A CsrA-Binding, trans-Acting sRNA of Coxiella burnetii is necessary for optimal intracellular growth and vacuole formation during early infection of host cells

Shaun Wachter, Matteo Bonazzi, Kyle Shifflett, Abraham S. Moses, Rahul Raghavan, Michael F. Minnick

Research output: Contribution to journalArticlepeer-review

Abstract

Coxiella burnetii is an obligate intracellular gammaproteobacterium and zoonotic agent of Q fever. We previously identified 15 small noncoding RNAs (sRNAs) of C. burnetii. One of them, CbsR12 (Coxiella burnetii small RNA 12), is highly transcribed during axenic growth and becomes more prominent during infection of cultured mammalian cells. Secondary structure predictions of CbsR12 revealed four putative CsrA-binding sites in stem loops with consensus AGGA/ANGGA motifs. We subsequently determined that CbsR12 binds to recombinant C. burnetii CsrA-2, but not CsrA-1, proteins in vitro. Moreover, through a combination of in vitro and cell culture assays, we identified several in trans mRNA targets of CbsR12. Of these, we determined that CbsR12 binds and upregulates translation of carA transcripts coding for carbamoyl phosphate synthetase A, an enzyme that catalyzes the first step of pyrimidine biosynthesis. In addition, CbsR12 binds and downregulates translation of metK transcripts coding for S-adenosylmethionine synthetase, a component of the methionine cycle. Furthermore, we found that CbsR12 binds to and downregulates the quantity of cvpD transcripts, coding for a type IVB effector protein, in mammalian cell culture. Finally, we found that CbsR12 is necessary for expansion of Coxiellacontaining vacuoles and affects growth rates in a dose-dependent manner in the early phase of infecting THP-1 cells. This is the first characterization of a trans-acting sRNA of C. burnetii and the first example of a bacterial sRNA that regulates both CarA and MetK synthesis. CbsR12 is one of only a few identified trans-acting sRNAs that interacts with CsrA.

Original languageEnglish
Article numbere00524-19
JournalJournal of Bacteriology
Volume201
Issue number22
DOIs
StatePublished - Nov 1 2019

Keywords

  • AhcY
  • CarA
  • Coxiella burnetii
  • CsrA
  • CvpD
  • MetK
  • Noncoding RNA
  • Posttranscriptional regulation
  • Small RNA

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