Cloning of chromosomal DNA from Haemophilus influenzae. Its use for studying the expression of type b capsule and virulence.
Cloning of chromosomal DNA from Haemophilus influenzae. Its use for studying the expression of type b capsule and virulence.
复制标题
流感嗜血杆菌染色体 DNA 的克隆。
DOI：
10.1172/jci111214
复制
发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Connelly,C
中科院分区：
文献类型：
--
作者：
Moxon,ER;Deich,RA;Connelly,C
流感嗜血杆菌可产生六种化学性质不同的荚膜多糖中的任何一种，但只有荚膜血清型b菌株通常会引起人类全身感染（如脑膜炎）。利用DNA的分子克隆技术研究b型胶囊的表达及其与流感嗜血杆菌毒力的关系。利用一株强毒株b型流感嗜血杆菌构建了Charon 4染色体DNA的lambda文库。从文库中分离出两个独立分离的重组噬菌体，发现它们具有表达b型胶囊所需的DNA。利用一个具有良好特征的流感嗜血杆菌全身感染大鼠模型，我们发现克隆DNA引发的b型转化体具有致病性，可引起菌血症和脑膜炎，而未转化的缺乏荚膜的流感嗜血杆菌则不会。通过Southern杂交分析，两个DNA克隆共有的4.4 kb EcoRI片段用于表征临床分离株，这些分离株代表了所有六种胶囊化血清型以及几种缺乏胶囊的流感嗜血杆菌。探针与八个独立分离的b型菌株的ecori消化的染色体DNA的相同大小（4.4 kb）片段杂交。在33例被包裹的非乙型流感嗜血杆菌的染色体DNA EcoRI片段中也发现了与探针同源的单条带。然而，这些EcoRI片段的大小被证明是不同荚膜血清型的特征。这些研究为开展致病性流感嗜血杆菌的流行病学和毒力分子分析提供了基础。图片
Haemophilus influenzae may make any one of six chemically distinct capsular polysaccharides, but only strains of capsular serotype b commonly cause systemic infection (e.g., meningitis) in humans. Molecular cloning of DNA was used to investigate the expression of type b capsule and its association with H. influenzae virulence. A virulent H. influenzae type b strain was used to construct a lambda library of chromosomal DNA in Charon 4. Two independently isolated recombinant phage were isolated from the library and were found to possess DNA necessary for expression of type b capsule. Using a well-characterized rat model of H. influenzae systemic infection, we showed that type b transformants elicited by the cloned DNA were pathogenic, causing bacteremia and meningitis, whereas the untransformed capsule-deficient H. influenzae organisms were not. A 4.4-kb EcoRI fragment, common to both DNA clones, was used to characterize clinical isolates representing all six encapsulated serotypes as well as several capsule-deficient H. influenzae by Southern hybridization analysis. The probe hybridized to an identical sized (4.4 kb) fragment of EcoRI-digested chromosomal DNA from eight independently isolated type b strains. Single bands of homology to the probe were also found in EcoRI fragments of chromosomal DNA obtained from 33 encapsulated, nontype b H. influenzae. However, the size of these EcoRI fragments proved to be characteristic for each of the different capsular serotypes. These studies provide a basis for pursuing the molecular analysis of the epidemiology and virulence of pathogenic H. influenzae.Images