Schroeder et al. BMC Genomics (2015) 16:1075 DOI 10.1186/s12864-015-2293-7 RESEARCH ARTICLE Open Access Bacterial small RNAs in the Genus Rickettsia Casey L. C. Schroeder1, Hema P. Narra1, Mark Rojas2, Abha Sahni1, Jignesh Patel1, Kamil Khanipov2, Thomas G. Wood3, Yuriy Fofanov2 and Sanjeev K. Sahni1* Abstract Background: 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. Results: 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. Conclusions: 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. Keywords: Endothelial cells, Rickettsia, Small RNAs, Spotted Fever, Typhus, Deep-Sequencing, Bioinformatics, SIPHT/sRNAPredict3 Background Listeria monocytogenes, Streptococcus pyogenes, Clostrid- Small noncoding RNAs (sRNAs) were first identified ium perfringens, and Staphylococcus aureus [2]. For ex- and described in the 1960s, but remained largely ignored ample, Helicobacter pylori, the causative agent of until recently when they were recognized as important chronic active, chronic persistent, and atrophic gastritis post-transcriptional regulators in both eukaryotic and in adults and children, and implicated in a majority of prokaryotic organisms [1]. These sRNAs have been duodenal and gastric ulcers, carries a repertoire of at found in a number of pathogenic bacteria belonging to least one anti-sense transcriptional start site on approxi- family Enterobacteriaceae, Pseudomonas aeruginosa, mately 46 % of its open-reading frames, 28 % of tRNAs, and the 5′ leader sequences for both 16S rRNA and 23S * Correspondence: [email protected] rRNA [3]. Barring a few exceptions, sRNAs are typically 1 Department of Pathology, the University of Texas Medical Branch, 301 50 to 500 nucleotides in length and do not code for pro- University Blvd, Galveston, TX 77555, USA – Full list of author information is available at the end of the article teins [4 6]. Regulatory functions can predominantly be © 2015 Schroeder et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Schroeder et al. BMC Genomics (2015) 16:1075 Page 2 of 18 attributed to their interactions with proteins or target large amount of non-coding DNA was projected to be the mRNA transcripts [7, 8]. The sRNAs in the latter category consequence of genomic reduction and pseudogenization manipulate RNA transcription through either cis-acting due to the loss or degradation of genes involved in several or trans-acting mechanisms [4, 9]. By definition, cis-acting mechanisms leading to obligate intracellular lifestyle of sRNAs are encoded on the anti-sense strand and generally this pathogen. A number of other rickettsial genomes, in- display perfect nucleotide complementarity with the target cluding those of R. rickettsii, R. conorii, R. typhi, and other sequence in the open reading frame. Trans-acting sRNAs, notable species have since been sequenced and either pub- on the other hand, are encoded within the intergenic re- lished or made available in biomedical databases [22–24]. gions, act on the targets elsewhere in the genome, and However, the presence of small, non-coding RNAs in dif- possess short segments of partial nucleotide complemen- ferent Rickettsia species still remains undetermined. With tarity to their target genes [6–8]. Accordingly, they require an aim to address this important knowledge gap, we used a known RNA chaperone, namely Hfq, encoded by nearly SIPHT/sRNAPredict2 to identify candidate novel sRNAs 50 % of all bacterial species to facilitate their binding in- within the intergenic regions of all four rickettsial groups, teractions with an mRNA transcript [2, 7]. In other or- leading to the prediction of a total of 1,785 novel sRNAs ganisms such as Listeria monocytogenes,however,most within 16 different strains representing 13 rickettsial spe- trans-acting small RNAs function independent of the cies. We further analyzed the predicted sRNAs in R. pro- chaperone activity of Hfq [10]. wazekii strain Brienl using other bioinformatic tools and The genus Rickettsia includes obligate, intracellular experimentally validated their expression using reverse Gram-negative bacteria belonging to the class Alphapro- transcriptase-polymerase chain reaction (RT-PCR) and teobacteria. Based on the transmitting natural vector, deep sequencing approaches. In tandem, these analyses disease presentation, and antigenicity, this genus was trad- constitute the very first evidence documenting the pres- itionally divided into spotted fever and typhus as two ence of sRNAs in rickettsial genomes. major groups, but sophisticated molecular phylogenetic analysis now classifies rickettsiae into four groups, namely ancestral (R. bellii and R. canadensis), typhus (R. prowaze- Results kii and R. typhi), transitional (R. australis, R. akari,andR. Bioinformatic prediction of Small RNAs felis), and spotted fever (R. rickettsii, R. conorii, R. massi- Ready availability of complete genome sequences render liae, and numerous more) [11]. Upon transmission into computational approaches a widely acceptable first step humans from the arthropod vector, vascular endothelial for identification of sRNAs [25–27]. Such bioinformatic cells are the primary targets of rickettsial infections, with approaches search the intergenic regions (IGRs) of a the notable exception of R. akari, which primarily invades bacterial genome for specific sRNA features. In general, macrophages [12, 13]. Among well-known human rickett- the strategy involves an in-depth search of IGRs for the sioses, Rocky Mountain spotted fever (RMSF) due to R. presence of Rho-independent terminators, promoters, rickettsii and epidemic typhus caused by R. prowazekii are and transcription factor binding sites, followed by the considered to be the most severe forms of disease. With- analysis of their secondary structure and comparison of out proper antibiotic treatment, mortality rate for RMSF such IGRs with closely related species [3, 26, 28]. In this is approximately 20 % [14, 15], while the same for epi- study, we employed the web-based program SIPHT to demic typhus reportedly ranges from 10 % to 60 % examine the genomes of 16 rickettsial strains, which rep- [12, 16]. Also, R. prowazekii is unique in that patients resent a total of 13 species spanning all four rickettsial can harbor sub-clinical infections after successful groups. Four known plasmids, representing three from treatment of the primary infection and later develop the spotted fever group strains and one from a transi- recrudescent typhus, also known as Brill-Zinsser dis- tional group strain, were also included. The R. felis ease, despite being symptom-free for years [16, 17]. pRFdelta plasmid (NC_007111.1) was excluded from the On the other hand, Mediterranean spotted fever and analysis as it was found to be an artifact from