA19ltgC基因的遗传修复株（FA19 ltgC-com）可回复为FA19的表型,上述结果表明LtgC是淋球菌内重要的、保守的功能性蛋白,该蛋白的缺失导致淋球菌细胞分裂不完全,生长缓慢。FA19ltgC_D393A和FA19ltgC_D405A菌落表型和FA19△ltgC菌落表型一致。FA19ltgC_D393A、FA19ltgC_D405A和FA19△ltgC细胞生长速度相近,均比FA19野生株生长缓慢,该结果暗示393位和405位的天冬氨酸共同作用切割肽聚糖糖苷键断裂。本实验采用分子克隆技术敲除LtgC结构域三163-244位氨基酸,以及用氨基酸RGR替换LtgC的167-244位氨基酸,重组表达纯化获得两个LtgC结构域三突变体蛋白(LtgC_△163-244和LtgC_△167RGR244). LtgC_△163-244突变体蛋白在大肠杆菌体内没有表达,但LtgC_△167RGR244蛋白表达量高且可溶性良好。将LtgC_△167RGR244基因同源重组到淋球菌基因组,获得LtgC结构域三缺失株(FA19ltgC_△DM3). FA19ltgC_△DM3的表型和FA19△ltgC表型相似,生长速度缓慢。但FA19ltgC_7M的表型和野生型未见显著性差异。该结果表明,结构域三是LtgC帮助淋球菌生长的关键结构区域,但不是其他蛋白结合位点。大肠杆菌溶解性转糖酶A（ MltA）和淋球菌LtgC是同源蛋白,在结构和功能方面有很多相似性.分别用mltA和ltgC基因对ltgC缺失株进行遗传修复,发现ltgC和MltA的互补株完全回复淋球菌野生株的表型,而且MltA对淋球菌生长的促进作用比LtgC更强.在检测MltA对淋球菌肽聚糖的切割作用时,观察到MltA酶切活性受到淋球菌高度乙酰化的肽聚糖的抑制,但可溶表达的LtgC蛋白切割未见酶切肽聚糖活性。在LtgC的N端加入19个氨基酸的脂蛋白后, LtgC蛋白在E.coli Bl21中表达量很低,可能该脂蛋白对E.coli产生毒性作用使表达受到抑制,暗示LtgC蛋白需要脂蛋白序列协助发挥作用;携带脂蛋白序列的LtgC_D393A和LtgC_D405A突变株蛋白在大肠杆菌中表达量和LtgC可溶蛋白表达量相近,说明Asp393和Asp405位点突变抑制LtgC对大肠杆菌的毒性作用;但结构域三表面的七重氨基酸突变体蛋白表达量仍然很低,证明结构域三可能不是其他协同作用蛋白和LtgC的结合部位。综上所述,本研究证实LtgC是淋球菌生长和分裂的重要功能性酶,首次证明Asp393和Asp405是LtgC影响淋球菌生长的重要功能部位;结构域三的缺失抑制淋球菌细胞分裂,但它不是多酶复合体的结合位点;首次发现MltA可修复由LtgC缺失引起的淋球菌表型变化,并促进淋球菌生长;无脂蛋白序列的LtgC无酶切活性,表明脂蛋白序列是LtgC的重要结构。首次鉴定MltA体外活性受到淋球菌肽聚糖乙酰化抑制,可能与淋球菌避免自溶有关。本论文初步证明LtgC可能是潜在的淋球菌药物作用靶点,尤其是LtgC的Asp393和Asp405可视为筛选抑制剂的潜在靶标,为研发新型抗淋球菌药物提供一定的理论依据。更多还原

【Abstract】 Gonorrhea, is a common sexually transmitted diseases worldwide caused by N.gonorrhoeae, which evokes the pyogenic infection of urogenital system. Even though Gonorrhea could be successfully cured with antibiotics, drug resistant strains are an increasing issue in many countries due to misuse and overuse of antibiotics. So therapeutic strategy need to be adjusted based on the bacterial sensitivity and there is an urgent need to identify new drug targets in anti-gonoccoci and develop new drugs. Lytic transglycosylase C（LtgC） is essential for growth and division of N.gonorrhoeae by cleavingβ-1,4 glycosidic bond of peptidoglycan, considered as a promising targets for antimicrobials. In the present study, the effect of active sites and domain 3 （DM3）of LtgC on Gonococcal growth and duplication was demonstrated by molecular cloning and homelogens DNA technology, the mechanism of LtgC catalysis was exhibited to provide a theoretical basis for research and development of novel drug.N.gonorrhoeae FA19△ltgC, FA19ltgC_D393A, FA19ltgC_D405A, FA19ltgC_△DM3 and FA19ltgC_7M（which includes 7 a.a. mutations in DM3 of LtgC） were generated by homologous recombination technology. Under light microscopic observation, colony morphologyof N. gonorrhoeae FA19 showed shiny and smooth, convex, however colonies of FA19△ltgC were winkled, dull, rough and flatat bacterium surface,exhibited abnormal growth,stopped growing after 4 h cultivation. And colony morphology of the complemented strains was indistinguishable from that of FA19 wild type strain. These results proved LtgC is a conservative and functional protein in N.gonorrhoeae. The colony morphology of FA19ltgC_D393A and FA19ltgC_D405A mutants are identity with that of FA19△ltgC. Growth of three strains showed the similar retarded growth characteristic, suggesting both Asp393 and Asp405 in LtgC are essential for cleaving peptidoglycan of LtgC.163-244 Amino acid deletion mutant of LtgC(LtgC_△163-244) and amino acid 167-244 replaced by RGR mutant of LtgC(LtgC_△167RGR244) were constructed, expressed and purified. Although LtgC_△163-244 couldn’t be expressed in E.coli, LtgC167RGR244 mutant protein was expressed considerable quantities and soluble. The gene of LtgC_△167RGR244was transformed into FA19 to generate the FA19 LtgC_△DM, which showed the similar phenotype with FA19△LtgC, grew very tardily. Seven-amino-acid sites mutation in LtgC domain 3 in FA19 (FA19 ltgC_7M) was generated, morphology of its colony was consistent with that of FA19’s. These result showed DM3 was the critical domain of LtgC involved in growth and division of N. gonorrhoeae, but it’s not the binding site of other proteins in the multi-enzyme system.Murein Lytic transglycosylase A （MltA） in E.coli and LtgC are homologous proteins, containing many similarity in the structure and function. MltA or LtgC was transformed into the genome of FA19△LtgC to generate two complemented strains. Both ltgC and mltA complemented strains totally recovery the morphology deficiency in FA19△LtgC, and MltA complemented strain grew and separated faster than ltgC complemented strain. This data coincided with the result of MltA and LtgC digesting PG in vitro. The truncated LtgC didn’t show any activity in PG digestion, but truncated MltA can cleave PG with low-density O-acetylated, but be inhibited by high-density acetylated PG.LtgC was also expressed in E.coli as lipoprotein, but its expression quantity was weak by western blot detection, possible LtgC was toxic to E.coli, and the lipoprotein-sequence collaborated LtgC to fulfill its function. The expression of LtgC_7M protein also was inhibited by E.coli, proving DM3 was not the binding position of other factors as 7-a.a mutation in DM3 still was toxic to E.coli.In conclusion, LtgC is a critical enzyme in gonococci growth and division. The residues of Asp 393 and Asp405 was firstly proved to collaborate to surport the growth of N.gonorhoeae; DM 3 deletion in LtgC inhibits cell separation, but DM3 is not the binding sites of multi-enzymatic complex; MltA could rescue the morphology of FA19?LtgC; We firstly identified the activity of MltA in vitro was inhibited by O-acetylated gonoccocal PG, and sLtgC didn’t show any enzymatic activity. In this thesis, we primarily proved LtgC is a crucial and concerved protein the residues of Asp393 and Asp405 could be the great inhibitors binding target of anti-gonoccoci.更多还原