The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages

Abstract

Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50* for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNA candidates and the absence of sRNA loci in genomes of naturally occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

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

“…In addition, the three mutants were attenuated when assessed for growth in mouse and insect models of infection. 156 These results reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.…”

“…The discovery of 150 putative regulatory RNAs was recently reported in Listeria. 156 A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are differentially expressed intracellularly. Mutants for three of these intracellularly expressed sRNA candidates revealed that their expression is required for efficient growth of bacteria in macrophages.…”

“…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. 156,187 Several expression analyses showed an overlap among regulons of key regulatory proteins of L. monocytogenes. In particular, PrfA and σ B were shown to jointly contribute to processes like intracellular growth and virulence.…”

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

“…An increasing amount of data shows that the Clostridia also contain sRNAs (Chen et al 2011;Mraheil et al 2011). For example, a great number and a large diversity of regulatory RNAs have been recently identified in the pathogenic clostridium Clostridium difficile (Soutourina et al 2013).…”

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

“…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.…”