CaMKⅡ Mediates The Mechanism Of Suilysininduced Apoptosis In PK-15 Cells

Suilysin（Sly）is the main virulence factor of Streptococcus suis（S.suis）,which plays a vital role in the pathogenesis of Streptococcus suis.Previous studies identified Ca²⁺/calmodulin-dependent protein kinase Ⅱ（CaMKⅡ）as one of the key host factors mediating the cytotoxicity of recombinant suilysin（rSly）,and this study aimed to investigate the mechanism of CaMKⅡ-mediated rSly-induced apoptosis in porcine kidney cells（PK-15）.1.Study on the construction of PK-15 cytotoxicity model induced by rSlyFirstly,the soluble rSly was successfully expressed.In this study,the Sly virulence gene（1455 bp）was amplified using the genomic DNA of Streptococcus suis serotype 2 as a template.The p Cold I-Sly recombinant plasmid was constructed,transferred into E.coli BL21,and induced to express for 24 h at 18℃,180 r/min.By SDS-PAGE,Western blotting and hemolytic activity assay,the rSly with hemolytic activity of 55 k Da size was successfully expressed.Secondly,a model of rSly-induced PK-15 cytotoxicity was constructed.PK-15 cells were treated with rSly at 0,0.3,0.6,1.2,2.4,and 4.8μg/m L for 6,12,and 24 h.By observing the cell morphology,measuring the cell survival rate and LDH release rate,it was found that rSly toxicity showed a significant dose effect.The model of rSly-induced cytotoxicity in PK-15 cells was constructed by:determining the concentration of rSly staining PK-15 cells at 0.3 and 0.6μg/m L;determining the treatment time of rSly staining PK-15 cells at 24 h;and determining the indicators of rSly staining PK-15 cells as follows:（1）cell growth characteristics:elongation of cell morphology and increase of gap;（2）cell survival rate:89.06%and 53.06%for rSly treated concentration of 0.3 and 0.6μg/m L,respectively;（3）LDH release rate:25.32%and33.61%for rSly treated concentration of 0.3 and 0.6μg/m L,respectively,33.61%.2.Study on CaMKⅡ-mediated rSly-induced apoptosis in PK-15 cellsFirstly,the cytotoxic effect of CaMKⅡ-mediated rSly was analyzed.First,PK-15 cells were treated with 0.3 and 0.6μg/m L of rSly for 24 h,respectively.Western blotting detected that rSly increased CaMKⅡ phosphorylation;second,both cell morphological observations and cell viability assays showed that inhibition of CaMKⅡ activity using KN93 significantly attenuated rSly-induced cytotoxicity and increased cell survival（P<0.01）,indicating that CaMKⅡ mediated the rSly-induced cytotoxic effects;then the CAMK2A,CAMK2B,CAMK2D,and CAMK2G genes encoding the four isoforms of CaMKⅡ proteins were knocked down on PK-15 cells using CRISPR/Cas9 gene editing technology,respectively.The resistance of PK-15^ΔCAMK2A and PK-15^ΔCAMK2B to the cytotoxicity of rSly was enhanced and cell survival was increased after rSly treatment of the four knockdown cells（P<0.01）,revealing that two isoforms of CaMKⅡ,αandβ,play a major role in rSly-induced cytotoxicity.Secondly,CaMKⅡ was found to mediate rSly-induced apoptosis in PK-15 cells.First,by measuring the characteristic indexes of apoptosis,necroptosis,autophagy and pyroptosis,it was found that the main pathway of rSly-induced cell death was apoptosis;second,after inhibition of CaMKⅡ activity by KN93,the AO-EB assay showed that rSly-induced apoptosis was inhibited,and the apoptosis rate determined by flow cytometry was reduced by 2.2-fold,revealing that CaMKⅡ promotes rSly-induced apoptosis in PK-15 cells.3.The mechanism of CaMKⅡ-mediated induction of apoptosis in PK-15 cells by rSlyFirstly,CaMKⅡ was found to mediate the binding of rSly to the cell membrane.Using the immunofluorescence technique,we found that inhibition of CaMKⅡ reduced the binding of rSly to the PK-15 cell membrane.Secondly,it was revealed that CaMKⅡ could promote rSly-induced apoptosis by promoting ROS generation and thus rSly-induced apoptosis.By measuring the content of pro-apoptotic factor ROS,the results indicated that inhibition of CaMKⅡ significantly reduced rSly-induced ROS production（P<0.001）,and pretreatment with ROS scavenger（N-acetyl-L-cysteine,NAC）proved that NAC was able to reduce apoptosis in PK-15 cells with a 3.2-fold reduction in apoptosis,illustrating CaMKⅡ can promote rSly-induced apoptosis by promoting ROS generation and thus rSly-induced apoptosis.In conclusion,this study found that CaMKⅡ could promote rSly-induced apoptosis in PK-15 cells,and CaMKⅡ promoted apoptosis in PK-15 cells by promoting the binding of rSly to cell membranes and the generation of ROS,which provides a corresponding theoretical basis to continue to elucidate the toxicity mechanism of Sly in depth in the future.