MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China.
PLoS Pathog. 2022 Aug 3;18(8):e1010765. doi: 10.1371/journal.ppat.1010765. eCollection 2022 Aug.
Streptococcus suis serotype 2 (SS2) is a major zoonotic pathogen resulting in manifestations as pneumonia and septic shock. The upper respiratory tract is typically thought to be the main colonization and entry site of SS2 in pigs, but the mechanism through which it penetrates the respiratory barrier is still unclear. In this study, a mutant with low invasive potential to swine tracheal epithelial cells (STECs) was screened from the TnYLB-1 transposon insertion mutant library of SS2, and the interrupted gene was identified as autolysin (atl). Compared to wild-type (WT) SS2, Δatl mutant exhibited lower ability to penetrate the tracheal epithelial barrier in a mouse model. Purified Atl also enhanced SS2 translocation across STEC monolayers in Transwell inserts. Furthermore, Atl redistributed the tight junctions (TJs) in STECs through myosin light chain kinase (MLCK) signaling, which led to increased barrier permeability. Using mass spectrometry, co-immunoprecipitation (co-IP), pull-down, bacterial two-hybrid and saturation binding experiments, we showed that Atl binds directly to vimentin. CRISPR/Cas9-targeted deletion of vimentin in STECs (VIM KO STECs) abrogated the capacity of SS2 to translocate across the monolayers, SS2-induced phosphorylation of myosin II regulatory light chain (MLC) and MLCK transcription, indicating that vimentin is indispensable for MLCK activation. Consistently, vimentin null mice were protected from SS2 infection and exhibited reduced tracheal and lung injury. Thus, MLCK-mediated epithelial barrier opening caused by the Atl-vimentin interaction is found to be likely the key mechanism by which SS2 penetrates the tracheal epithelium.
猪链球菌 2 型（SS2）是一种主要的人畜共患病病原体，可引起肺炎和感染性休克。通常认为上呼吸道是 SS2 在猪体内定植和进入的主要部位，但它穿透呼吸道屏障的机制尚不清楚。在本研究中，从 SS2 的 TnYLB-1 转座子插入突变体文库中筛选出一株对猪气管上皮细胞（STEC）侵袭能力较低的突变株，并鉴定出中断的基因为自溶素（atl）。与野生型（WT）SS2 相比，Δatl 突变株在小鼠模型中穿透气管上皮屏障的能力较低。纯化的 Atl 也增强了 SS2 在 Transwell 插入物中穿过 STEC 单层的易位。此外，Atl 通过肌球蛋白轻链激酶（MLCK）信号重新分布 STEC 中的紧密连接（TJ），导致屏障通透性增加。通过质谱、共免疫沉淀（co-IP）、下拉、细菌双杂交和饱和结合实验，我们表明 Atl 直接与波形蛋白结合。在 STEC 中靶向敲除波形蛋白（VIM KO STECs）消除了 SS2 穿过单层的易位能力、SS2 诱导的肌球蛋白 II 调节轻链（MLC）和 MLCK 转录的磷酸化，表明波形蛋白对于 MLCK 激活是必不可少的。一致地，波形蛋白缺失小鼠免受 SS2 感染，并表现出减轻的气管和肺部损伤。因此，发现 Atl-波形蛋白相互作用引起的 MLCK 介导的上皮屏障开放可能是 SS2 穿透气管上皮的关键机制。
