Translocated Legionella pneumophila small RNAs mimic eukaryotic miRNAs to dampen the host immune response

Extracellular vesicles (EVs) produced by bacteria, archaea and eukaryotic cells, are increasingly recognized as important mediators of intercellular communication via transfer of a wide variety of molecular cargoes. To characterize and identify the RNA molecules enriched in Legionella pneumophila extracellular vesicles (Lp-EVs) we performed RNAseq analyses. Four independent RNAseq libraries were constructed from RNA isolated from purified Lp-EVs and as control from the bacterial pellets, from which the Lp-EVs had been shed and deep sequenced using an Illumina platform. The sequences obtained from the Lp-EVs RNAseq libraries were compared to those obtained from the RNA extracted from the bacterial pellets (Figure 1g). The Lp-EV-sRNA cargo was defined as RNAs for which after normalization at least 1000 reads were sequenced and that showed an enrichment of a log2FC > 5 as compared to the RNAs from the bacterial pellets were. Using these parameters the analysis identified 47 different sRNAs enriched in the Lp-EVs. The 20 highest enriched sRNA that were present in all four biological replicates comprised 8 tRNAs, 7 sRNAs and 5 fragments of mRNA located in the CDS or UTR. Overall design: For each RNAseq experiment, the RNA extracted from purified Legionella-vesicles was compared to the total RNA extracted from the correspondent bacterial cultures. In total n=4 independent experiments were performed