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International audience; The roles of bacterial extracellular vesicles (EVs) in cell-to-cell signaling are progressively being unraveled. These membranous spheres released by many living cells carry various macromolecules, some of which influence host-pathogen interactions. Bacterial EVs contain RNA, which may serve in communicating with their infected hosts. Staphylococcus aureus, an opportunistic human and animal pathogen, produces EVs whose RNA content is still poorly characterized. Here, we investigated in depth the RNA content of S. aureus EVs. A high-throughput RNA sequencing approach identified RNAs in EVs produced by the clinical S. aureus strain HG003 under different environmental conditions: early- and late-stationary growth phases, and presence or absence of a sublethal vancomycin concentration. On average, sequences corresponding to 78.0% of the annotated transcripts in HG003 genome were identified in HG003 EVs. However, only ~5% of them were highly covered by reads (≥90% coverage) indicating that a large fraction of EV RNAs, notably mRNAs and sRNAs, were fragmented in EVs. According to growth conditions, from 86 to 273 highly covered RNAs were identified into the EVs. They corresponded to 286 unique RNAs, including 220 mRNAs. They coded for numerous virulence-associated factors (hld encoded by the multifunctional sRNA RNAIII, agrBCD, psmβ1, sbi, spa, and isaB), ribosomal proteins, transcriptional regulators, and metabolic enzymes. Twenty-eight sRNAs were also detected, including bona fide RsaC. The presence of 22 RNAs within HG003 EVs was confirmed by reverse transcription quantitative PCR (RT-qPCR) experiments. Several of these 286 RNAs were shown to belong to the same transcriptional units in S. aureus. Both nature and abundance of the EV RNAs were dramatically affected depending on the growth phase and the presence of vancomycin, whereas much less variations were found in the pool of cellular RNAs of the parent cells. Moreover, the RNA abundance pattern differed between EVs and EV-producing cells according to the growth conditions. Altogether, our findings show that the environment shapes the RNA cargo of the S. aureus EVs. Although the composition of EVs is impacted by the physiological state of the producing cells, our findings suggest a selective packaging of RNAs into EVs, as proposed for EV protein cargo. Our study shedds light to the possible roles of potentially functional RNAs in S. aureus EVs, notably in host-pathogen interactions.

BactInflam IJL focuses on the bacterial components involved in some inflammatory diseases. In particular, in two of them: chronic inflammatory bowel disease (IBD) and mastitis, affecting human health and animal health, respectively.; International audience; Introduction: Bacterial extracellular vesicles (EVs) carry various macromolecules able to affect host-pathogen interactions, such as RNAs. Staphylococcus aureus, an important human and animal pathogen, releases EVs whose RNA content is still unkown. Here, we adress what classes of RNAs compose S. aureus EVs. Methods: S. aureus strain HG003 was cultured in Brain Heart Infusion medium under different in vitro conditions: early- and late-stationary phases, in the presence or absence of a sublethal concentration of vancomycin (0.5 μg/mL). EVswere purified from cell-free culture supernatants using density gradient ultracentrifugation. Bacterial and EV samples were submitted to phenolchloroform RNA extraction, DNAse treatment, and library preparation (Ovation Prokaryotic RNA-Seq, Nugen, rRNA depletion). Sequencing was performed using Illumina, NextSeq500, 75 cycles, single reading, High Output. Results: Particle yields were similar between conditions, however, EVs from late-stationary phases were ∼55% ,larger. On average, 78.0% of HG003 annotated genes were identified in EVs, while only ∼5% presented - 90% read coverage. Highly covered EV RNAs included mRNAs coding for virulence-factors (hld, agrBCD, psmB1, sbi, spa, isaB), ribosomal proteins, transcriptional regulators, and metabolic enzymes. sRNAs were also detected, including the bona fide rsaC. Interestingly, several of these RNAs were shown to belong to the same transcriptional units in S. aureus. Both nature and abundance of the RNAs in EVs were dramatically affected by growth conditions, whereas much less in the parent cells. Finally, the RNA abundance pattern differed between EVs and parent cells. Summary/Conclusion: To our knowledge, this is the first work characterizing the RNA cargo of S. aureus EVs. Our findings show that EV RNAs are shaped by the environment, and suggest the selective packaging of RNAs into EVs. Finally, this study also shedds light to the possible roles of potentially functional RNAs in S. aureus EVs, notably in host-pathogen interactions.