Endocytosis‒Mediated Invasion and Pathogenicity of <i>Streptococcus agalactiae</i> in Rat Cardiomyocyte (H9C2)

Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse‒ transcription PCR (RT‒PCR) and quantitative real time‒PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae‒infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose‒dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy‒dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction.无乳链球菌（Streptococcus agalactiae）感染会导致心血管疾病（cardiovascular disease, CVD）患者死亡率居高不下，在心脏手术期间植入人工瓣膜的患者中尤为显著。然而，无乳链球菌与心血管疾病相关的致病机制尚未得到充分研究。本研究证实了无乳链球菌在大鼠心肌细胞（H9C2）中的致病性。值得注意的是，H9C2细胞可同时摄取无乳链球菌的活菌与死菌菌体。为进一步解析无乳链球菌的内化过程，我们使用染料木黄酮（genistein）、氯丙嗪（chlorpromazine）、诺考达唑（nocodazole）与细胞松弛素B（cytochalasin B），对H9C2细胞的细胞摄取通路的不同环节进行了化学抑制。经诺考达唑与细胞松弛素B分别抑制微管与肌动蛋白形成后，无乳链球菌对H9C2细胞的内化能力受到显著损伤。与之相符的是，逆转录聚合酶链式反应（reverse-transcription PCR, RT-PCR）与实时定量聚合酶链式反应（quantitative real time-PCR, RT-qPCR）分析结果显示，无乳链球菌感染的H9C2细胞中，细胞骨架形成相关基因Acta1与Tubb5的转录水平显著升高，这提示功能性细胞骨架在致病过程中不可或缺。宿主存活实验证实，无乳链球菌的内化可诱导H9C2细胞产生细胞毒性。经苄青霉素（benzyl penicillin）培养的无乳链球菌，其内化能力与诱导H9C2细胞产生细胞毒性的能力均有所降低，该现象可归因于该菌表面的脂磷壁酸（lipoteichoic acid, LTA）被去除。进一步研究发现，从无乳链球菌中提取的LTA同样可在H9C2细胞中呈现剂量依赖性的细胞毒性。综上，本研究数据表明，无乳链球菌通过能量依赖的内吞过程实现对H9C2细胞的内化，且该菌的LTA在宿主细胞内化与细胞毒性诱导过程中发挥关键作用。