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Coxiella burnetii small 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

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 burnetii small 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 summary C. 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.


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Coxiella burnetii is a Gram-negative, obligate intracellular bacterium and the etiological displayed a prolonged lag phase from 1-3 days post-inoculation that was not observed in Tn1832 193 or Tn327-Comp strains (Fig 2A). Following lag phase, Tn327 grew at a slightly increased rate 194 relative to the other strains (days 6-9 post-infection), but failed to reach cell numbers seen in the 195 other two strains throughout the assay. The "wild-type" (Tn1832) and complemented  Comp) strains produced essentially indistinguishable growth curves. The qRT-PCR results 197 showed that the Tn327 insertion completely abrogated CbsR12 expression (Fig. 2B). The results 198 also confirmed CbsR12's increased expression in LCVs compared to SCVs as the copies of 199 CbsR12 per C. burnetii genome were highest at 3 days post-inoculation. and CbsR12 levels directly correlates to growth rates of the respective strains between these two 209 time points. However, we observed a dysregulation of CbsR12 in Tn327-Comp infecting THP-1 210 cells that was strikingly different than what was seen during axenic growth. Specifically, in THP-211 1s, we observed a maintenance of CbsR12 expression throughout the infection (Fig. 3B), 212 whereas in axenic growth there was a progressive drop-off in expression after 3dpi (see Fig 2B). 213 These results suggest that CbsR12 regulation differs in this host-cell type and that a CCVs with relatively unclear boundaries (Fig 4A). In contrast, the Tn327-Comp strain produced 226 CCVs that were similar in size to those produced by the Tn1832 strain, reflecting the trend 227 observed in their respective growth curves (see Fig 3A). Quantitatively, there were significantly 228 fewer Tn327 colonies per THP-1 cell at 3dpi as compared to the other two strains, suggesting a 229 possible defect in adhesion / internalization (Fig 4B). In addition, we observed significantly 230 smaller CCVs produced by Tn327 at 3dpi compared to the other strains (Fig 4C) Hybridized RNAs in lysates from both Tn1832 and Tn327 strains were cross-linked, and upon 264 RNA-Seq analysis used to identify RNAs that were enriched in Tn1832 vs. Tn327 strains (Fig 6). 265 Crosslink-Seq results confirmed that CbsR12 targets carA, metK, and cvpD transcripts in vivo, as 266 demonstrated in vitro (Fig 5). We also discovered an additional mRNA target,   and Tn327 levels were not significantly different, presumably due to reduced CbsR12 expression 296 in Tn1832 SCVs (see Fig 3B). However, Tn327-Comp cvpD expression was significantly lower, 297 most likely due to the maintained expression of CbsR12 in Tn327-Comp SCVs (see Fig 3B). 298 Whether or not two different forms of CvpD protein are produced from the cvpD gene is 299 unknown, although it appears that CbsR12 may negatively regulate the full-length cvpD 300 transcript in vivo.

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In order to determine if CbsR12 binds to carA and metK transcripts in vivo, we devised a 302 novel reporter assay in E. coli. 5' RACE results for both Tn1832 and Tn327 strains revealed that 303 carA has two potential TSSs, a finding that is consistent with transcription of E. coli carA [42]. of CbsR12 relative to a strain lacking the sRNA (Fig 7C). 312 In contrast, CbsR12 was predicted to downregulate MetK translation by binding to the 313 coding region of the transcript, immediately downstream of its start codon (Fig 8A). As is often 314 the case with this type of sRNA-mediated regulation, RNase III would likely be recruited and the 315 metK transcript cleaved, resulting in downregulation of the encoded protein product.

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Unexpectedly, 5' RACE analyses of metK mRNA also identified apparent alternative "TSSs" 317 within the CbsR12-binding region, suggesting that the truncated mRNAs resulted from CbsR12-mediated RNase III processing (Figs 8A, 8B). Indeed, 5' RACE analysis of RNA from strain 319 Tn327 infecting THP-1 cells did not detect the "TSSs", suggesting they are a product of RNase 320 III processing (data not shown). Results of the reporter assays in E. coli confirmed our 321 hypothesis, as the presence of CbsR12 significantly down-regulated translation of luciferase 322 enzyme from the metK-luc fusion construct compared to a strain lacking the sRNA (Fig 8C). 323 Although we determined that CbsR12 targets carA and metK transcripts in vitro and in vivo, 324 we were curious whether the absence of CbsR12 would also result in differential amounts of MetK. As predicted, when proteins from whole-cell lysates of Tn1832, Tn327, and Tn327-Comp 328 strains were compared, we found that CarA was expressed in the control (Tn1832) and 329 complemented (Tn327-Comp) strains at comparable levels, but was undetectable in protein 330 profiles of the cbsR12 mutant (strain Tn327) (Fig 9A). In sharp contrast, MetK was highly 331 expressed in the cbsR12 mutant (strain Tn327) but expressed at relatively lower and comparable 332 levels in the Tn1832 and Tn327-Comp strains (Fig 9B). cells. 344 We initially became interested in CbsR12 based on its remarkably high expression in LCVs   Table 1 arginine/pyrimidine in accordance with carA regulation in E. coli. 415 We also showed that CbsR12 binds to metK transcripts (see

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This study also highlights the existence of CsrA-binding sites in the secondary structure of 492 CbsR12 (see S1A Fig). We showed that CbsR12 binds rCsrA-2, but not rCsrA- sizes as the infection progressed (see Fig 4C). 526 CbsR12's high level of expression during infection likely facilitates and regulates the many 527 functions we have described. We predict that expression of cbsR12 is itself regulated by an  Fig 2B) that is not apparent 534 during axenic growth (see Fig 1B). Alternatively, CbsR12 expression could be upregulated by CbsR12 is one of few identified trans-acting sRNAs that also binds CsrA [reviewed in [73]].

626
In order to purify total RNA from C. burnetii grown in THP-1 cell lines, growth medium 627 was first removed and replaced with 1 mL TRI Reagent. Flasks containing TRI Reagent were 628 then rocked for 1 hour at room temperature, after which cells were mechanically scraped and 629 collected into a 15-mL conical tube. The mixture was frozen overnight at -80°C, thawed to room 630 temperature for 30 minutes, and the RNA purification procedure continued as described above.

631
Genomic DNA was purified from TRI Reagent mixtures according to manufacturer 632 protocols (Ambion). Briefly, after the aqueous phase was removed in the total RNA extraction,