Malabirade Antoine, Habier Janine, Heintz-Buschart Anna, May Patrick, Godet Julien, Halder Rashi, Etheridge Alton, Galas David, Wilmes Paul, Fritz Joëlle V
Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
UMR CNRS 7021, Laboratoire de BioImagerie et Pathologies, Université de Strasbourg, Strasbourg, France.
Front Microbiol. 2018 Aug 30;9:2015. doi: 10.3389/fmicb.2018.02015. eCollection 2018.
Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host-pathogen interactions. The presence of larger RNA transcripts in OMVs has been less studied and their potential role in host-pathogen interactions remains largely unknown. Here we analyze RNA from OMVs secreted by serovar Typhimurium ( Typhimurium) cultured under different conditions, which mimic host-pathogen interactions. Typhimurium was grown to exponential and stationary growth phases in minimal growth control medium (phosphate-carbon-nitrogen, PCN), as well as in acidic and phosphate-depleted PCN, comparable to the macrophage environment and inducing therefore the expression of pathogenicity island 2 (SPI-2) genes. Moreover, pathogenicity island 1 (SPI-1), which is required for virulence during the intestinal phase of infection, was induced by culturing Typhimurium to the stationary phase in Lysogeny Broth (LB). For each condition, we identified OMV-associated RNAs that are enriched in the extracellular environment relative to the intracellular space. All RNA classes could be observed, but a vast majority of rRNA was exported in all conditions in variable proportions with a notable decrease in LB SPI-1 inducing media. Several mRNAs and ncRNAs were specifically enriched in/on OMVs dependent on the growth conditions. Important to note is that some RNAs showed identical read coverage profiles intracellularly and extracellularly, whereas distinct coverage patterns were observed for other transcripts, suggesting a specific processing or degradation. Moreover, PCR experiments confirmed that distinct RNAs were present in or on OMVs as full-length transcripts (; ; ; ; ; ; ; ; ; ; ), whereas others seemed to be rather present in a processed or degraded form. Finally, we show by a digestion protection assay that OMVs are able to prevent enzymatic degradation of given full-length transcripts (, , , and . In summary, we show that OMV-associated RNA is clearly different in distinct culture conditions and that at least a fraction of the extracellular RNA is associated as a full-length transcripts with OMVs, indicating that some RNAs are protected by OMVs and thereby leaving open the possibility that those might be functionally active.
细菌外膜囊泡（OMV）以及与OMV相关的小RNA已被证明在宿主与病原体的相互作用中发挥作用。关于OMV中较大RNA转录本的研究较少，其在宿主与病原体相互作用中的潜在作用在很大程度上仍不清楚。在此，我们分析了在不同条件下培养的鼠伤寒血清型（鼠伤寒沙门氏菌）分泌的OMV中的RNA，这些条件模拟了宿主与病原体的相互作用。鼠伤寒沙门氏菌在基本生长对照培养基（磷酸盐 - 碳 - 氮，PCN）中生长至指数生长期和稳定期，以及在酸性和磷酸盐耗尽的PCN中生长，这与巨噬细胞环境相当，因此诱导了毒力岛2（SPI - 2）基因的表达。此外，感染肠道阶段毒力所需的毒力岛1（SPI - 1）通过在溶原肉汤（LB）中将鼠伤寒沙门氏菌培养至稳定期来诱导。对于每种条件，我们鉴定了相对于细胞内空间在细胞外环境中富集的与OMV相关的RNA。可以观察到所有RNA类别，但绝大多数rRNA在所有条件下以不同比例输出，在LB SPI - 1诱导培养基中显著减少。几种mRNA和ncRNA根据生长条件在OMV中/上特异性富集。需要注意的是，一些RNA在细胞内和细胞外显示出相同的读取覆盖图谱，而其他转录本则观察到不同的覆盖模式，这表明存在特定的加工或降解。此外，PCR实验证实不同的RNA以全长转录本的形式存在于OMV中或其上（;;;;;;;;;），而其他一些似乎以加工或降解的形式存在。最后，我们通过消化保护试验表明OMV能够防止给定全长转录本（、、和）的酶促降解。总之，我们表明在不同培养条件下与OMV相关的RNA明显不同，并且至少一部分细胞外RNA以全长转录本的形式与OMV相关联，这表明一些RNA受到OMV的保护，从而留下了它们可能具有功能活性的可能性。
