Table_1_Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland.xls

Staphylococcus aureus is a major pathogen responsible for bovine mastitis, the most common and costly disease affecting dairy cattle. S. aureus naturally releases extracellular vesicles (EVs) during its growth. EVs play an important role in the bacteria-bacteria and bacteria-host interactions and are notably considered as nanocarriers that deliver virulence factors to the host tissues. Whether EVs play a role in a mastitis context is still unknown. In this work, we showed that S. aureus Newbould 305 (N305), a bovine mastitis isolate, has the ability to generate EVs in vitro with a designated protein content. Purified S. aureus N305-secreted EVs were not cytotoxic when tested in vitro on MAC-T and PS, two bovine mammary epithelial cell lines. However, they induced the gene expression of inflammatory cytokines at levels similar to those induced by live S. aureus N305. The in vivo immune response to purified S. aureus N305-secreted EVs was tested in a mouse model for bovine mastitis and their immunogenic effect was compared to that of live S. aureus N305, heat-killed S. aureus N305 and to S. aureus lipoteichoic acid (LTA). Clinical and histopathological signs were evaluated and pro-inflammatory and chemotactic cytokine levels were measured in the mammary gland 24 h post-inoculation. Live S. aureus induced a significantly stronger inflammatory response than that of any other condition tested. Nevertheless, S. aureus N305-secreted EVs induced a dose-dependent neutrophil recruitment and the production of a selected set of pro-inflammatory mediators as well as chemokines. This immune response elicited by intramammary S. aureus N305-secreted EVs was comparable to that of heat-killed S. aureus N305 and, partly, by LTA. These results demonstrated that S. aureus N305-secreted EVs induce a mild inflammatory response distinct from the live pathogen after intramammary injection. Overall, our combined in vitro and in vivo data suggest that EVs are worth to be investigated to better understand the S. aureus pathogenesis and are relevant tools to develop strategies against bovine S. aureus mastitis.

金黄色葡萄球菌是引起奶牛乳腺炎的主要病原体，该疾病是影响奶牛最常见且代价最高的疾病。金黄色葡萄球菌在其生长过程中自然释放细胞外囊泡（EVs）。EVs在细菌-细菌和细菌-宿主相互作用中发挥着重要作用，并被视为将致病因子递送至宿主组织的纳米载体。EVs在乳腺炎背景下是否发挥作用尚不清楚。在本研究中，我们展示了奶牛乳腺炎分离株金黄色葡萄球菌Newbould 305（N305）能够在体外产生具有特定蛋白质含量的EVs。经纯化的金黄色葡萄球菌N305分泌的EVs在体外对MAC-T和PS两种奶牛乳腺上皮细胞系进行测试时，未表现出细胞毒性。然而，它们引起的炎症细胞因子基因表达水平与活化的金黄色葡萄球菌N305引起的水平相似。在奶牛乳腺炎小鼠模型中测试了纯化的金黄色葡萄球菌N305分泌的EVs的体内免疫反应，并将其免疫原性与活化的金黄色葡萄球菌N305、热杀死的金黄色葡萄球菌N305以及金黄色葡萄球菌脂肽酸（LTA）进行了比较。在注射后24小时，对乳腺的临床和病理学迹象进行了评估，并测量了乳腺中促炎和趋化细胞因子的水平。活化的金黄色葡萄球菌引起的炎症反应显著强于其他任何测试条件。然而，金黄色葡萄球菌N305分泌的EVs诱导了剂量依赖性的中性粒细胞募集以及一组特定的促炎介质和趋化因子的产生。这种由乳腺内金黄色葡萄球菌N305分泌的EVs引发的免疫反应与热杀死的金黄色葡萄球菌以及部分脂肽酸引起的免疫反应相当。这些结果表明，金黄色葡萄球菌N305分泌的EVs在乳腺内注射后可诱导一种与活化的病原体不同的轻度炎症反应。总的来说，我们的体外和体内数据表明，EVs值得进一步研究以更好地理解金黄色葡萄球菌的致病机制，并且是开发针对奶牛金黄色葡萄球菌乳腺炎策略的相关工具。