Enterococcus faecalis rnjB Is Required for Pilin Gene Expression and Biofilm Formation
Enterococcus faecalis rnjB Is Required for Pilin Gene Expression and Biofilm Formation
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DOI：
10.1128/jb.00725-10
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发表时间：
2010-10-01
影响因子：
3.2
通讯作者：
Harvey, Barrett R.
中科院分区：
文献类型：
--
作者：
Gao, Peng;Pinkston, Kenneth L.;Harvey, Barrett R.
革兰氏阳性菌中的菌毛在宿主组织的定植和生物膜的形成中起着重要作用。它们是很有希望的疫苗或药物靶标，因为它们具有高度的免疫原性，并且在各种革兰氏阳性病原体中具有共同的结构和功能特征。许多文献已经帮助建立了对菌毛表面组装的详细了解，但对菌毛基因表达的调控还没有很好的定义。利用开发的针对粪肠球菌主要菌毛蛋白EbpC的单抗，我们从缺乏表面暴露的EBP菌毛的转座子(TN)插入文库中鉴定了突变株。除了在EBP调节子中插入外，在EF1184(DAPA)中的插入还显著降低了EbpC的水平。对框内DAPA缺失突变体和下游基因rnjB缺失突变体的分析进一步证明，rnjB是EbpC缺失的原因。序列分析表明，rnjB编码一个可能的RNaseJ2。随后的实时定量聚合酶链式反应(qRT-PCR)和Northern blotting结果表明，rnjB缺失突变体的ebpABC基因转录水平显著降低。此外，利用报告基因分析，我们证实了rnjB影响ebpABC操纵子的表达。在功能上，rnjB缺失突变体的生物被膜能力减弱，与缺乏EBP菌毛的ebpABC缺失突变体相似。综上所述，这些结果证明了rnjB参与粪肠球菌菌毛基因的表达，并为调节革兰氏阳性病原菌菌毛产生的新机制提供了洞察力。
Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface-exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 (dapA) significantly reduced levels of EbpC. Analysis of in-frame dapA deletion mutants and mutants with the downstream gene rnjB deleted further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent quantitative real-time PCR (qRT-PCR) and Northern blotting demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects the expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pilus production in Gram-positive pathogens.