Identification and Characterization of Novel Small RNAs in Rickettsia prowazekii.

Casey L C Schroeder,Jignesh Patel,Sanjeev K Sahni,Yuriy Fofanov,Mark Rojas,Kamil Khanipov,Riya Shah,Hema P Narra,Abha Sahni

doi:10.3389/fmicb.2016.00859

Abstract

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.

Highlights

As critical post-transcriptional regulators of gene expression, regulatory RNAs have been found in a wide array of organisms from all branches of life and considered to be ubiquitous in nature
RNA sequencing of human microvascular endothelial cells (HMECs) infected with R. prowazekii for 3 h resulted in ∼42 to 46 million total reads, whereas 27 to 29 million total reads were obtained for RNA isolated from cells infected for 24 h
This is in agreement with a recent demonstration that intracellular organisms, such as Rickettsia species, constitute only 5% of extracted total RNA, whereas the remaining 95% belongs to eukaryotic host cells

Summary

Introduction

As critical post-transcriptional regulators of gene expression, regulatory RNAs have been found in a wide array of organisms from all branches of life and considered to be ubiquitous in nature. Small regulatory RNAs (sRNAs) in pathogenic bacteria have garnered immense recent attention due to their ability to control diverse, physiologically important lifecycle processes such as quorum sensing, metabolism, stress responses, and virulence. Despite being longer in length, these sRNAs are considered to be analogous to eukaryotic sRNAs in the context of certain functional implications. Posttranscriptional sRNA-mediated regulatory mechanisms are broadly categorized into sRNAprotein and sRNA-mRNA interactions. Interactions with the protein-coding transcripts are further categorized into two groups, namely, trans-acting, and cis-acting (Liu and Camilli, 2010; Gottesman and Storz, 2011).

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Conclusion