Bacterial small RNAs in the Genus Rickettsia.
Bacterial small RNAs in the Genus Rickettsia.
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DOI：
10.1186/s12864-015-2293-7
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发表时间：
2015-12-18
期刊：
影响因子：
4.4
通讯作者：
Sahni SK
中科院分区：
文献类型：
--
作者：
Schroeder CL;Narra HP;Rojas M;Sahni A;Patel J;Khanipov K;Wood TG;Fofanov Y;Sahni SK
立克次体是专性细胞内革兰氏阴性致病菌和疾病病原，如落基山斑疹热（RMSF），地中海斑疹热，流行性斑疹伤寒和鼠斑疹伤寒。基因组测序结果显示，prawazekii具有约25%的非编码DNA，其中大部分被认为是“垃圾DNA”或由于基因组减少而产生的假基因。这些特征也定义了其他立克次体基因组。细菌小rna的生物发生主要归因于基因间区（反式作用）或开放阅读框的反义链（顺式作用），现在被认为是细菌毒力和生长的最重要的转录后调节剂之一。我们假设立克次体物种的基因间区编码参与转录组调控的小的非编码rna (sRNAs)，导致根据宿主生态位改变毒力和适应性。我们采用生物信息学和体外方法相结合来探索立克次体属中许多物种中sRNAs的存在。利用SIPHT网络界面的sRNA鉴定协议，我们预测了13种立克次体16种不同菌株的基因间区存在超过1700个小rna。我们进一步利用细菌启动子预测程序（BPROM）和TransTermHP预测算法分别鉴定了斑疹伤寒（R. prowazekii和R. typhi）和斑点热（R. rickettsii和R. conorii）群体的启动子和rho独立终止子的新sRNAs。所有新型小rna的上游均存在强σ70启动子，表明其具有潜在的转录活性。接下来，我们用R. prowazekii感染人微血管内皮细胞（HMECs） 3 h和24 h，并进行Next Generation测序以实验验证R. prowazekii预测的26个sRNA候选物的表达。我们还利用逆转录酶PCR进一步验证了6个新的sRNA候选物在鼠僵菌中的表达。我们的结果提供了新的R. prowazekii sRNA候选物在hmec感染期间表达的明确证据。这是对立克次体高致病性物种的新型小rna的首次描述，这将导致对立克次体毒力和适应机制的新见解。本文的在线版本（doi:10.1186/s12864-015-2293-7）包含补充材料，可供授权用户使用。
Rickettsia species are obligate intracellular Gram-negative pathogenic bacteria and the etiologic agents of diseases such as Rocky Mountain spotted fever (RMSF), Mediterranean spotted fever, epidemic typhus, and murine typhus. Genome sequencing revealed that R. prowazekii has ~25 % non-coding DNA, the majority of which is thought to be either “junk DNA” or pseudogenes resulting from genomic reduction. These characteristics also define other Rickettsia genomes. Bacterial small RNAs, whose biogenesis is predominantly attributed to either the intergenic regions (trans-acting) or to the antisense strand of an open reading frame (cis-acting), are now appreciated to be among the most important post-transcriptional regulators of bacterial virulence and growth. We hypothesize that intergenic regions in rickettsial species encode for small, non-coding RNAs (sRNAs) involved in the regulation of its transcriptome, leading to altered virulence and adaptation depending on the host niche. We employed a combination of bioinformatics and in vitro approaches to explore the presence of sRNAs in a number of species within Genus Rickettsia. Using the sRNA Identification Protocol using High-throughput Technology (SIPHT) web interface, we predicted over 1,700 small RNAs present in the intergenic regions of 16 different strains representing 13 rickettsial species. We further characterized novel sRNAs from typhus (R. prowazekii and R. typhi) and spotted fever (R. rickettsii and R. conorii) groups for their promoters and Rho-independent terminators using Bacterial Promoter Prediction Program (BPROM) and TransTermHP prediction algorithms, respectively. Strong σ70 promoters were predicted upstream of all novel small RNAs, indicating the potential for transcriptional activity. Next, we infected human microvascular endothelial cells (HMECs) with R. prowazekii for 3 h and 24 h and performed Next Generation Sequencing to experimentally validate the expression of 26 sRNA candidates predicted in R. prowazekii. Reverse transcriptase PCR was also used to further verify the expression of six putative novel sRNA candidates in R. prowazekii. Our results yield clear evidence for the expression of novel R. prowazekii sRNA candidates during infection of HMECs. This is the first description of novel small RNAs for a highly pathogenic species of Rickettsia, which should lead to new insights into rickettsial virulence and adaptation mechanisms. The online version of this article (doi:10.1186/s12864-015-2293-7) contains supplementary material, which is available to authorized users.