Identification and Characterization of Novel Small RNAs in Rickettsia prowazekii.
Identification and Characterization of Novel Small RNAs in Rickettsia prowazekii.
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DOI：
10.3389/fmicb.2016.00859
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发表时间：
2016
影响因子：
5.2
通讯作者：
Sahni SK
中科院分区：
文献类型：
--
作者：
Schroeder CL;Narra HP;Sahni A;Rojas M;Khanipov K;Patel J;Shah R;Fofanov Y;Sahni SK
新出现的证据暗示细菌小RNA（sRNA）作为生理学、代谢、应激/适应性反应和毒力的转录后调节因子具有至关重要的作用，但sRNA在致病性立克次体物种中的作用仍然知之甚少。在这里，我们报告的鉴定新的和众所周知的细菌sRNAs普氏立克次体，已知引起流行性斑疹伤寒在人类。人微血管内皮细胞（HMEC），在人类立克次体病的首选目标，感染了R。prowazekii的结果显示，分别存在35个反式作用和23个顺式作用的sRNA。其中，通过北方印迹或RT-PCR分析，进一步证实了两种反式作用（Rp_sR17和Rp_sR60）和一种顺式作用（Rp_sR47）新sRNA以及四种充分表征的细菌sRNA（RNaseP_bact_a、α-tmRNA、4.5S RNA、6S RNA）的表达。利用5′ RLM-RACE技术对5个新的立克次体sRNA和6S RNA的转录起始位点进行了测定，为它们在立克次体中的独立生物起源提供了证据。prowazekii。最后，采用计算方法来确定新型sRNA的二级结构和潜在的mRNA靶点。总之，这些结果确立了sRNA在R. prowazekii在宿主细胞感染，并建议潜在的功能作用，这些重要的转录后调节立克次体的生物学和发病机制。
Emerging evidence implicates a critically important role for bacterial small RNAs (sRNAs) as post-transcriptional regulators of physiology, metabolism, stress/adaptive responses, and virulence, but the roles of sRNAs in pathogenic Rickettsia species remain poorly understood. Here, we report on the identification of both novel and well-known bacterial sRNAs in Rickettsia prowazekii, known to cause epidemic typhus in humans. RNA sequencing of human microvascular endothelial cells (HMECs), the preferred targets during human rickettsioses, infected with R. prowazekii revealed the presence of 35 trans-acting and 23 cis-acting sRNAs, respectively. Of these, expression of two trans-acting (Rp_sR17 and Rp_sR60) and one cis-acting (Rp_sR47) novel sRNAs and four well-characterized bacterial sRNAs (RNaseP_bact_a, α-tmRNA, 4.5S RNA, 6S RNA) was further confirmed by Northern blot or RT-PCR analyses. The transcriptional start sites of five novel rickettsial sRNAs and 6S RNA were next determined using 5′ RLM-RACE yielding evidence for their independent biogenesis in R. prowazekii. Finally, computational approaches were employed to determine the secondary structures and potential mRNA targets of novel sRNAs. Together, these results establish the presence and expression of sRNAs in R. prowazekii during host cell infection and suggest potential functional roles for these important post-transcriptional regulators in rickettsial biology and pathogenesis.