Coxiella burnetiismall RNA 12 binds CsrA regulatory protein and transcripts for the CvpD type IV effector, regulates pyrimidine and methionine metabolism, and is necessary for optimal intracellular growth and vacuole formation during infection

Abstract

Coxiella burnetii is an obligate intracellular gammaproteobacterium and zoonotic agent of Q fever. We previously identified 15 small non-coding RNAs (sRNAs) of C. burnetii. One of them, named CbsR12 (Coxiella burnetiismall RNA 12) is highly expressed during growth in axenic medium and becomes even more dominant during infection of cultured mammalian cells. Secondary structure predictions of CbsR12 revealed four putative CsrA-binding sites in single-stranded segments of stem loops with consensus AGGA/ANGGA motifs. From this foundation, we 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 in vivo assays, we identified several in trans mRNA targets of CbsR12. Of these, we determined that CbsR12 binds to 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-adenosyl methionine (SAM) synthase, 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 vitro and in vivo. Finally, we found that CbsR12 is necessary for full expansion of Coxiella-containing vacuoles (CCVs) and affects bacterial growth rates in a dose-dependent manner in the early phase of infecting THP-1 cells. This is the first detailed characterization of a trans-acting sRNA of C. burnetii and the first example of a bacterial sRNA that regulates both CarA and MetK expression. CbsR12 is also one of only a few identified trans-acting sRNAs that interacts with CsrA. Results illustrate the importance of sRNA-mediated regulation in establishment of the intracellular CCV niche.Author summaryC. burnetii is an obligate intracellular bacterial pathogen that is transmitted to humans from animal reservoirs. Upon inhalation of aerosolized C. burnetii, the agent is phagocytosed by macrophages in the lung. The pathogen subverts macrophage-mediated degradation and resides in a large, intracellular, acidic vacuole, termed the Coxiella-containing vacuole (CCV). Small RNAs (sRNAs) are not translated into proteins. Instead, they target mRNAs in order to up- or down-regulate their stability and translation. Alternatively, some sRNAs bind to regulatory proteins and serve as “sponges” that effectively sequester the proteins and inhibit their function. C. burnetii’s CbsR12 sRNA is highly expressed during infection in order to expand the CCV, and it works by a variety of mechanisms, including: 1) directly regulating transcripts of several metabolic genes that aid in bacterial replication, 2) binding to and regulating transcripts of a type IV effector protein that aids in infection, and 3) indirectly regulating an unknown number of genes by binding to a homolog of the global regulatory protein, CsrA. CbsR12 represents one of only a few sRNAs known to bind and sequester CsrA while also directly regulating mRNAs.