Small Regulatory RNAs of Rickettsia conorii

Abstract

Small regulatory RNAs comprise critically important modulators of gene expression in bacteria, yet very little is known about their prevalence and functions in Rickettsia species. R. conorii, the causative agent of Mediterranean spotted fever, is a tick-borne pathogen that primarily infects microvascular endothelium in humans. We have determined the transcriptional landscape of R. conorii during infection of Human Microvascular Endothelial Cells (HMECs) by strand-specific RNA sequencing to identify 4 riboswitches, 13 trans-acting (intergenic), and 22 cis-acting (antisense) small RNAs (termed ‘Rc_sR’s). Independent expression of four novel trans-acting sRNAs (Rc_sR31, Rc_sR33, Rc_sR35, and Rc_sR42) and known bacterial sRNAs (6S, RNaseP_bact_a, ffs, and α-tmRNA) was next confirmed by Northern hybridization. Comparative analysis during infection of HMECs vis-à-vis tick AAE2 cells revealed significantly higher expression of Rc_sR35 and Rc_sR42 in HMECs, whereas Rc_sR31 and Rc_sR33 were expressed at similar levels in both cell types. We further predicted a total of 502 genes involved in all important biological processes as potential targets of Rc_sRs and validated the interaction of Rc_sR42 with cydA (cytochrome d ubiquinol oxidase subunit I). Our findings constitute the first evidence of the existence of post-transcriptional riboregulatory mechanisms in R. conorii and interactions between a novel Rc_sR and its target mRNA.

Transcriptional profiling of Rickettsia prowazekii coding and non-coding transcripts during in vitro host-pathogen and vector-pathogen interactions

“…As previously reported, bacterial sRNA expression can not only differ among tissues and organ systems within an infected host, but also between genders of a particular host species (Woolfit et al, 2015) and evidence from our laboratory has suggested differential regulation of R. conorii sRNAs in tick and human host cells (Narra et al, 2016). To further investigate whether R. prowazekii selectively and/or differentially express sRNAs in different host cells, we measured the expression levels of five novel candidate sRNAs in HMECs infected at either 37°C or 34°C and AAE2 cells infected at 34°C.…”

“…Recently, we have shown R. conorii trans-acting sRNA Rc _sR42 to directly bind to cydA mRNA in vitro , implicating the possibility of a chaperone-independent mechanism of sRNA-mRNA interaction in Rickettsia species (Narra et al, 2016). Using bioinformatics based approach, this study further identified several target genes involved in signaling, DNA repair, and cold shock response to be regulated by Rp _sR’s selectively and differentially expressed during tick cell infection.…”

“…Approximately 24h prior to infection, the medium in each flask was replaced with L-15B infection medium (pH 7.5) containing 25mM sodium bicarbonate and HEPES (Munderloh and Kurtti, 1989). In vitro infection of HMECs with R. prowazekii stocks was carried out at 37°C or 34°C according to our standard protocols and procedures (Rydkina et al, 2005; Narra et. al., 2016).…”

“…To gain insight into the post-transcriptional regulation by Rp _sRs, targets genes potentially regulated by Rp _sR76, 83, 86 and 159 were predicted using IntaRNA algorithm (Busch et al, 2008) using default parameters with the only exception that the region of interrogation for identification of seed region was set to +150 and −100 bases with respective to the translational start codon of an ORF (Narra et. al., 2016).…”

Evolution, purification, and characterization of RC0497: a peptidoglycan amidase from the prototypical spotted fever species Rickettsia conorii

“…The single monolayer of microvascular endothelium lining the blood vessels represents the primary target of pathogenic Rickettsia species for establishment and dissemination of infection (Sahni et al, 2013). Therefore, we recently mapped the global transcriptional landscape of R. conorii during in vitro infection of HMECs (Narra et al, 2016). In this analysis, RC0497 emerged as one of the highly expressed rickettsial genes along with a number of others previously implicated in adhesion and invasion (ompA,ompB,Sca2) and metabolism (gltA and gapdh) (Narra and Sahni, unpublished data).…”