plcR在炭疽杆菌中的功能研究

plcR was detected in many Bacillus cereus and B.thuringiensis soil isolates and from strain collections. The plcR gene was also found to be present in the related bacteria B.anthracis and B.mycoides. Therefore, plcR is probably present in all members of the B.cereus group. However, the GenBank protein databases of finished and unfinished bacterial genomes were screened for sequences similar to that of the deduced amino acid sequence of plcR and no significant similarity was found, suggesting that plcR might be restricted to the members of the B.cereus group..In B. cereus and B.thuringiensis, PlcR acts by binding to a well-defined site (the PlcR-box), consisting of a-16bp consensus sequence present in promoter regions of PlcR-regulated genes. B. cereus virulence is thought to be a consequence of its secretion of diverse non-specific virulence factors such as phospholipases and enterotoxins. Conversely, in B.anthracis, a nonsense mutation in plcR results in a premature translational stop and consequently gene expression in B.anthracis differs drastically from that in B.cereus. .Moreover, the PlcR-regulated genes are dispersed on the chromosome. This observation is not consistent with the recent acquisition of the PlcR regulon by horizontal transfer, but instead favours the notion that B.anthracis, B.cereus and B.thuringiensis are derived from a common ancestral bacterium containing the plcR regulon. During the evolution, B.anthracis acquire the virulence large plasmid pXO1. The global regulator atxA, which is responsible for the virulence and spore formation of the B.anthracis, is thought incompatible with the functional PlcR. So, the PlcR-controlled regulon in B.anthracis was counterselected on account of its disadvantageous effects. It is just these observations motivated us to investigate “If the AtxA cannot coexist with the active plcR system?”..In this study, we will generate two plasmid-free strains on wild strain A16. Three kinds of plcR and papR DNA fragments were inserted into an expression plasmid pBE2A to construct three plcR-expression plasmids, and then they were introduced into the A16D2 and A16DD to constructed six recombinant B.anthracis strains. Then the “omics” technologies and mass spectrum will be used to carry out the “comparative researches” about the “transcriptome” and “proteomic profiling” between the six strains to elucidate the global effect that the PlcR on the B.anthracis. We also will investigate the effect of the expression of a functional PlcR on the virulence and the sporulation of B.anthracis using mouse infection model, cell proliferation assay and sporulation efficiency assay. We also will investigate if the functional PlcR will activate the hemolytic activity in B.anthracis and could the phenotype be kept stably after several continuously passage culture in in nutrient medium. All the results will be used to elucidate the compatibility of the pXO1-encoded regulator AtxA with the functional PlcR.

在蜡样芽胞杆菌（Bc）和苏云金芽胞杆菌（Bt）中，plcR是一个多效调控子。炭疽杆菌（Ba）与Bc和Bt同属于蜡样芽胞杆菌群，但是，所有的Ba的plcR基因都发生了一个特有的无义突变，使其编码的PlcR蛋白失去功能。研究者推测Ba、Bc和Bt可能起源于一个含有plcR的共同祖先，进化中Ba获得大质粒pXO1，在它上面的另一个全局调控基因atxA与plcR具有不相容性，导致了plcR被选择性失活。因此本研究提出了“在炭疽杆菌中plcR是否与atxA不相容？”这一科学问题。.本课题将以Ba野生株A16为出发菌，构建两个突变株，然后把插入了不同的plcR片段的表达载体导入突变株中，实现plcR在炭疽杆菌中功能性表达，然后对它们的转录谱、蛋白表达谱、对巨噬细胞及小鼠的毒力、芽胞形成能力已及激活表型稳定传代能力等方面进行比较研究，来探讨“plcR与atxA的相容性”。

在蜡样芽胞杆菌群中，关于两个全局调控plcR、atxA及芽胞形成能力三者的不相容性一直是有争议和令人感兴趣的问题。plcR基因是蜡样芽胞杆菌杆菌群中特有的重要毒力调控因子，但是为什么在炭疽杆菌中的plcR基因选择性失活，一直吸引着研究者的关注，也有很多互相矛盾的研究结果。.本研究通过质粒携带把外源plcR基因， plcRpapR基因，炭疽杆菌本身plcR基因（只把640位碱基突变为G）导入不同的炭疽杆菌菌株中（atxA+/-），并通过CRISPR/cas9基因编辑系统实现对不同炭疽杆菌菌株（atxA+/-）染色体上的plcR基因实现原位点突变，从而恢复plcR基因在炭疽杆菌中的活性，通过对这些菌株生长的差异、溶血酶、卵磷脂酶等毒力因子的差异、对炭疽杆菌atxA，pagA,lef等毒力因子的影响、对芽胞形成的影响的比较、对细胞及小鼠毒力的影响以及对炭疽杆菌蛋白表达谱的影响的比较，深入研究了炭疽杆菌中plcR的激活对atxA及芽胞形成的影响。我们的结果显示炭疽杆菌中plcR的激活使炭疽杆菌对小鼠的毒力减弱，同时芽胞形成能力也减弱。我们推测，由于炭疽杆菌的plcR的激活，导致炭疽杆菌表达溶血酶、卵磷脂酶等毒力因子，这些毒力因子与炭疽杆菌固有的炭疽毒素（致死毒素和水肿毒素）相比，毒力较弱，对于增强炭疽杆菌毒力方面作用不大，但是由于这些因子可以更显著的激活宿主菌的免疫反应，从而更容易被宿主菌激活，因而表现出plcR毒力调控因子激活的菌株毒力反而减弱这种看似非常奇怪的现象。同时plcR的激活也微弱的影响了炭疽杆菌芽胞的形成。这些都导致炭疽菌更容易被清除，不利于炭疽菌的感染和传播，因而在长期的进化和自然选择中产生plcR突变的菌株具有更好的竞争优势，因而被选择和保留下来。