Study On Two Kinds Of PSM Aggregates Purified From The Supernatant Of Staphylococcus Aureus

Staphylococcus aureus is a common pathogenic bacteria.In contrast to hospitalassociated Methicillin-resistant S.aureus(HA-MRSA)infections,for which there is a predisposing risk factor or condition,community-associated MRSA(CA-MRSA)infections can occur in otherwise healthy individuals.CA-MRSA is highly virulent and has transmission capacity.Therefore,CA-MRSA has become a major focus of public health professionals.Phenol-soluble modulins(PSM)are key factors in increased virulence of CA-MRSA.The PSM family includes PSM?,PSM?,PSM? and PSM-mec.Most of the members are ?-helical amphiphilic peptides.Due to the nature of peptides,they tend to aggregate in solution,but few studies related to PSM aggregation in the functional studies on PSM family proteins.It has been found that some peptides in the PSM family,such as PSM?1 and PSM?3,can aggregate to form amyloid fibers,and their fiber state and monomer state play different or even opposite functions.Therefore,in order to understand how PSM functions,it is an essential project about PSM aggregation.Purpose: At present,the research of PSM aggregate can use a certain synthetic peptide to simulate in vitro,but this does not reflect the true nature very well.There are many members of the PSM family,and some peptides such as PSM? and ?-toxin has copious secretion.What is important is that some peptides such as PSM?4 highly hydrophobic in water but are present in the S.aureus supernatant with a soluble form.Thus,we supposed that the PSM peptides in the supernatant are more likely to aggregate and they will bind to each other.Therefore,we hope that we can directly obtain PSM components from the S.aureus supernatant,and study its aggregation.Subsequently,we can use synthetic peptides to verify our conjecture,and finally obtain information about the structure and function of PSM aggregates.Content and method: We first obtained relatively pure PSM components from the supernatant of S.aureus MW2 through purification,used size exclusion chromatography to divide the PSM aggregates according to the molecular weight,and used Transmission electron microscopy,Liquid chromatography-tandem mass spectrometry,Thioflavin T staining experiment,lysis of Neutrophil,Analytical ultracentrifugation,Static light scattering,bis(sulfosuccinic)suberic acid crosslinking experiment,Matrix-assisted laser desorption time-of-flight mass spectrometry,Co-immunoprecipitation and other methods to characterize the composition,morphology,function and other properties of the separated PSM aggregates.Subsequently,we used High performance liquid chromatography(HPLC)to analyze the PSM aggregates which separated from the supernatant and reproduced them in vitro by synthetic peptides.Then we analyzed the aggregates by Nuclear magnetic resonance,Circular dichroism spectroscopy,Microfluidic spectroscopy,Atomic force microscopy and other methods,trying to explain the reasons for the different aggregation forms of PSM.Finally,functional experiments such as biofilm formation,cell lysis,bacteriostasis,bacterial survival in blood,lethality of mouse bacteremia model,and pathological section of mouse kidney cyst model were used to illustrate the corresponding functions of different aggregates of PSM.Result: We used size exclusion chromatography to divide the PSM obtained from the S.aureus MW2 supernatant into two aggregates: amyloid fibrillar Aggregates1 with larger molecular weight(more than 400 k Da)composed of ?-toxin,and the small molecular weight(20-30 k Da)oligomer complex Aggregate 2 composed of ?-toxin and PSM?1-4.The two kinds of aggregates have obvious differences in molecular weight,morphology,and function.The HPLC results showed that the fibrillar Aggregate1 was mainly composed of formyl-modified ?-toxin(f?-toxin),while the oligomer complex Aggregate2 was mainly composed of deformylated ?-toxin(df?-toxin).The peptide experiment proved that f?-toxin tends to form fibrils and df?-toxin tends to form oligomers.The results of structural analysis of f?-toxin and df?-toxin aggregates showed that f?-toxin and df?-toxin both exhibit ?-helix structure when they aggregate,but the proportion of ?-helix of f?-toxin is lower.When they form oligomers,f?-toxin oligomers are smaller,tighter and tend to be connected into filaments,while df?-toxin oligomers are larger and looser to maintain the oligomer state.In functional experiments,f?-toxin fibrils make S.aureus biofilms more stable,while df?-toxin oligomers hinder the formation of S.aureus biofilms and assist biofilm disassembly.In addition,since df?-toxin and PSM?can form a complex,the functions of complex was verified in cell lysis,bacteriostasis,bacterial survival in blood and in vivo experiments.Conclusion: We supposed that S.aureus has different forms of PSM aggregates by producing f?-toxin and df?-toxin.f?-Toxin tends to connect to fibrils after forming oligomers,showing fibrous PSM Aggregates1;while df?-toxin forms oligomer complexes with PSM? and tends to maintain the form of oligomers,showing globular protein PSM Aggregate2 with cytolytic activity.Through structural analysis,we supposed that the formyl modification of ?-toxin makes the secondary structure of f?-toxin and df?-toxin obvious differences.This differences further affects the ability of aggregation: the oligomers formed by f?-toxin are smaller and more compact,and tend to continue to aggregate into fibrils,while the oligomers formed by df?-toxin are larger and looser,so they maintain the oligomeric form.Due to the difference in the aggregation ability of f?-toxin and df?-toxin,the effects of the two on the biofilm of S.aureus showed opposite functions: f?-toxin fibrils make the biofilm of S.aureus more stable,while the oligomer df?-toxin hinders the formation of biofilm and assist biofilm disassembly.In addition,df?-toxin can form a complex with PSM?,which greatly enhances the function of PSM?,and this is also reflected in the mouse bacteremia model of S.aureus.In this project,we discovered and studied the properties,structure information and functions of the two kinds of PSM aggregates isolated from the supernatant of S.aureus,subsequently analyzed and verified the reasons and laws of the formation of the two kinds of aggregates.Our research opened up a new perspective for the study of CA-MRSA and PSM virulence—the existence and function of PSM oligomer complexes were isolated and reported for the first time.In addition,we discovered for the first time the difference between f?-toxin and df?-toxin in the aggregation ability,and speculated that the regulation of f?-toxin and df?-toxin is likely to be a low-cost way to regulate S.aureus physiological state.What's more,the stable df?-toxin oligomer has certain reference significance for the oligomer research of other amyloid diseases.