Staphylococcus pseudintermedius induces pyroptosis of canine corneal epithelial cells by activating the ROS–NLRP3 signalling pathway

ABSTRACT

Staphylococcus pseudintermedius (S. pseudintermedius) is a common pathogen that causes canine corneal ulcers. However, the pathogenesis remained unclear. In this study, it has been demonstrated that S. pseudintermedius invaded canine corneal epithelial cells (CCECs) intracellularly, mediating oxidative damage and pyroptosis by promoting the accumulation of intracellular reactive oxygen species (ROS) and activating the NLRP3 inflammasome. The canine corneal stroma was infected with S. pseudintermedius to establish the canine corneal ulcer model in vivo. The intracellular infectious model in CCECs was established in vitro to explore the mechanism of the ROS – NLRP3 signalling pathway during the S. pseudintermedius infection by adding NAC or MCC950. Results showed that the expression of NLRP3 and gasdermin D (GSDMD) proteins increased significantly in the infected corneas (p < 0.01). The intracellular infection of S. pseudintermedius was confirmed by transmission electron microscopy and immunofluorescent 3D imaging. Flow cytometry analysis revealed that ROS and pyroptosis rates increased in the experimental group in contrast to the control group (p < 0.01). Furthermore, NAC or MCC950 inhibited activation of the ROS – NLRP3 signalling pathway and pyroptosis rate significantly, by suppressing pro-IL-1β, cleaved-IL-1β, pro-caspase-1, cleaved-caspase-1, NLRP3, GSDMD, GSDMD-N, and HMGB1 proteins. Thus, the research confirmed that oxidative damage and pyroptosis were involved in the process of CCECs infected with S. pseudintermedius intracellularly by the ROS – NLRP3 signalling pathway. The results enrich the understanding of the mechanisms of canine corneal ulcers and facilitate the development of new medicines and prevention measures.

KEYWORDS:

• Staphylococcus pseudintermedius
• canine corneal epithelial cells
• intracellular infection
• pyroptosis
• NLRP3 inflammasome
• reactive oxygen species

Acknowledgements

The authors would like to thank the technical staff of the Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses for their excellent technical assistance.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

• Conceptualization: Heng Wang, Zhihao Wang, Long Guo.

• Data curation: Zhihao Wang, Long Guo, Chengcheng Zhu, Changning Yuan, Peng Mao, Kangjun Liu.

• Funding acquisition: Heng Wang, Jianji Li, Luying Cui, Junsheng Dong, Guoqiang Zhu, Jun Li, Xia Meng, Long Guo, Zhihao Wang.

• Methodology: Zhihao Wang, Long Guo, Haoran Zhong, Chengcheng Zhu, Changning Yuan, Peng Mao.

• Writing – original draft: Zhihao Wang, Long Guo, Haoran Zhong

• Writing – review & editing: Heng Wang, Jianji Li, Luying Cui, Junsheng Dong, Guoqiang Zhu, Xia Meng, Jun Li.

Data Availability statement

Data is openly available in a public repository.

supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2024.2333271

Additional information

Funding

This work was supported financially by 333 High-level Talent Training Project of Jiangsu Province (CN), the Jiangsu Postgraduate Research and Innovation Plan [KYCX21_3273], Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD), the 111 Project D18007, the Open Project of International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions, and Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (TAPP).