Small Regulatory RNAs of Rickettsia conorii

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 alpha-tmRNA) was next confirmed by Northern hybridization. Comparative analysis during infection of HMECs vis-a-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.