Clinical Microbiology, Department of Translational Medicine, Faculty of Medicine, Lund University, Malmö, Sweden.
Front Immunol. 2018 Dec 18;9:2910. doi: 10.3389/fimmu.2018.02910. eCollection 2018.
Non-typeable (NTHi), a commensal organism in pre-school children, is an opportunistic pathogen causing respiratory tract infections including acute otitis media. Adults suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized by NTHi. Previous research has suggested that, in some bacterial species, the intracellular elongation factor thermo-unstable (EF-Tu) can moonlight as a surface protein upon host encounter. The aim of this study was to determine whether EF-Tu localizes to the surface of , and if such surface-associated EF-Tu is a target for bactericidal antibodies. Using flow cytometry, transmission immunoelectron microscopy, and epitope mapping, we demonstrated that EF-Tu is exposed at the surface of NTHi, and identified immunodominant epitopes of this protein. Rabbits immunized with whole-cell NTHi produced significantly more immunoglobulin G (IgG) directed against EF-Tu than against the NTHi outer membrane proteins D and F as revealed by enzyme-linked immunosorbent assays. Chemical cleavage of NTHi EF-Tu by cyanogen bromide (CNBr) followed by immunoblotting showed that the immunodominant epitopes were located within the central and C-terminal regions of the protein. Peptide epitope mapping by dot blot analysis further revealed four different immunodominant peptide sequences; EF-Tu, EF-Tu, EF-Tu, and EF-Tu. These epitopes were confirmed to be surface-exposed and accessible by peptide-specific antibodies in flow cytometry. We also analyzed whether antibodies raised against NTHi EF-Tu cross-react with other respiratory tract pathogens. Anti-EF-Tu IgG significantly detected EF-Tu on unencapsulated bacteria, including the Gram-negative and various Gram-positive of the oral microbiome. In contrast, considerably less EF-Tu was observed at the surface of encapsulated bacteria including serotype b (Hib) and (e.g., serotype 3 and 4). Removal of the capsule, as exemplified by Hib RM804, resulted in increased EF-Tu surface density. Finally, anti-NTHi EF-Tu IgG promoted complement-dependent bacterial killing of NTHi and other unencapsulated Gram-negative bacteria as well as opsonophagocytosis of Gram-positive bacteria. In conclusion, our data demonstrate that NTHi EF-Tu is surface-exposed and recognized by antibodies mediating host innate immunity against NTHi in addition to other unencapsulated respiratory tract bacteria.
无乳链球菌（NTHi）是学龄前儿童共生体，也是一种机会性病原体，可引起包括急性中耳炎在内的呼吸道感染。患有慢性阻塞性肺疾病（COPD）的成年人会持续被 NTHi 定植。先前的研究表明，在某些细菌物种中，细胞内延伸因子热不稳定（EF-Tu）在遇到宿主时可以兼作表面蛋白。本研究旨在确定 EF-Tu 是否定位于 NTHi 的表面，如果是，那么这种表面相关的 EF-Tu 是否是杀菌抗体的靶标。我们使用流式细胞术、透射免疫电子显微镜和表位作图，证明 EF-Tu 暴露在 NTHi 的表面，并鉴定了该蛋白的免疫显性表位。用全细胞 NTHi 免疫的兔子产生的针对 EF-Tu 的免疫球蛋白 G（IgG）明显多于针对 NTHi 外膜蛋白 D 和 F 的 IgG，这一点通过酶联免疫吸附试验揭示。用氰化溴（CNBr）对 NTHi EF-Tu 进行化学切割，然后进行免疫印迹显示，免疫显性表位位于该蛋白的中央和 C 末端区域。通过斑点印迹分析进行的肽表位作图进一步显示了四个不同的免疫显性肽序列；EF-Tu、EF-Tu、EF-Tu 和 EF-Tu。这些表位通过流式细胞术的肽特异性抗体被证实是表面暴露和可及的。我们还分析了针对 NTHi EF-Tu 的抗体是否与其他呼吸道病原体发生交叉反应。针对 NTHi EF-Tu 的 IgG 显著检测到未包裹细菌（包括革兰氏阴性菌和口腔微生物组中的各种革兰氏阳性菌）上的 EF-Tu。相比之下，在包裹细菌（包括乙型流感嗜血杆菌（Hib）和 ）的表面观察到的 EF-Tu 要少得多。如 Hib RM804 所示，去除荚膜会导致 EF-Tu 表面密度增加。最后，针对 NTHi EF-Tu 的 IgG 促进了针对 NTHi 和其他未包裹的革兰氏阴性细菌的补体依赖性细菌杀伤，以及对革兰氏阳性细菌的调理吞噬作用。总之，我们的数据表明，NTHi EF-Tu 是表面暴露的，并被介导针对 NTHi 宿主固有免疫的抗体识别，除了其他未包裹的呼吸道细菌之外。
