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中华实验和临床感染病杂志(电子版) ›› 2020, Vol. 14 ›› Issue (05) : 411 -417. doi: 10.3877/cma.j.issn.1674-1358.2020.05.010

所属专题： 文献；

论著

金黄色葡萄球菌和纤维连接蛋白结合蛋白A对血管内皮细胞紧密连接的破坏作用
冯洒然1, 李德志2,†(), 林殿杰2, 朱玲2   
  1. 1. 250014　济南市，山东第一医科大学第一附属医院血液科
    2. 250014　济南市，山东第一医科大学附属省立医院呼吸与危重症医学科
  • 收稿日期:2019-10-29 出版日期:2020-10-20
  • 通信作者: 李德志
  • 基金资助:
    山东省自然科学基金项目(No. ZR2014HQ050，No. ZR2015HL38)

Disruption of Staphylococcus aureus and staphylococcal Fn-binding protein A to the tight junction of human microvascular endothelial cells

Saran Feng1, Dezhi Li2,†(), Dianjie Lin2, Ling Zhu2   

  1. 1. Department of Hematology, First Affiliated Hospital of Shandong First Medical University, Jinan 250014, China
    2. Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250014, China
  • Received:2019-10-29 Published:2020-10-20
  • Corresponding author: Dezhi Li
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引用本文：

冯洒然, 李德志, 林殿杰, 朱玲. 金黄色葡萄球菌和纤维连接蛋白结合蛋白A对血管内皮细胞紧密连接的破坏作用[J]. 中华实验和临床感染病杂志(电子版), 2020, 14(05): 411-417.

Saran Feng, Dezhi Li, Dianjie Lin, Ling Zhu. Disruption of Staphylococcus aureus and staphylococcal Fn-binding protein A to the tight junction of human microvascular endothelial cells[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2020, 14(05): 411-417.

目的

观察金黄色葡萄球菌和纤维连接蛋白结合蛋白A基因（fnBA）敲除金黄色葡萄球菌菌株对人微血管内皮细胞（HMEC-1）紧密连接蛋白ZO-1、Claudin-5的影响，并探讨金黄色葡萄球菌侵袭血管内皮细胞的机制。

方法

将野生金黄色葡萄球菌菌株NCTC8325与HMEC-1按100︰1比例共培养，实时定量RT-PCR检测共培养30 min、60 min和120 min时HMEC-1紧密连接成份ZO-1和Claudin-5 mRNA的表达，同时应用Western blot分析和免疫组织化学染色观察共培养不同时间紧密连接蛋白ZO-1和Claudin-5的表达。构建fnBA基因敲除突变菌株NCTC8325ΔfnbA，以野生金黄色葡萄球菌菌株NCTC8325为阳性对照，观察突变株NCTC8325ΔfnbA与HMEC-1共培养120 min后紧密连接蛋白ZO-1和Claudin-5的变化。

结果

金黄色葡萄球菌NCTC8325与HMEC-1共培养30 min、60 min和120 min后紧密连接蛋白ZO-1、Claudin-5 mRNA的表达在30 min、120 min时较对照组显著下降[30 min时与对照组比较：ZO-1：t = 4.104、P = 0.015，Claudin-5 mRNA：t = 2.802、P = 0.049；120 min时与对照组比较：ZO-1：t = 3.478、P = 0.025，Claudin-5 mRNA：t = 1.802、P = 0.261]，但60 min时ZO-1、Claudin-5 mRNA的表达有一过性升高。与金黄色葡萄球菌NCTC8325共培养后，免疫组织化学结果发现在30 min时ZO-1和Claudin-5两种紧密连接蛋白较对照组表达显著下降（t = 33.6、59.03，P均< 0.001），120 min时ZO-1和Claudin-5两种紧密连接蛋白较对照组表达亦显著下降（t = 31.8、60.75，P均< 0.001）；Western blot与免疫组组织化学结果一致。与突变菌株NCTC8325ΔfnbA共培养30 min、60 min和120 min后，在30 min和60 min时ZO-1、Claudin-5蛋白的表达与NCTC8325组差异无统计学意义（P均> 0.05），在120 min时ZO-1和Claudin-5蛋白的表达较NCTC8325组显著升高，差异均有统计学意义（ZO-1：t = 14.89、P < 0.001，Claudin-5：t = 7.008、P = 0.002）。

结论

金黄色葡萄球菌能通过下调紧密连接蛋白破坏HMEC-1紧密连接屏障，且其表面蛋白FnBPA发挥了重要作用。

关键词: 金黄色葡萄球菌, 基因敲除, 纤维连接蛋白结合蛋白A基因, 紧密连接, 闭锁小带蛋白1, 紧密连接整合膜蛋白5
Objective

To investigate the changes of tight junction protein ZO-1 and Claudin-5 of human microvascular endothelial cells (HMEC-1) after incubation with Staphylococcus aureus (S. aureus) and fnbA knockout strains, and to explore the mechanism of S. aureus invading the vascular endothelial cells.

Methods

Wild NCTC8325 S. aureus strain were co-cultured with HMEC-1 in 100:1 ratio for 30 min, 60 min and 120 min. Quantitative real-time-PCR analyses were performed to examine the expressions of ZO-1 and Claudin-5 mRNA. At the same time, the expressions of protein ZO-1 and Claudin-5 were also analyzed by western blot and immunohistochemistry methods. The strain NCTC8325Δ fnbA with fnbA gene knockout was generated and co-cultured with HMEC-1 for 120 min. Western blot and immunohistochemistry were used to measure the expressions of ZO-1 and Claudin-5.

Results

The mRNA expression at different times showed a pronounced transcription of ZO-1 and Claudin-5 (P = 0.0017). The transcriptional level at 60 min was higher. At 30 min, transcriptional levels of ZO-1 and Claudin-5 were higher than 0 min (ZO-1: t = 4.104, P = 0.0148; Claudin-5: t = 2.802, P = 0.0487). At 120 min, transcriptional levels of ZO-1 and Claudin-5 were higher than 0 min (ZO-1: t = 3.478, P = 0.0254; Claudin-5: t = 1.802, P = 0.2611). After incubation with S. aureus NCTC8325, the levels of tight junction proteins ZO-1 and Claudin-5 were significantly down-regulated at 30 min and 120 min examined by immunohistochemistry (30 min: ZO-1: t = 33.6, P = 0.0001; Claudin-5: t = 59.03, P = 0.0001; 120min: ZO-1: t = 31.8, P = 0.0001; Claudin-5: t = 60.75, P = 0.0001). The levels of tight junction proteins ZO-1 and Claudin-5 were consistent with the results of immunohistochemistry. After incubation with S. aureus NCTC8325Δ fnbA for 30 min, 60 min and 120 min, there were no differences in S. aureus NCTC8325 and S. aureus NCTC8325Δ fnbA group at 30 min and 60 min, but the levels of tight junction proteins ZO-1 and Claudin-5 were significantly up-regulated than the control at 120 min (ZO-1: t = 14.89, P = 0.0001; Claudin-5: t = 7.008, P = 0.0022).

Conclusions

S. aureus could breach the microvascular endothelial cells so as to favor themselves shuttling by disrupting tight junction proteins ZO-1 and Claudin-5, and FnBPA might play an important role in the process.

Key words: Staphylococcus aureus, Gene knockout, Fn-binding protein A gene, Tight junction, Zonula occludens-1, Claudin-5
图1 金黄色葡萄球菌NCTC8325在不同时间对HMEC-1 ZO-1和Claudin-5 mRNA的影响（免疫组织化学染色）
图2 金黄色葡萄球菌NCTC8325在不同时间对HMEC-1紧密连接蛋白ZO-1和Claudin-5的影响（免疫组织化学染色）
图3 金黄色葡萄球菌NCTC8325在不同时间对HMEC-1紧密连接蛋白ZO-1和Claudin-5的影响（200 ×）
图4 金黄色葡萄球菌NCTC8325在不同时间对HMEC-1紧密连接蛋白ZO-1和Claudin-5的影响（Western blot分析）
图5 金黄色葡萄球菌NCTC8325和NCTC8325ΔfnbA在120 min时对HMEC-1紧密连接蛋白ZO-1和Claudin-5的影响（免疫组织化学染色）
图6 金黄色葡萄球菌NCTC8325和NCTC8325ΔfnbA在120 min时对HMEC-1紧密连接蛋白ZO-1和Claudin-5的影响（Western blot分析）
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