A CsrA-Binding, trans-Acting sRNA of Coxiella burnetii Is Necessary for Optimal Intracellular Growth and Vacuole Formation during Early Infection of Host Cells.
A CsrA-Binding, trans-Acting sRNA of Coxiella burnetii Is Necessary for Optimal Intracellular Growth and Vacuole Formation during Early Infection of Host Cells.
复制标题
伯内氏立克次体的 CsrA 结合、反式作用 sRNA 对于宿主细胞早期感染期间的最佳细胞内生长和液泡形成是必要的。
DOI：
10.1128/jb.00524-19
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发表时间：
2019
影响因子：
3.2
通讯作者：
Minnick,MichaelF
中科院分区：
文献类型：
--
作者：
Wachter,Shaun;Bonazzi,Matteo;Shifflett,Kyle;Moses,AbrahamS;Raghavan,Rahul;Minnick,MichaelF
贝氏柯克斯体（Coxiella burnetii）是一种专性胞内γ-蛋白菌，是Q热的人畜共患病病原体。我们以前鉴定了15个小的非编码RNA（sRNA）的C。伯内特氏菌其中之一，CbsR 12（Coxiella burnetiismallRNA 12），在无菌生长期间高度转录，并在感染培养的哺乳动物细胞期间变得更加突出。CbsR 12的二级结构预测揭示了四个推定的CsRA结合位点的干环与共识AGGA/ANGGA基序。我们随后确定CbsR 12结合重组C. Burnetii CsrA-2蛋白，而不是CsrA-1蛋白。此外，通过体外和细胞培养试验的结合，我们确定了几个CbsR 12的intransmRNA靶点。其中，我们确定CbsR 12结合并上调编码氨甲酰磷酸合成酶A（一种催化嘧啶生物合成第一步的酶）的carA转录物的翻译。此外，CbsR 12结合并下调编码S-腺苷甲硫氨酸合成酶（甲硫氨酸循环的一个组成部分）的metK转录物的翻译。此外，我们还发现，在哺乳动物细胞培养中，CbsR 12与cvpD转录物结合并下调cvpD转录物的数量，cvpD转录物编码IVB型效应蛋白。最后，我们发现，CbsR 12是必要的扩展ofCoxiella含有空泡和影响生长速率在感染THP-1细胞的早期阶段，在剂量依赖性的方式。这是首次对C. burnetii和细菌sRNA的第一个例子，调节CarA和MetK的合成。CbsR 12是目前发现的与CsrA相互作用的反式作用sRNAs之一。Burnetii不是很好理解。在这里，我们表明，C。与非复制型小细胞变体相比，贝氏小RNA 12（CbsR 12）在代谢活性大细胞变体中高度转录。我们发现，CbsR 12直接调节参与代谢的几个基因，沿着IV型效应基因，intrans.此外，我们证明了CbsR 12在体外与CsrA-2结合，并在大肠杆菌中异位转录时诱导自聚集和生物膜形成，与其他CsrA螯合sRNA一致。这些结果暗示CbsR 12通过CsRA介导的调节活性间接调节许多基因。这些结果也支持CbsR 12作为哺乳动物细胞感染早期的关键调控成分。
Coxiella burnetii is an obligate intracellular gammaproteobacterium and zoonotic agent of Q fever. We previously identified 15 small noncoding RNAs (sRNAs) of C. burnetii. One of them, CbsR12 (Coxiella burnetiismallRNA12), is highly transcribed during axenic growth and becomes more prominent during infection of cultured mammalian cells. Secondary structure predictions of CbsR12 revealed four putative CsrA-binding sites in stem loops with consensus AGGA/ANGGA motifs. We subsequently determined that CbsR12 binds to recombinant C. burnetii CsrA-2, but not CsrA-1, proteinsin vitro. Moreover, through a combination ofin vitroand cell culture assays, we identified several intransmRNA targets of CbsR12. Of these, we determined that CbsR12 binds and upregulates translation ofcarAtranscripts coding for carbamoyl phosphate synthetase A, an enzyme that catalyzes the first step of pyrimidine biosynthesis. In addition, CbsR12 binds and downregulates translation ofmetKtranscripts coding forS-adenosylmethionine synthetase, a component of the methionine cycle. Furthermore, we found that CbsR12 binds to and downregulates the quantity ofcvpDtranscripts, coding for a type IVB effector protein, in mammalian cell culture. Finally, we found that CbsR12 is necessary for expansion ofCoxiella-containing vacuoles and affects growth rates in a dose-dependent manner in the early phase of infecting THP-1 cells. This is the first characterization of atrans-acting sRNA of C. burnetii and the first example of a bacterial sRNA that regulates both CarA and MetK synthesis. CbsR12 is one of only a few identifiedtrans-acting sRNAs that interacts with CsrA.IMPORTANCERegulation of metabolism and virulence in C. burnetii is not well understood. Here, we show that C. burnetii small RNA 12 (CbsR12) is highly transcribed in the metabolically active large-cell variant compared to the nonreplicative small-cell variant. We show that CbsR12 directly regulates several genes involved in metabolism, along with a type IV effector gene, intrans. In addition, we demonstrate that CbsR12 binds to CsrA-2in vitroand induces autoaggregation and biofilm formation when transcribed ectopically in Escherichia coli, consistent with other CsrA-sequestering sRNAs. These results implicate CbsR12 in the indirect regulation of a number of genes via CsrA-mediated regulatory activities. The results also support CbsR12 as a crucial regulatory component early on in a mammalian cell infection.