National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, 102206, Beijing, China.
Huadong Medical Institute of Biotechniques, 210002, Nanjing, China.
Sci Rep. 2016 Jul 1;6:28827. doi: 10.1038/srep28827.
Anthrax is a disease caused by Bacillus anthracis. Specifically, the anthrax toxins and capsules encoded by the pXO1 and pXO2 plasmids, respectively, are the major virulence factors. We previously reported that the pXO1 plasmid was retained in the attenuated strain of B. anthracis vaccine strains even after subculturing at high temperatures. In the present study, we reinvestigate the attenuation mechanism of Pasteur II. Sequencing of pXO1 and pXO2 from Pasteur II strain revealed mutations in these plasmids as compared to the reference sequences. Two deletions on these plasmids, one each on pXO1 and pXO2, were confirmed to be unique to the Pasteur II strain as compared to the wild-type strains. Gene replacement with homologous recombination revealed that the mutation in the promoter region of the pagR gene on pXO2, but not the mutation on pXO1, contributes to lethal levels of toxin production. This result was further confirmed by RT-PCR, western blot, and animal toxicity assays. Taken together, our results signify that the attenuation of the Pasteur II vaccine strain is caused by a mutation in the pagR gene on its pXO2 plasmid. Moreover, these data suggest that pXO2 plasmid encoded proteins are involved in the virulence of B. anthracis.
炭疽是由炭疽芽孢杆菌引起的疾病。具体来说，分别由 pXO1 和 pXO2 质粒编码的炭疽毒素和荚膜是主要的毒力因子。我们之前报道过，即使在高温下进行传代培养，pXO1 质粒仍保留在减毒的炭疽芽孢杆菌疫苗菌株中。在本研究中，我们重新研究了巴斯德二世菌株的减毒机制。与参考序列相比，从巴斯德二世菌株的 pXO1 和 pXO2 测序发现这些质粒发生了突变。与野生型菌株相比，这两个质粒（pXO1 和 pXO2 各有一个）上的两个缺失被证实是巴斯德二世菌株所特有的。同源重组的基因替换表明，pXO2 上 pagR 基因启动子区域的突变而不是 pXO1 上的突变导致了毒素产生的致死水平。这一结果通过 RT-PCR、western blot 和动物毒性试验进一步得到证实。综上所述，我们的结果表明，巴斯德二世疫苗菌株的减毒是由其 pXO2 质粒上 pagR 基因的突变引起的。此外，这些数据表明，pXO2 质粒编码的蛋白参与了炭疽芽孢杆菌的毒力。
