A CsrA-Binding, trans -Acting sRNA of Coxiella burnetii Is Necessary for Optimal Intracellular Growth and Vacuole Formation during Early Infection of Host Cells

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 Coxiella-containing 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. IMPORTANCE Regulation of metabolism and virulence in C. burnetii is not well understood. Here, we show that C. burnetii small RNA 12 (CbsR12) is highly transcribed in the metabolically active large-cell variant compared to the nonreplicative small-cell variant. We show that CbsR12 directly regulates several genes involved in metabolism, along with a type IV effector gene, in trans. In addition, we demonstrate that CbsR12 binds to CsrA-2 in vitro and induces autoaggregation and biofilm formation when transcribed ectopically in Escherichia coli, consistent with other CsrA-sequestering sRNAs. These results implicate CbsR12 in the indirect regulation of a number of genes via CsrA-mediated regulatory activities. The results also support CbsR12 as a crucial regulatory component early on in a mammalian cell infection.

RNA-Sequencing Analyses of Small Bacterial RNAs and their Emergence as Virulence Factors in Host-Pathogen Interactions

“…In a recent study performed by the same group, functional C. burnetii small RNA 12 (CbsR12) has been characterized. CbsR12 is involved in the regulation of the methionine cycle, pyrimidine biosynthesis and Coxiella vacuolar protein D (CvpD) [95], a protein acting as a crucial component in parasitophorous vacuole generation (required for successful intracellular parasitism) [96].…”

Novel small RNAs expressed byBartonella bacilliformisunder multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host

“…Raw reads were quality filtered and aligned as previously described [35]. Briefly, raw fastq files were concatenated, quality filtered with the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/), and then clipped, aligned, and filtered with Nesoni version 0.128 tools (http://www.vicbioinformatics.com/software.nesoni.shtml).…”

“…Finally, cells were harvested into TRI Reagent (Ambion; Austin, TX), as previously described for infected Vero cells [35].…”

“…The cells were incubated at room temperature for 1 h then frozen at -80⁰C for ≥ 2 h. The cells were thawed, centrifuged at 10,000 x g at 4⁰C for 10 min, and resuspended in 1 ml of TRI Reagent (Sigma-Aldrich; St. Louis, MO). The cells were incubated at room temperature for 1 h then frozen at -80⁰C for ≥ 2 h. Finally, cells were thawed and total RNA and genomic DNA isolation were done as previously described [35]. Total RNA pools from human blood infections were globin-depleted using a GLOBINclear kit (Ambion) according to manufacturer's specifications.…”

“…Q5 clones were sequenced, re-amplified with T7-engineered primers, and in vitro transcribed using the MAXIscript T7 Transcription kit (Invitrogen) with or without 0.5 mM Bio-16-UTP (Invitrogen), as required. Dose-dependent RNA-RNA EMSAs were performed as previously described [35]…”