The Molecular Mechanism Of RAB8B Mediated Streptococcus Suis Sly Cytotoxicity And Immunoprotective Efficacy Of Actinobacillus Pleuropenumoniae Apx Fusion Antigen

Pore-forming toxins（PFTs）are virulence factors produced by many pathogenic bacteria.PFTs cause cell lysis by forming pores in the host cell plasma membrane.Although the process by which PFTs form pores in the plasma membrane is well known,the host factors on which PFTs depend and their molecular mechanisms are rarely reported.Suilysin（Sly）from Streptococcus suis（S.suis）is a representative of PFTs with strong cytotoxicity.In this study,we used CRISPR/Cas9 technology to screen the host factors which mediated cytotoxicity of Sly and explored the molecular mechanisms.In addition,PFTs have good immunogenicity and are potential candidates for associated pathogenic subunit vaccines.However,the cytotoxicity of PFTs limits the development and utilization of related vaccines.Actinobacillus pleuropenumoniae-RTX-Toxin（Apx）from Actinobacillus pleuropenumoniae（APP）is a representative of PFTs with strong immunogenicity.This study constructed Apx fusion protein based on Apx structural protein and evaluated its immunoprotective efficacy.A summary of our research is presented below.1.CRISPR/Cas9 screening identifies host factors for Sly.Firstly,we successfully screened and identified seven host factors for Sly.This study used the CRISPR/Cas9 library-based screening method to identify disruptions in porcine coding genes that confer resistance to the cytolytic action of Sly on PK15 cells.We ranked the candidate genes based on the number of unique sg RNA versus NGS reads and selected the top seven genes for validation.We generated PK15?CAMK2B（PK15 derivative,CAMK2B deletion）,PK15?RASAL1,PK15?RAB8B,PK15?DOCK9,PK15?PTEN,PK15?AP2S1and PK15?VPS54 cell lines using the CRISPR/Cas9 approach.These knockout cells were challenged with Sly,and the cell viability was measured by a CCK-8 assay.All seven knockout cells were confirmed to be resistant to Sly compared with wild-type PK15 cells,especially PK15?RAB8B cells,which were highly resistant（～5.5-fold）to Sly compared with wild-type cells.Secondly,we successfully demonstrated that RAB8B was one of the vital host factors for Sly.Monoclonal cells(PK15^△RAB8B)without expression of RAB8B protein were obtained by a limited dilution method.PK15^?RAB8B cells were more resistant to Sly than wild-type cells（～5-fold）.Furthermore,after being treated with Sly,PK15^?RAB8B cells showed an intact plasma membrane,but significant membrane damage was observed in PK15 cells under a scanning electron microscope.Cells incubated with Sly,and probed with a Sly specific mouse polyclonal antibody,showed that the binding of Sly to PK15^?RAB8B cells was substantially decreased compared to that of parental cells.Cell intoxication with Sly specifically induced RAB8B m RNA transcription（P<0.01）.2.Molecular mechanism of RAB8B mediated Sly cytotoxicity.Firstly,we demonstrated that RAB8B interacts with Sly,but the interaction does not influence Sly cytotoxicity.In this study,we expressed RAB8B（aa 1-207）/RAB8B-I（aa 1-110）/Sly（aa 29-497）/Sly-D1-3（aa 29-387）and Sly D4（aa 388-497）recombinant proteins.RAB8B（1-208 aa）was divided into RAB8B-I（1-110 aa）and RAB8B-II（111-208 aa）,which were fused with GFP and expressed in HEK293T cells.Immunoprecipitation and ELISA assays were used.The results showed that Sly interacts with RAB8B,and RAB8B-I and Sly-D1-3 are the critical regions for interaction.Unexpectedly,preincubation with recombinant RAB8B or RAB8B-I could not inhibit Sly binding to cells,even at high concentrations.Moreover,preincubation with recombinant RAB8B or RAB8B-I could not protect PK15 cells and erythrocytes from Sly cytotoxicity.In addition,preincubated with RAB8 antibody could not protect PK15 against the cytolysis induced with Sly.Secondly,we demonstrated that RAB8B mediated Sly cytotoxicity by regulating membrane accessible cholesterol.Sly is a member of the cholesterol-dependent cytolysins（CDCs）,and the membrane accessible cholesterol is a receptor for CDCs.GFP-D4,a membrane accessible cholesterol probe that can detect the membrane accessible cholesterol,was used to treat PK15 and PK15^△RAB8B cells.These results showed that the content of membrane accessible cholesterol of PK15^△RAB8B was significantly lower than that of PK15.MβCD-cholesterol can increase the content of membrane accessible cholesterol.Sly was added to PK15^△RAB8B cells treated with MβCD-cholesterol.Compared with untreated PK15^△RAB8B cells,the sensitivity of treated PK15^△RAB8B cells to Sly was significantly increased（P<0.001）,and the binding of Sly to PK15^△RAB8B cells was significantly increased.These results indicated that RAB8B facilitated Sly cytotoxicity by increasing membrane accessible cholesterol.In addition,MβCD can decrease the content of membrane accessible cholesterol.We found that MβCD was effective in protecting PK15 against the cytolysis induced with Sly,but MβCD-cholesterol increased the sensitivity of cells to Sly.Consistently,MβCD treatment completely reduced Sly binding to cells.Still,MβCD-cholesterol treatment significantly increased Sly binding affinity to target cells,indicating that the cytotoxicity of Sly depends on the membrane accessible cholesterol.Third,we demonstrated that RAB8B mediated Sly cytotoxicity by regulating the transportation of low-density lipoprotein（LDL）-derived cholesterol.Treating PK15 and PK15^△RAB8B with lipoprotein-free serum（LPDS）to starve cells of lipids and added LDL to LPDS-treated cells to mimic the transport of LDL-derived cholesterol within cells.Treatment of LDL-deficient PK15 cells with LDL supplement was sufficient to increase high levels of membrane accessible cholesterol.Likewise,LDL incubation with LDL-deficient PK15 cells increased Sly binding and sensitivity to target cells（P<0.001）.However,the membrane accessible cholesterol in PK15^?RAB8B cells was at deficient levels even with high LDL-supplement concentrations.Correspondingly,LDL incubation with LDL-deficient PK15^?RAB8B cells did not increase Sly binding to cells or the sensitivity of target cells to Sly.These results suggested that RAB8B-deficient inhibited the transport of LDL-derived cholesterol to the plasma membrane to increase membrane accessible cholesterol,thereby increasing resistance to Sly in cells.Quantification of cellular total cholesterol content indicated similar cholesterol levels in PK15^?RAB8B and parental cells after LDL deficiency and LDL loading,suggesting no difference in the efficiency of LDL uptake upon RAB8B depletion.However,the amount of lysosomal cholesterol in parental cells was significantly lower than that in PK15^?RAB8B cells in normal medium or LDL supplement medium,indicating that RAB8B depletion inhibited LDL-derived cholesterol leaving from lysosomal organelles under our experimental conditions.In conclusion,RAB8B promotes LDL-derived cholesterol leaving from lysosomes to the plasma membrane and increasing the content of membrane accessible cholesterol,and increases the sensitivity of target cells to Sly.3.Immunoprotective efficacy of APP Apx-fusion antigenFirstly,we successfully designed and prepared Apx-fusion protein delivered by outer membrane vesicles（Apxr-OMVs）.Cly A（909 bp）gene fragment of E.coli and Apx I structural gene fragment（Apx IA,651 bp）,Apx II structural gene fragment（Apx IIA,594 bp）,Apx III structural gene fragment（Apx IIIA,702 bp）of Actinobacillus pleuropenumoniae（APP）were combined with the p BAD18-CM expression vector.The recombinant vector was transfected into E.coli JC8031 and the outer membrane vesicles secreted by the recombinant strain JC8031were collected.The vesicle structure with a diameter of 20-200 nm was observed under transmission electron microscopy.The Apx-fusion protein bands with the expected molecular weight（about 100 k Da）were successfully detected by Western-blot.Secondly,we confirmed that Apx-fusion protein（Apxr-OMVs）had cross-protection against APP serotypes 1 and 7.We assessed the cellular immune,humoral immune,pathological changes and cross-protective rate in a mouse model.Results showed that the specific Ig G antibody level of the Apxr-OMVs group was significantly higher than that of the PBS group（P<0.001）.In addition,the proliferation of lymphocytes and IFN-γ,IL-2,and IL-4 cytokines in the Apxr-OMVs group were significantly higher than those in the PBS group（P<0.01）.APP challenge assay showed that Apxr-OMVs could provide 87.5%and 62.5%protection against APP serotype 7（strain 1516）and serotype 1（strain 2701）,respectively.Results of the pathological analysis showed that the degree of lung lesions in the Apxr-OMVs group was significantly lower than that in the PBS group.In conclusion,this study firstly found that RAB8B mediated Sly cytotoxicity and demonstrated that RAB8B promotes LDL-derived cholesterol leaving from lysosomes to the plasma membrane and increased the content of membrane accessible cholesterol,and increased the sensitivity of target cells to Sly.These results further elucidate host factors and their molecular mechanisms that mediate PFTs cytotoxicity.Meanwhile,we constructed Apx fusion protein（Apxr-OMVs）with cross-protection against APP serotypes 1 and 7.It is expected to play a vital role in providing technical support for developing PFTs appropriate multivalent subunit vaccines.