Regulated antisense RNA eliminates alpha-toxin virulence in Staphylococcus aureus infection
Regulated antisense RNA eliminates alpha-toxin virulence in Staphylococcus aureus infection
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DOI：
10.1128/jb.181.21.6585-6590.1999
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发表时间：
1999-11-01
影响因子：
3.2
通讯作者：
Woodnutt, G
中科院分区：
文献类型：
--
作者：
Ji, YD;Marra, A;Woodnutt, G
选择性干扰基因功能的能力在阐明基因对细菌生长和/或致病的重要性方面仍然是一个关键因素。在这项研究中，我们将TET调节表达系统用于金黄色葡萄球菌，目的是通过诱导反义RNA来下调基因表达。我们证明，在口服诱导剂后，该系统在培养(即体外)培养的细菌细胞中显示出50至100倍的剂量依赖的诱导水平，并在小鼠(即体内)中发挥作用。为了确定诱导的反义RNA是否会干扰染色体来源的基因表达，我们在tet启动子系统的下游克隆了S金黄色葡萄球菌α毒素基因(Hla)的反义片段，并将其导入金黄色葡萄球菌。体外诱导的反义人类白细胞抗原RNA可下调染色体来源的人类白细胞抗原基因的表达约14倍。同样，在体内诱导人类白细胞抗原反义RNA显著降低了两种不同金黄色葡萄球菌感染小鼠模型中α-毒素的表达。最重要的是，这种减少完全消除了感染的致命性。这些结果表明，TET调节系统在金黄色葡萄球菌中有效地发挥作用，诱导的反义RNA在体内外都能有效地下调染色体基因的表达。
The ability to selectively disrupt gene function remains a critical element in elucidating information regarding gene essentiality for bacterial growth and/or pathogenesis. In this study,we adapted a tet regulatory expression system for use in Staphylococcus aureus,,vith the goal of downregulating gene expression via induction of antisense RNA. We demonstrate that this system exhibits a 50- to 100-fold dose-dependent level of induction in bacterial cells grown in culture (i.e., in vitro) and also functions in mice (i.e., in vivo) following oral administration of inducer. To determine whether induced antisense RNA could interfere with chromosomally derived gene expression, we cloned a fragment of the S, aureus alpha-toxin gene (hla) in antisense orientation downstream of the tet promoter system and introduced the construct into S. aureus. Induced antisense hla RNA downregulated chromosomally derived hla gene expression in vitro approximately 14-fold. Similarly, induction of hla antisense RNA in vivo dramatically reduced alpha-toxin expression in two different murine models of S. aureus infection. Most importantly, this reduction completely eliminated the lethality of the infection. These results indicate that the tet regulatory system functions efficiently in S. aureus and induced antisense RNA can effectively do downregulate chromosomal gene expression both in vitro and in vivo.