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

doi:10.3877/cma.j.issn.1674-1358.2020.05.010

Abstract

Abstract:

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

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.

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Figures/Tables 6

References 25

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