REDUCED VIRULENCE OF A DEFINED PNEUMOLYSIN-NEGATIVE MUTANT OF STREPTOCOCCUS-PNEUMONIAE
REDUCED VIRULENCE OF A DEFINED PNEUMOLYSIN-NEGATIVE MUTANT OF STREPTOCOCCUS-PNEUMONIAE
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DOI：
10.1128/iai.57.7.2037-2042.1989
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发表时间：
1989-07-01
影响因子：
3.1
通讯作者：
PATON, JC
中科院分区：
文献类型：
--
作者：
BERRY, AM;YOTHER, J;PATON, JC
采用插入重复突变构建肺炎链球菌溶血素阴性衍生物。这是通过首先用载体pVA 891的衍生物转化未包封的菌株Rx 1来实现的，所述载体pVA 891携带来自肺炎球菌溶血素结构基因中间的690个碱基对的DNA片段。从产生的红霉素抗性、肺炎球菌溶血素阴性的粗糙型肺炎球菌中提取DNA，并用于转化S。pneumoniae D39，2型强毒株。从两个独立的实验中获得了几个红霉素抗性转化体，并且这些转化体都不产生肺炎球菌溶血素。Southern印迹分析证实，这些转化体中的肺炎球菌溶血素基因已被质粒衍生的序列中断。与D39相比，肺炎球菌溶血素阴性突变体对小鼠的毒力降低，如通过鼻内攻击后的存活时间、腹膜内50%致死剂量和血液清除研究所判断的。在一个突变体中，通过用克隆的肺炎球菌溶血素基因转化，恢复了肺炎球菌溶血素的生产，同时丧失了红霉素抗性;该分离株在小鼠中的毒力与D39的毒力没有区别。这些结果证实了肺炎球菌溶血素参与肺炎球菌的发病机制。
Insertion-duplication mutagenesis was used to construct a pneumolysin-negative derivative of Streptococcus pneumoniae. This was achieved by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 690-base-pair DNA fragment from the middle of the pneumolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, pneumolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these produced pneumolysin. Southern blot analysis confirmed that the pneumolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The pneumolysin-negative mutants showed reduced virulence for mice compared with D39, as judged by survival time after intranasal challenge, intraperitoneal 50% lethal dose, and blood clearance studies. Pneumolysin production was reinstated in one of the mutants by transformation with the cloned pneumolysin gene, with the concomitant loss of erythromycin resistance; the virulence in mice of this isolate was indistinguishable from that of D39. These results confirm the involvement of pneumolysin in pneumococcal pathogenesis.