Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host

Abstract

Bartonella bacilliformis, the etiological agent of Carrión’s disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión’s disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador.B.bacilliformisis spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized inB.bacilliformis, to date. We therefore performed total RNA-sequencing analyses onB.bacilliformisgrownin vitrothen shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component,ffHleader RNA, and the alphaproteobacterial sRNAs αr45 andspeFleader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized aBartonellabacilliformisgroup Iintron (BbgpI) that disrupts an un-annotated tRNACCUArggene and determined that the intron splicesin vivoand self-splicesin vitro. Furthermore, we demonstrated the molecular targeting ofBartonellabacilliformissmallRNA9(BbsR9) to transcripts of theftsH,nuoF, andgcvTgenes,in vitro.