Environmental plasticity of the RNA content of Staphylococcus aureus Extracellular Vesicles. S. aureus EV RNA cargo

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 deepth 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 multifunctional sRNA RNAIII, agrBCD, psm1, sbi, spa, isaB), ribosomal proteins, transcriptional regulators, and metabolic enzymes. 28 sRNAs were also detected, including bona fide RsaC. The presence of 22 RNAs within HG003 EVs was confirmed by 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.

细菌细胞外囊泡（extracellular vesicles，EVs）在细胞间信号传导中的作用正逐步被阐明。这类由多种活细胞分泌的膜性囊泡可携带多种大分子物质，其中部分可影响宿主与病原体间的相互作用。细菌细胞外囊泡含有RNA，这类RNA或可参与与感染宿主的信息交流。金黄色葡萄球菌（Staphylococcus aureus）作为一种人畜机会致病菌，其产生的细胞外囊泡的RNA组成仍未得到充分解析。本研究深入解析了金黄色葡萄球菌细胞外囊泡的RNA组成：采用高通量RNA测序技术，对临床分离的金黄色葡萄球菌菌株HG003在不同环境条件下（包括生长早期及晚期静止期、有无亚致死浓度万古霉素处理）产生的细胞外囊泡中的RNA进行了鉴定。平均而言，HG003基因组中78.0%的已注释转录本的序列均可在其细胞外囊泡中被检测到。然而其中仅有约5%的转录本可被测序读段高度覆盖（覆盖度≥90%），这表明细胞外囊泡中的大部分RNA（尤其是信使RNA（messenger RNA，mRNA）和小RNA（small RNA，sRNA））在囊泡内发生了片段化。根据不同生长条件，细胞外囊泡中可鉴定出86至273条高度覆盖的RNA，共计286种独特转录本，其中包含220条mRNA。这些转录本编码多种毒力相关因子（如由多功能sRNA RNAIII编码的hld、agrBCD、psmβ1、sbi、spa、isaB）、核糖体蛋白、转录调控因子以及代谢酶类。此外还检测到28条sRNA，其中包括真实存在的RsaC。通过实时定量逆转录PCR（reverse transcription quantitative PCR，RT-qPCR）实验，验证了HG003细胞外囊泡中22条RNA的存在。这286条RNA中有多条被证实属于金黄色葡萄球菌中同一转录单元。细胞外囊泡RNA的组成与丰度均会随生长阶段及万古霉素处理情况发生显著变化，而产囊泡宿主细胞的胞内RNA库则仅存在较少的变异。此外，不同生长条件下，细胞外囊泡与产囊泡宿主细胞的RNA丰度模式亦存在差异。综上，本研究结果表明环境因素可调控金黄色葡萄球菌细胞外囊泡的RNA载荷组成。尽管细胞外囊泡的组成会受产囊泡细胞的生理状态影响，但本研究结果表明，RNA会被选择性包装进入细胞外囊泡，这与此前针对细胞外囊泡蛋白载荷提出的假说一致。本研究为揭示金黄色葡萄球菌细胞外囊泡中功能性RNA的潜在作用（尤其是在宿主-病原体相互作用中的作用）提供了新的见解。