A Small Non-Coding RNA Mediates Transcript Stability and Expression of Cytochrome bd Ubiquinol Oxidase Subunit I in...

Small regulatory RNAs (sRNAs) are now widely recognized for their role in the post-transcriptional regulation of bacterial virulence and growth. We have previously demonstrated the biogenesis and differential expression of several sRNAs in Rickettsia conorii during interactions with the human host and arthropod vector, as well as the in vitro binding of Rickettsia conorii sRNA Rc_sR42 to bicistronic cytochrome bd ubiquinol oxidase subunits I and II (cydAB) mRNA. However, the mechanism of regulation and the effect of sRNA binding on the stability of the cydAB bicistronic transcript and the expression of the cydA and cydB genes are still unknown. In this study, we determined the expression dynamics of Rc_sR42 and its cognate target genes, cydA and cydB, in mouse lung and brain tissues during R. conorii infection in vivo and employed fluorescent and reporter assays to decode the role of sRNA in regulating cognate gene transcripts. Quantitative RT-PCR revealed significant changes in the expression of sRNA and its cognate target gene transcripts during R. conorii infection in vivo, and a greater abundance of these transcripts was observed in the lungs compared to brain tissue. Interestingly, while Rc_sR42 and cydA exhibited similar patterns of change in their expression, indicating the influence of sRNA on the mRNA target, the expression of cydB was independent of sRNA expression. Further, we constructed reporter plasmids of sRNA and cydAB bicistronic mRNA to decipher the role of sRNA on CydA and CydB expression. We observed increased expression of CydA in the presence of sRNA but detected no change in CydB expression in the presence or absence of sRNA. In sum, our results demonstrate that the binding of Rc_sR42 is required for the regulation of cydA but not cydB. Further studies on understanding the influence of this interaction on the mammalian host and tick vector during R. conorii infection are in progress.