Study of Apoptosis Mode of Porcine Kidney Cells Induced by Low Concentration Suilysin
DAI Shi-Xin, ZHAO Qin, XU Kui, MOU Xin-Ran, DU Sen-Yan, WU Rui, WEN Yi-Ping, HUANG Xiao-Bo, YAN Qi-Gui, CAO San-Jie*
College of Veterinary Medicine/Research Center of Swine Diseases/National Demonstration Center for Experimental Animal Education/Sichuan Science-Observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technique, Ministry of Agriculture and Rural Affairs, Sichuan Agricultural University, Chengdu 611130, China
Abstract：Streptococcus suis is a serious zoonotic pathogen. Suilysin (Sly) plays a key role in the invasion and pathogenesis of S. suis. It has toxic effects on various types of cells, but there are few studies on the cytotoxic mechanism of Sly. In order to explore the death modes of the porcine kidney cells 15 (PK-15) induced by low concentration of Sly (0.3~0.6 μg/mL), in this study, recombinant suilysin (rSly) was obtained by prokaryotic expression and purification, the toxicity of rSly on PK-15 cells was detected by lactate dehydrogenase (LDH) release assay. Then, for 4 common programmed cell death modes, qPCR, immunofluorescence, Western blot, and flow cytometry assay were used to verify the ways of rSly-induced cell death in PK-15 cells for 12 and 24 h. The results showed that rSly with high hemolytic activity (214 hemolytic units) was successfully expressed, and its toxic effect on PK-15 cells was confirmed to be concentration-dependent. The transcription levels of apoptosis-related genes-cysteine aspartic acid specific protease 3 (Caspase 3) and Caspase 9 were significantly increased after rSly treatment for 12 and 24 h (P<0.05). The apoptosis rate was positively correlated with the concentration of rSly, and the apoptosis rate of PK-15 cells increased from 9.35% to 49.9% after 0.6 μg/mL rSly treatment for 24 h. There was no significant change in the transcription level of microtubule associated protein 1 light chain 3β (LC3β), a key autophagy gene, and no autophagosome formation. The transcription level of gasdermin D (GSDMD), a pyroptosis-related gene had no significant change, and no obviously cleavage of GSDMD protein. Necroptosis-related genes were down-regulated, and the expression level of phosphorylated mixed lineage kinase domain-like protein (p-MLKL) was inhibited. The results suggested that low concentration of rSly induced PK-15 cell death mainly by apoptosis. This study enriches the cellular model of Sly toxicity and helps to reveal the molecular mechanisms underlying the development of S.suis infection.
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