Study On The Mechanism Of Vibrio Vulnificus Hemolysin Activating Platelets And Participating In The Development Of Sepsis

Vibrio vulnificus（V.vulnificus）is a moderately halophilic,gram-negative bacterium which is more common in marine environments.V.vulnificus can infect fish and cause disease in humans.People are mainly infected by eating uncooked seafood with bacteria or contacting seawater with wounds.Patients with ingestive infections will develop gastroenteritis-type symptoms at an early stage,and the fatality rate of patients who develop sepsis is more than 50%.Epidemiological studies have shown that people with low immunity such as liver disease and diabetes are susceptible to V.vulnificus.Among patients with sepsis after infection,liver disease accounts for the first place.Alcoholic liver disease（ALD）accounts for a larger proportion.high.However,there are also some patients with V.vulnificus infection without underlying diseases.In 2006,the American"Emerging Infectious Diseases"magazine first reported cases of healthy people infected with V.vulnificus and listed it as the most dangerous bacteria.V.vulnificus is widely distributed in the oceans of China.Naval soldiers have been exposed to sea water and marine life for a long time,so it is necessary to be vigilant against V.vulnificus infection at all times.Primary sepsis caused by V.vulnificus usually shows thrombocytopenia,and platelet activation-related indicators are closely related to the prognosis of sepsis.The number of platelets in the circulatory system is second only to red blood cells.In addition to the well-known hemostatic function,platelets can also act as an inflammatory cell,which can secrete alpha particles and dense particles after activation.These particles contain a large number of active molecules such as chemokines,platelet activating factor（PAF）,P-selectin（CD62P）and adhesion molecules.Activated platelets can also produce important immunoregulatory factors such as CD40 ligand（CD40L）.These molecules can be expressed on the surface of platelets or secreted into the plasma in the form of exosomes or microvesicles.Among them,CD62P can bind to the ligand of P-selectin on neutrophils,the mucin-like cell surface glycoprotein PSGL-1 to form a platelet-neutrophil complex,which plays an important role in the chemotaxis of leukocytes in the early stage of inflammation.Besides,CD62P and CD40L participate in the recruitment of neutrophils,lymphocytes,macrophages and endothelial cells,can activate immune cells to secrete IL-1β,IL-6,TNF-αand other pro-inflammatory factors,and participate in causing“cytokine storm”,which plays an important role in the occurrence of sepsis.In addition to cytokines,procalcitonin（PCT）can also reflect the level of inflammation in the body,and can be used as an auxiliary diagnostic indicator for sepsis.Studies have shown that Staphylococcus aureus,Streptococcus pneumoniae,Streptococcus sanguis,and Helicobacter pylori can directly or indirectly activate human platelets and participate in causing sepsis.In V.vulnificus,the interaction between bacteria and platelets and its role in sepsis have not been reported yet.At present,research on the pathogenic mechanism of V.vulnificus mainly focuses on cytotoxicity,evasion of host immune defense.V.vulnificus hemolysin（VVH）is a cytotoxin encoded by the vvhA gene and secreted to the outside of the cell,which,and can produce toxic effects on a variety of cells depends on cholesterol.Studies have shown that cytolysin is beneficial for V.vulnificus to invade into the blood from the intestine.In addition to VVH,RTX（repeats in toxin）toxins also play an important cytotoxic effect.V.vulnificus RTX toxin A1（Rtx A1）can cause damage to intestinal epithelial cells and intestinal microvilli structure,causing intestinal epithelial tissue necrosis.It has been reported that V.vulnificus lacking two secretory factors,Rtx A1 and VVH,cannot cause intestinal infection in mice.In addition,V.vulnificus can also produce extracellular proteases including metalloproteinases,chondroitinase and hyaluronidase to exert cytotoxic effects.However,the toxicity of V.vulnificus to platelets and whether it can activate platelets is unclear.Can the platelets be activated after V.vulnificus infection?Which virulence factors are involved in activating platelets?Is there a connection between platelet activation and V.vulnificus sepsis?In the process of V.vulnificus infection in ALD patients and healthy people,do these virulence factors play the same role?In response to the above problems,this study used the"V.vulnificus-platelet interaction"as a research starting point to explore the pathogenic mechanism of V.vulnificus to activate platelets and participate in causing sepsis.Relevant research results are conducive to promoting the logistics and sanitation prevention and control of the Chinese Navy,and provide a theoretical basis for the development of treatment drugs for V.vulnificus sepsis.The research content mainly includes three parts:1.Analysis of transcriptome and platelet activation in the mouse model of V.vulnificus infection（1）Transcriptome analysis of V.vulnificus mouse infection model:（1）Through chronic alcohol feeding plus acute alcohol gavage and then chronic alcohol feeding,we constructed an ALD model of C57BL/6N mice.Serum ALT and AST activities were significantly increased after acute alcohol gavage.After chronic feeding,HE staining and oil red O staining showed obvious fatty degeneration in the liver After the end of chronic feeding,HE staining and Oil Red O staining showed obvious fatty degeneration in the liver.Plasma cytokines IL-1β,IL-6,IFN-γ,and TNF-αshowed no significant difference between ALD and control group.The results showed that the ALD model was successfully established and can be used to study the pathogenic mechanism of V.vulnificus.（2）We simulated the ingestion infection of V.vulnificus by gavage the clinical strain YJ016（or LBS）of V.vulnificus to ALD and control mice.After 12 hours of gavage,small intestine and whole blood samples were collected for RNA-seq analysis.The results showed that:compared with LBS gavage,after YJ016 gavage,ALD and control mice had different expression levels of small plate activation-related genes in small intestine tissues.The corresponding GO,KEGG,and Reactome enrichment analysis results were:blood particles,Plateletαparticles（existing only in ALD mice）,complement and coagulation cascade reaction,platelet degranulation,reaction to platelet cytoplasmic Ca²⁺increase,respectively.（2）Analysis of platelet activation in the mouse model of V.vulnificus infection:（1）We found that after YJ016 gavage,CD62P in the small intestine tissues of ALD and control mice both increased,through the immunohistochemistry of CD62P in small intestine tissue.Plasma soluble CD62P（s CD62P）and soluble CD40L（s CD40L）were detected by enzyme-linked immunosorbent assay.s CD62P and s CD40L in plasma of ALD and control mice increased after YJ016 gavage,and the ALD group was higher than the control group.（2）Detecting plasma PCT by ELISA,we found that PCT in plasma of ALD and control mice increased after YJ016 gavage,and the ALD group was higher than the control group.Correlation analysis showed that plasma s CD62P and s CD40L were positively correlated with PCT.2.The molecular mechanism of V.vulnificus activating human platelets（1）Identification of platelet activation stimulators:（1）In order to determine which components of V.vulnificus activate platelets,we separately incubated YJ016 cells or log phase culture supernatants with human platelets,and passed the flow Cytometry detected the expression of CD62P on the surface of platelets,and found that YJ016culture supernatant can activate platelets to release CD62P,while the supernatant after incubation with cholesterol and anti-VVH antibodies cannot activate platelets.Therefore,we speculate that it is the cholesterol-dependent pore-forming toxin VVH of V.vulnificus that activates human platelets.（2）We constructed the vvhA gene knockout strain YJ016-ΔvvhA based on the p DS132 traceless mutation technology,and detected the expression of CD62P on the platelet surface and the formation of PNC by flow cytometry,and detected platelet-derived microvesicles by flow cytometry It was found that YJ016 culture supernatant after the vvhA gene was knocked out could not activate platelets,and it was found that VVH was the only bacterial stimulator in YJ016secretory supernatant that could activate platelets.（3）Using ammonium sulfate precipitation,anion chromatography,hydrophobic chromatography,ultrafiltration and other methods,we purified the natural VVH from the supernatant of YJ016 culture.It was found that VVH can activate platelets to release microvesicles,express CD62P,and induce the formation of PNC.These results again clarified the activation effect of VVH on platelets.（2）The mechanism of VVH activating human platelets:By detecting the effect of inhibitors of important platelet signaling pathway molecules on VVH-induced CD62P expression and the detection of phosphorylation of myosin light chain（MLC）,an important molecule involved in platelet degranulation,we explored the molecular mechanism of VVH activation of human platelets.（1）We found that U73122 and ML-7can block VVH from activating platelets,that is,VVH activates platelets through PLC-IP3/DAG-MLCK(rather than Rho-ROCK-MLC or external Ca²⁺influx)pathway,and through PLC specific inhibitor U73122,MLCK specific inhibitor ML-7,ROCK specific inhibitor Y27632,etc.（2）By EGTA chelating Ca²⁺in plasma and adjusting Ca²⁺in buffer,we found that extracellular Ca²⁺is a necessary condition for VVH to induce platelet activation.Combining the results of（1）（2）with the pore-forming characteristics of VVH,we speculate that VVH may form pores on the cell membrane surface to cause Ca²⁺influx,and then activate the PLC-IP₃/DAG-MLCK pathway through G protein-coupled receptors,etc.,leading to platelet activation.3.Research on the role of VVH in in vivo infection（1）The role of VVH in sepsis:（1）After 12 and 72 hours of intragastric administration of LBS,YJ016-ΔvvhA and YJ016 with ALD and Pair mice,we used Luminex technology to detect IL-1β,IL-6,TNF-αand other 17 cytokines,The results showed that:YJ016 and YJ016-ΔvvhA had differences in the cytokine inflammatory storm induced by the early stage of infection（12 h）.（2）Through blood plate count and ELISA,we measured the blood bacterial load and plasma PCT of the mice in the early stage of infection（12 h）.The results showed that:YJ016 and YJ016-ΔvvhA did not see the blood bacterial load in the early stage of infection（12 h）Significant difference;and after ALD mice were infected with YJ016,the plasma PCT content was significantly higher than YJ016-ΔvvhA.In addition,the bacterial load and PCT of ALD mice infected with YJ016 were higher than those of Pair group.（2）The role of VVH in platelets activation:（1）Through the immunohistochemistry of CD62P in the small intestine tissue,we found that after YJ016 gavage,CD62P in the small intestine tissue of ALD and control mice both increased;the plasma solubility was detected by enzyme-linked immunosorbent assay CD62P（s CD62P）,soluble CD40L（s CD40L）,we found that s CD62P and s CD40L in the plasma of ALD and control mice increased after YJ016 gavage,and the ALD group was higher than the control group.（2）The platelet count of the mice was performed by a blood cell analyzer.We found that there was no significant difference in the number of platelets in the mice after LBS,YJ016-ΔvvhA and YJ016 were gavage.The platelet activation markers such as s CD62P and s CD40L in the plasma of mice infected with YJ016 were all higher than YJ016-ΔvvhA by ELISA,and the ALD mice were higher than those in the control group.（3）The relationship between platelet activation and sepsis:We selected 3 main pro-inflammatory factors IL-1β,IL-6,TNF-αand PCT as indicators of the severity of sepsis;s CD62P and s CD40L are used as indicators to reflect the level of platelet activation.Correlation analysis showed that s CD62P and s CD40L were positively correlated with IL-1β,TNF-α,IL-6,and PCT.In summary,we found that VVH,as an important platelet activation stimulator of V.vulnificus,can activate human platelets through the PLC-IP3/DAG-MLCK pathway.And in the early stage of V.vulnificus infection,it is involved in the activation of platelets and the occurrence of sepsis,and the severity of sepsis is positively correlated with the level of platelet activation.This study is the first to use two models of ALD and healthy mice to reveal the mechanism of V.vulnificus-platelet interaction that is involved in causing sepsis.The results provide clues for the susceptible population of V.vulnificus-the critical treatment of patients with liver disease.It provides a theoretical basis for the research and development of sepsis treatment drugs targeting platelets.