Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA.
Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA
Infect Immun. 2021 Mar 17;89(4). doi: 10.1128/IAI.00614-20.
Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe delivers its SLs to host cells and that the ability of to synthesize SLs limits the elicited host inflammatory response during cellular infection. As robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyperinflammatory immune response from THP-1 macrophage-like cells with elevated tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing beta interferon) by SL-null No significant differences in gingipain activity were observed in our infection models, and both strains produced OMVs of similar sizes. Using comparative two-dimensional gel electrophoresis, we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response.
鞘脂（SLs）是哺乳动物细胞膜的重要结构成分。我们的研究小组最近确定，口腔厌氧菌 向宿主细胞输送 SLs，并且 合成 SLs 的能力限制了细胞感染期间宿主炎症反应的激发。由于 能够大量产生外膜囊泡（OMVs），我们假设 OMVs 是 SLs 的一种输送载体，OMVs 的 SL 状态可能会影响到 OMVs 的货物装载，并且含有 SL 的 OMVs 可以限制激发宿主炎症，这与直接细菌挑战观察到的结果相似。Transwell 细胞培养实验确定，与亲本菌株 W83 相比，SL 缺失突变体从 THP-1 巨噬样细胞中引发了过度炎症的免疫反应，导致肿瘤坏死因子 alpha（TNF-α）、白细胞介素 1β（IL-1β）和白细胞介素 6（IL-6）水平升高。对 Toll 样受体（TLRs）的靶向评估表明，TLR2 的表达上调，TLR4 不变，而衔接分子 MyD88 和 TRIF（Toll/IL-1 受体域包含衔接诱导β干扰素）的表达上调。在我们的感染模型中，未观察到牙龈蛋白酶活性的显著差异，两种菌株产生的 OMVs 大小相似。使用比较二维凝胶电泳，我们在亲本和 SL 缺失菌株的 OMV 蛋白货物中鉴定出差异。重要的是，使用纯化的 OMVs 可以重现整个细菌在 Transwell 系统中观察到的细胞炎症反应。这些发现为 SLs 在 OMV 货物组装中的作用提供了新的见解，并扩展了我们对 SL-OMVs 作为调节宿主炎症反应的细菌结构的理解。
