Disruption of the genes encoding antigen 85A and antigen 85B of Mycobacterium tuberculosis H37Rv:: Effect on growth in culture and in macrophages
Disruption of the genes encoding antigen 85A and antigen 85B of Mycobacterium tuberculosis H37Rv:: Effect on growth in culture and in macrophages
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DOI：
10.1128/iai.68.2.767-778.2000
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发表时间：
2000-02-01
影响因子：
3.1
通讯作者：
Norris, SJ
中科院分区：
文献类型：
--
作者：
Armitige, LY;Jagannath, C;Norris, SJ
结核分枝杆菌的致病机制被认为是多因素的，其中假定的毒力因子是抗原85（Ag 85）复合物。该输出纤连蛋白结合蛋白家族由成员Ag 85 A、Ag 85 B和Ag 85 C组成，并且最突出地由85 A和85 B代表。这些蛋白质最近被证明具有分枝菌酰转移酶活性，并可能在细胞壁合成中发挥作用。本研究的目的是获得M.结核分枝杆菌缺乏该复合物的主要成员的表达，以确定它们在结核分枝杆菌发病机制中的作用。然后通过电穿孔将用卡那霉素抗性标记Omega Km破坏并含有不同量的侧翼基因和质粒载体序列的fbpA和fbpB的构建体作为线性片段引入H37 Rv。Southern印迹和PCR分析显示在一个fbpA：：Omega Km和一个fbpB：：Omega Km中的同源基因位点被破坏。fbpA：：Omega Km突变体LAa 1是由双交换整合事件引起的，而fbpB：：Omega Km变体LAb 1是导致Omega Km和质粒序列插入的单交换类型事件的产物。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western印迹分析证实，在fbpA和fbpB突变体中未检测到被破坏的基因的表达。生长速率的分析表明，fbpB突变LAb 1的生长速度与野生型亲本相似，在丰富和营养贫乏的实验室培养基中，以及在人类（THP-1）和小鼠（J774.1A）巨噬细胞样细胞系。fbpA突变体LAa 1在丰富的实验室培养基中生长类似于亲本H37 Rv，但在营养不良的培养基和巨噬细胞样细胞系中几乎没有生长。靶向破坏结核分枝杆菌中编码分枝菌酰转移酶和纤连蛋白结合活性的两个基因将允许系统地确定它们在该生物体的生理学和发病机制中的作用。
The mechanism of pathogenesis of Mycobacterium tuberculosis is thought to be multifactorial, Among the putative virulence factors is the antigen 85 (Ag85) complex. This family of exported fibronectin-binding proteins consists of members Ag85A, Ag85B, and Ag85C and is most prominently represented by 85A and 85B. These proteins have recently been shown to possess mycolyl transferase activity and likely play a role in cell wall synthesis. The purpose of this study was to generate strains of M. tuberculosis deficient in expression of the principal members of this complex in order to determine their role in the pathogenesis of M, tuberculosis. Constructs of fbpA and fbpB disrupted with the kanamycin resistance marker Omega Km and containing varying amounts of flanking gene and plasmid vector sequences were then introduced as linear fragments into H37Rv by electroporation. Southern blot and PCR analyses revealed disruption of the homologous gene locus in one fbpA::Omega Km transformant and one fbpB::Omega Km transformant. The fbpA::Omega Km mutant, LAa1, resulted from a double-crossover integration event, whereas the fbpB::Omega Km variant, LAb1, was the product of a single-crossover type event that resulted in insertion of both Omega Km and plasmid sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis confirmed that expression of the disrupted gene was not detectable in the fbpA and fbpB mutants. Analysis of growth rates demonstrated that the fbpB mutant LAb1 grew at a rate similar to that of the wild-type parent in enriched and nutrient-poor laboratory media as well as in human (THP-1) and mouse (J774.1A) macrophage-like cell lines. The fbpA mutant LAa1 grew similarly to the parent H37Rv in enriched laboratory media but exhibited little or no growth in nutrient-poor media and macrophage-like cell lines, The targeted disruption of two genes encoding mycolyl transferase and fibronectin-binding activities in M, tuberculosis will permit the systematic determination of their roles in the physiology and pathogenesis of this organism.