Identification of small Hfq-binding RNAs in Listeria monocytogenes

Abstract

The RNA-binding protein Hfq plays important roles in bacterial physiology and is required for the activity of many small regulatory RNAs in prokaryotes. We have previously shown that Hfq contributes to stress tolerance and virulence in the Grampositive human pathogen Listeria monocytogenes. In the present study, we performed coimmunoprecipitations followed by enzymatic RNA sequencing to identify Hfq-binding RNA molecules in L. monocytogenes. The approach resulted in the discovery of three small RNAs (sRNAs). The sRNAs are conserved between Listeria species, but were not identified in other bacterial species. The initial characterization revealed a number of unique features displayed by each individual sRNA. The first sRNA is encoded from within an annotated gene in the L. monocytogenes EGD-e genome. Analogous to most regulatory sRNAs in Escherichia coli, the stability of this sRNA is highly dependent on the presence of Hfq. The second sRNA appears to be produced by a transcription attenuation mechanism, and the third sRNA is present in five copies at two different locations within the L. monocytogenes EGD-e genome. The cellular levels of the sRNAs are growth phase dependent and vary in response to growth medium. All three sRNAs are expressed when L. monocytogenes multiplies within mammalian cells. This study represents the first attempt to identify sRNAs in L. monocytogenes.

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…In L. monocytogenes, Hfq regulates multiple processes, such as stress tolerance and virulence and its responsiveness to stress is regulated by σ B . 197 From the 150 putative regulatory RNAs reported thus far, three Hfqinteracting small RNAs (sRNAs) were identified, 198 and one functionally characterized. 199 Three other appear to be required for efficient bacterial growth in macrophages (see intracellular multiplication section) and at least two are involved in virulence in mice.…”

Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function

“…Although numerous sRNAs have been detected in many Gram-positive bacteria, it is not yet clear whether Hfq is implicated in sRNA network. In Listeria monocytogenes, Hfq has been shown to play a role in stress tolerance and virulence (Christiansen et al 2004) and its inactivation does not generally affect sRNAs abundance, only three sRNAs coimmunoprecipitated with Hfq (Christiansen et al 2006;Mandin et al 2007;ToledoArana et al 2009) with facilitation of LhrA binding to its targets (Nielsen et al 2010(Nielsen et al , 2011. In S. aureus, Hfq is neither required for RNAIII function nor for sRNA stabilization (Boisset et al 2007;Geissmann et al 2009).…”

How the Study of Listeria Monocytogenes has Led to New Concepts in Biology

“…Regulatory RNAs are increasingly being recognized as important players in many physiological and adaptive responses in pathogenic bacteria [18][19][20]. The first noncoding RNAs (ncRNAs) in L. monocytogenes were identified by coimmunoprecipitation with Hfq, a small RNA-binding protein required for the activity of small regulatory RNAs in prokaryotes [21] and by an in silico-based approaches [22]. Since then, the use of high-density tiling arrays and RNA deep sequencing provided a picture of the whole L. monocytogenes transcriptome in multiple conditions, allowing the discovery of many regulatory RNAs [23][24][25][26][27][28] and the annotation of hundreds of regulatory RNAs in L.…”

“…Since then, L. monocytogenes has become a useful model for evaluation of the cellular 21 interactions that are crucial for the induction and establishment of the host T-cell response.…”