Study On The Interaction Mechanism Of Streptococcus Suis And Phospholipase A₂ Group ?A

Streptococcus suis?S.suis?is a Gram-positive facultative anaerobic bacterium.It is a new emerging and important zoonotic pathogen.According to the antigenicity of capsular polysaccharide,Streptococcus suis can be divided into 35 serotypes?serotypes1 to 34 and serotype 1/2?,of which Streptococcus suis serotype 2?S.suis 2?is the most virulent and the most widely distributed serotype.Infection in humans and pigs can cause meningitis,arthritis,endocarditis,sepsis and even acute death.In 1998 and 2005,two large-scale infections were caused in Jiangsu Province and Sichuan Province.Streptococcus suis is seriously threatening the development of the pig industry and human health.Studies in recent years have found that capsular polysaccharide?CPS?,muramidase released protein?MRP?,extracellular factor?EF?,Suilysin?SLY?and other substances are the main virulence factors of Streptococcus suis.Phospholipase A₂?PLA₂?is an enzyme that catalyzes the hydrolysis of phospholipids at the sn-2 position,which can produce lysophospholipids and free fatty acids.Mammalian PLA₂ can be divided into secreted PLA₂?s PLA₂?,cytosolic PLA₂?c PLA₂?and Ca²⁺independent?i PLA₂?.Among them,PLA₂ group IIA?PLA₂G2A?is the most studied enzyme in secreted PLA₂.PLA₂G2A can affect or combine with bactericidal/permeability-increasing protein?BPI?synergistically and penetrate the bacterial cell wall,hydrolyze the phospholipid of the cell membrane,damage and rupture the bacterial cell membrane to kill the bacteria.When there is inflammation or bacterial infection in a certain part of the host,the concentration of PLA₂G2A in that part increases rapidly.At this concentration,PLA₂G2A can effectively kill the infected bacteria,which plays an important role in the host's defense against bacterial infections.PLA₂G2A is considered to be part of innate immunity.At present,the identification of the precise roles of PLA₂G2A has remained a challenge and the research on the effect of Streptococcus suis and PLA₂G2A is relatively blank,so studying the effect of Streptococcus suis and PLA₂G2A can help improve the mechanism of the interaction between Streptococcus suis and the host.In this study,we first confirmed the bactericidal effect of PLA₂G2A on Streptococcus suis by in vitro experiments.In the first chapter of the study,we first compared the bactericidal activity of PLA₂G2A against different pathogenic bacteria strains.PLA₂G2A has no obvious bactericidal activity against Gram-negative pathogenic bacteria.Compared with it,Gram-positive pathogenic Streptococcus suis can be killed by it,and its survival rate gradually decreases with increasing concentration.The other two Gram-positive pathogens,Streptococcus pyogenes and Streptococcus agalactiae,are more sensitive to PLA₂G2A.Prove that Streptococcus suis can be effectively killed by PLA₂G2A,but it needs a higher concentration.The2148hk/rr gene is a two-component signal transduction system in Streptococcus suis.Compared with Streptococcus suis serotype 2 strains 05ZYH33 and 261,the mutant strain?2148hk/rr is more sensitive to PLA₂G2A.In addition,the bactericidal effect of PLA₂G2A on different serotypes of Streptococcus suis was compared.The results showed that the effects of PLA₂G2A on four serotypes of Streptococcus suis showed concentration and time dependence and different sensitivities,and the sensitivities were S7>S2>S9>S1?from high to low?,combined with transmission electron microscopy to observe the morphology of the corresponding strains,preliminarily inferred that the capsule of Streptococcus suis affects the bactericidal effect of PLA₂G2A.Atomic force microscopy observation and measurement results showed that the surface potential of Streptococcus suis has changed,presuming that the two were combined through electrostatic interaction.The results of the action of specific inhibitors and divalent metal cations prove that PLA₂G2A relies on its own phospholipase enzyme activity to exert a bactericidal effect on Streptococcus suis,in which divalent metal cations play an important role.The capsular polysaccharide is an important virulence factor of Streptococcus suis and plays an important role in resisting the phagocytosis of host immune phagocytes.In order to study the effect of the S.suis capsule on the bactericidal effect of PLA₂G2A,in the second chapter of the study,we first constructed the S.suis serotype 2 CPS gene cluster deletion mutant strain?CPS.Combined with PCR identification and transmission electron microscope morphology observation,the results proved that the mutant strain?CPS was constructed successfully.Compared with the wild strain,?CPS's surface capsule is missing.The challenge experiment of Balb/c mice proved that the mutant strain?CPS had reduced virulence.Blood survival experiments show that the mutant strain?CPS has a reduced ability to survive in blood and is more easily eliminated.Then PLA₂G2A was added to whole blood,the survival rate of wild strain05ZYH33 decreased compared with control,while the survival rate of mutant strain?CPS did not change significantly.In vitro sterilization experiments show that the mutant strain?CPS to get the same sterilization rate as the wild strain 05ZYH33,which requires higher PLA₂G2A concentration and longer action time.Capsule strains are more sensitive than those lacking them.We infer that the negatively charged capsules have a higher affinity for the positively charged basic protein PLA₂G2A and are easier to bind.BLI experiments proved that PLA₂G2A and capsular strains have a stronger affinity.The research in this chapter proves that the Streptococcus suis capsule has a positive effect on the bactericidal effect of PLA₂G2A.In order to further study the effect of Streptococcus suis and PLA₂G2A,in the third chapter,we study the effect of Streptococcus suis on the expression of the host PLA₂G2A.After Streptococcus suis stimulated,type I,III,and IV PLA₂ were expressed at transcription level in human brain microvascular endothelial cells,but no PLA₂G2A expression was detected.ELISA experiments showed that the expression level of the mouse peritoneal macrophage cell line RAW 264.7 PLA₂G2A was low.At the same time,Dot-Blot experiments proved that after C3H/He N mice were infected with bacteria,the circulating PLA₂G2A content was also low.Therefore,we selected guinea pig alveolar macrophages?AMs?which have been applied to study the interaction between bacteria and host PLA₂G2A to further study the effect of Streptococcus suis on the expression of host PLA₂G2A.After the interaction between Streptococcus suis and alveolar macrophages,real-time quantitative PCR results showed that Streptococcus suis can cause the up-regulation of PLA₂G2A expression levels.But compared with wild strain 05ZYH33 and complementary strain C?SLY,mutant strain?SLY induced AMs to express higher levels of PLA₂G2A.Recombinant Streptococcus suis Suilysin r SLY acted on AMs alone,there was no significant change in the expression levels of PLA₂G2A.LPS could cause the up-regulation of PLA₂G2A expression.When LPS and r SLY were incubated with AMs,the results showed that the expression levels of PLA₂G2A gradually decreased with the increase of r SLY concentration,indicating that SLY has a negative effect on the expression of PLA₂G2A.In addition,S.suis type 2adenosine synthase deletion mutants?Ssads and?SLY had similar effects.In summary,this study showed the bactericidal effect of PLA₂G2A on Streptococcus suis,indicated the positive effect of the capsule on the surface of Streptococcus suis on the bactericidal effect of PLA₂G2A,proved that Streptococcus suis could induce the host to express PLA₂G2A,Streptococcus suis's related virulence factors can limit the expression of PLA₂G2A,and the results show the complexity of the interaction between S.suis and PLA₂G2A.