Random inducible controlled expression (RICE) for identification of mycobacterial virulence genes.
Random inducible controlled expression (RICE) for identification of mycobacterial virulence genes.
复制标题
DOI：
10.1016/j.tube.2011.10.013
复制
发表时间：
2011-12
期刊：
影响因子：
--
通讯作者：
Cirillo JD
中科院分区：
文献类型：
--
作者：
Janagama HK;Hassounah HA;Cirillo SL;Cirillo JD
我们开发了随机诱导控制表达（RICE），这是一种高通量遗传学方法，用于鉴定致病性分枝杆菌中受调控的毒力途径。RICE允许细菌基因在它们通常关闭的条件下表达，例如在实验室生长条件下，通过使用诱导型或组成型启动子以及由于质粒上基因的存在而产生的基因剂量效应。可以消化分枝杆菌基因组DNA以产生随机片段，用于克隆到分枝杆菌启动子下游的自杀表达载体中，或者用它们自己的启动子在复制质粒上通过基因剂量效应增加表达。质粒DNA通常在E.大肠杆菌中，并递送到分枝杆菌中以选择重组体或质粒转化体。然后在功能测定中直接筛选所得文库的增强的宿主细胞相互作用，所述功能测定评价宿主细胞内粘附、进入和复制的效率。这种方法已经导致从致病性分枝杆菌中鉴定出几种毒力因子。我们对RICE鉴定的一个这样的位点，即分枝杆菌增强进入位点（mel 2）的分析发现，存在的基因通过保护病原体免受氧化损伤而促进宿主内的细菌持久性。因此，我们已经开发了一种遗传策略，该策略具有以下几个优点：（i）它允许鉴定在宿主-病原体相互作用期间具有直接作用的细菌遗传元件;（ii）它可以用于鉴定广泛病原体中的毒力因子;以及（iii）它可以揭示仅在感染的特定阶段诱导的基因。
We have developed Random Inducible Controlled Expression (RICE), a high throughput genetic approach to identify regulated virulence pathways in pathogenic mycobacteria. RICE allows expression of bacterial genes under conditions where they are normally off, e.g. under laboratory growth conditions, via the use of an inducible or constitutive promoter as well as gene dosage effects due to the presence of the gene on a plasmid. Mycobacterial genomic DNA can be digested to yield random fragments for cloning into a suicide expression vector downstream of a mycobacterial promoter or with their own promoter on a replicating plasmid increasing expression by gene dosage effects. The plasmid DNA is normally amplified in E. coli and delivered into mycobacteria to select for recombinants or plasmid transformants. The resulting library is then directly screened for enhanced host cell interactions in functional assays that evaluate the efficiency of adherence, entry and replication inside host cells. This approach has resulted in identification of several virulence factors from pathogenic mycobacteria. Our analysis of one such locus identified by RICE, the mycobacterial enhanced entry locus (mel2), found that the genes present facilitate bacterial persistence inside the host by protecting the pathogen against oxidative damage. Thus, we have developed a genetic strategy that offers several advantages: (i) it allows identification of bacterial genetic elements that have a direct role during host-pathogen interactions (ii) it can be used to identify virulence factors in a broad range of pathogens and (iii) it can reveal genes that are only induced at specific stages of infection.
登录
查看更多内容
影响因子：
4.8
作者：
Eichhorn, E;van der Ploeg, JR;Leisinger, T
通讯作者：
Leisinger, T
影响因子：
4.2
作者：
Subbian S;Mehta PK;Cirillo SL;Cirillo JD
通讯作者：
Cirillo JD
影响因子：
30.8
作者：
通讯作者：
--
影响因子：
56.9
作者：
HENSEL, M;SHEA, JE;HOLDEN, DW
通讯作者：
HOLDEN, DW
影响因子：
3.1
作者：
El-Etr, SH;Subbian, S;Cirillo, JD
通讯作者：
Cirillo, JD