Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids
Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids
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DOI：
10.1016/j.ymthe.2004.07.006
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发表时间：
2004-10-01
影响因子：
12.4
通讯作者：
Good, L
中科院分区：
文献类型：
--
作者：
Nekhotiaeva, N;Awasthi, SK;Good, L
细菌基因功能的研究落后于基因组测序的进展，部分原因是目前基于基因组破坏的反向遗传学技术不允许对一系列物种中所有基因的基因表达进行微妙的控制。关键基因和聚集区域尤其成问题。反义技术为微生物基因组学提供了一个有吸引力的选择。不幸的是，细菌缺乏RNAi机制，传统的寡核苷酸不能被有效地吸收。然而，在大肠杆菌中，利用附着在载体肽（KFFKFFKFFK）上的反义肽核酸（PNAs）可以实现高效和基因特异性的反义敲除。载体肽可以进入一系列微生物物种，在本研究中，我们询问肽- pnas是否可以介导金黄色葡萄球菌的反义作用。使用低微摩尔浓度，我们观察到报告基因gfp和内源性基因phoB的剂量和序列依赖性抑制。此外，针对mhb、gyrA和hmrB必需基因的反义肽- pnas具有生长抑制作用。对照肽- pnas的效果要差得多，PNA和靶mRNA序列的序列改变减少或消除了抑制作用。PNAs的抗菌潜力有待进一步开发，但目前的结果表明，该方法可以用于研究这种重要病原体的基因功能和需求。
Gene function studies in bacteria lag behind progress in genome sequencing, in part because current reverse genetics technology based on genome disruption does not allow subtle control of gene expression for all genes in a range of species. Essential genes and clustered regions are particularly problematic. Antisense technology offers an attractive alternative for microbial genomics. Unfortunately, bacteria lack RNAi mechanisms and conventional oligonucleotides are not taken up efficiently. However, in Escherichia coli, efficient and gene-specific antisense knock down is possible using antisense peptide nucleic acids (PNAs) attached to carrier peptides (KFFKFFKFFK). Carrier peptides can enter a range of microbial species, and in this study we asked whether peptide-PNAs could mediate antisense effects in Staphylococcus aureus. Using low micromolar concentrations we observed dose- and sequence-dependent inhibition of the reporter gene gfp and endogenous gene phoB. Also, antisense peptide-PNAs targeted to the essential genes fmhB, gyrA, and hmrB were growth inhibitory. Control peptide-PNAs were much less effective, and sequence alterations within the PNA and target mRNA sequences reduced or eliminated inhibition. Further development is needed to raise the antibacterial potential of PNAs, but the present results show that the approach can be used to study gene function and requirement in this important pathogen.