Inhibition Mechanism Of MRSA By Platelets

Due to the widespread use of antibiotics,the current clinical familiar pathogens such as Staphylococcus aureus,Enterococcus,Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa,have developed resistance to most of the frequently-used antibiotics.Bacterial resistance is considered a major threat to global health in the 21st century,with the world health organization（WHO）recently making the treatment of drug-resistant bacteria a major global human health priority.Methicillin-resistant Staphylococcus aureus（MRSA）is one of the most common drug-resistant bacteria and difficult for treatment.Currently,the detection rate of MRSA accounts for 35.3%of Staphylococcus aureus clinically isolated in China.In addition to methicillin resistance,MRSA is also resistant to a variety of antibiotics such as lactamins,erythromycin,clindamycin,tetracycline,norfloxacin,etc.Vancomycin has become the only effective antibiotic.However,after vancomycin-resistant staphylococcus aureus（VRSA）occurred in the United States in 2002,"superbacteria"began to update people’s understanding of the pathogen of infection,and new"superbacteria"continuously emerged,posing a huge threat to human health.Therefore,it is urgent to research and develop new antibacterial strategies and antibacterial agents.Platelets are small,seedless cells produced by the degranulation of megakaryocytes in the bone marrow and are second only to red blood cells in number in human circulation.The classic physiological role of platelets is to adhere,activate,and aggregate damaged vascular endothelial cells to play a key role of anticoagulant.Recent research has shown that in addition to the classic anticoagulant function,platelets also play an important role in inflammatory and immune responses.Platelets contain a variety of pro-inflammatory cytokines（such as P-selectin,CD40L,IL-1β,etc.）and antimicrobial peptides,which can not only directly kill pathogens with the antimicrobial peptides secreted by themselves,but also assist white blood cells to migrate and gather to the infected site to exert immune effects.Previous studies on the antibacterial effect of platelets mainly focused on drug-sensitive bacteria such as Staphylococcus epidermis,Faecalis,Staphylococcus aureus,etc.Whether platelets can also play an antibacterial effect on drug-resistant bacteria has not been reported,and the mechanism of the antibacterial effect of platelets is not completely yet.In this study,MRSA were used to explore the inhibition and mechanism of platelets on MRSA inhibition in vitro and in vivo,as well as the influence of platelets on MRSA virulence.The main research contents included:1.Study on the MRSA inhibition by plateletsAppropriate amount of purified platelets and MRSA bacterial solution were taken and incubated at 37℃for 10h.The cultured bacterial solution was tested for OD_600nm and counted as colony coating plate.The results showed that in vitro platelets could significantly inhibit the proliferation of MRSA and the antibacterial effect lasted for a long time.Flow cytometry with CFSE staining was further used to detect the co-culture bacterial fluid.The results also showed that platelets inhibited the proliferation of MRSA.The MRSA infection model of mice was established to observe the changes of infection status and survival after fresh isolated platelets were injected into the mice and the platelets were reduced by antibody neutralization.The results showed that platelet infusion could significantly relieve the symptoms of infection and improve the survival of mice.After the depletion of platelets,the mice’s infection symptoms were aggravated,and the survival was also significantly reduced.These results suggest that platelets also inhibit MRSA proliferation and reduce its pathogenicity in vivo.2.Mechanism of platelet inhibiting MRSAAppropriate amount of purified platelets and MRSA bacterial solution were taken and incubated at 37℃for 10h.The cultured bacteria were respectively observed by transmission electron microscopy,detected by RNA-sequence,verified by q RT-PCR,and detected by flow cytometry for hydroxyl radical（OH^●）generation and apoptosis-related indicators.The results of transmission electron microscopy showed that the nuclear chromatin of MRSA contracted after the treatment of platelets.The results of RNA-sequence and q RT-PCR showed that the expression of DNA damage-repairing and oxidative phosphorylation related genes were up-regulated at the m RNA level after the treatment of MRSA by platelets.A series of apoptotic characteristics of MRSA were found after platelets treatment by flow cytometry,including DNA fragmentation,PS valgus of cell membrane,potential depolarization of cell membrane,increased intracellular Caspase activity,and significantly increased intracellular OH^●production of MRSA.In order to further confirm that the increase of OH^●was the cause of apoptotic death of MRSA,the inhibitors NAC and Bpy of OH^●were added to the co-culture system of platelets and MRSA,and the cultured bacterial solution was detected by OD_600nm detection and flow cytometry detection of OH^●generation and apoptosis-related indicators,respectively.The results showed that the inhibition effect of platelets on MRSA was significantly reduced and the generation of intracellular OH^●was significantly reduced after the addition of OH^●inhibitor.Meanwhile,various apoptotic indicators of MRSA were also inhibited.These results indicated that platelet induced an increase in intracellular OH^●production and led to apoptosis-like death of MRSA.In order to explore which components of platelets play a bacteriostatic role,the superprotein of platelets co-cultured with MRSA was collected for mass spectrometry.The results showed that the platelet-derived Dermcidin（DCD）in the supernatant was down-regulated by more than half（P<0.05）.These results hinted that antimicrobial peptide DCD may reduce in the supernatant due to large consumption during the antibacterial effect,and it may play a key role in the antibacterial effect of platelets on MRSA.3.Effect of platelets on MRSA toxicityIn order to further improve the anti-MRSA function of platelets,an appropriate amount of purified platelets and MRSA bacterial solution were taken and incubated at 37℃for 10h.The results of RNA-sequence and q RT-PCR showed that the hemolysin genes hlg A,hlg B,hlg C and the bicomponent genes agr C,agr A,sae S,sae R of MRSA were down-regulated after platelet treatment.Also,the results of mass spectrum showed that 19toxic proteins secreted by MRSA were decreased after platelet treatment.These results suggest that platelets may reduce the expression and secretion of MRSA virulence proteins.Then the mice were infected with the same amount of MRSA and the MRSA bacterial solution after co-culture with platelets,and the infection status and survival of the mice were observed.The results showed that compared with the MRSA group,the platelet-treated mice had lighter symptoms of infection and longer survival,suggesting that platelets may inhibit the virulence of MRSA.Furthermore,the clotting time of human plasma was observed by adding the same amount of MRSA and the MRSA bacterial solution after co-culture with platelets into the plasma.The results showed that the coagulation time was significantly prolonged after the platelet treated MRSA solution was added to the plasma,suggesting that the plasma coagulase secretion of MRSA decreased after platelet treated.These findings indicated that platelets could inhibit the secretion of MRSA virulence protein in vivo and in vitro.4.ConclusionIn vivo and in vitro,this study confirmed that platelets could inhibit the proliferation of MRSA,and the mechanism of platelet’s antibacterial effect may be that the antibacterial peptides secreted by platelets can induce the increase of intracellular OH^●production,leading to the apoptosis-like death of MRSA.Furthermore,the finding that platelets could weakened virulence of MRSA was confirmed from the levels of m RNA,protein and phenotype.These enriched the mechanism of platelet inhibiting MRSA.This study deepened and expanded the understanding of platelet function,and provided a new theoretical and experimental basis for the research and clinical use of comprehensive platelet therapy.