| Group A streptococcus?GAS?is one of the most common pathogenic bacteria,which can depend on its own structural proteins?such as:M protein,FbaA protein,etc.?and secretory proteins?such as:streptolysin O,streptococcus pyrogen exotoxin B,etc.?to invade epithelial or endothelial cells,posing a threat to the host.However,in the game between host cells and pathogens,pathogens can be eliminated by autophagy,which is currently considered to be the most effective way for non-phagocytic cells to clear pathogens.Autophagy is a highly conserved process of cellular catabolism and plays a key role in cellular homeostasis.It can be significantly induced in response to various environmental pressures,such as starvation,hypoxia,oxidative stress and radiation.The process of autophagy involves the formation of bilayer membrane vesicles,which are called autophagosomes.The autophagosome itself does not degrade,but its various contents will be degraded with the combination of the autophagosome and lysosome,and the resulting nutrients will be recovered and involved in the synthesis of new substances or energy production.Basal autophagy maintains normal homeostasis by continuously removing damaged organelles,protein aggregates,and long-lived proteins.In addition,when many pathogens,including Salmonella,Shigella,Listeria,Legionella,Mycobacterium,Francis and Group A streptococci,etc.,invade the host cells,they can also induce autophagy.Targeting invading pathogens,this process is called xenophagy.A growing body of research has shown that xenophagy,as a key mechanism of innate immunity in cells,can control the fate of many foreign microorganisms,including bacteria,viruses and parasites.Given the prevalent role of xenophagy in innate cellular defense,a key aspect of this process relies on the host cell's perception of invading bacteria,thereby triggering autophagy.Mammalian cells are capable of expressing a variety of cell surface or cytosolic pattern recognition receptors?PRRs?,such as Toll-like receptors?TLRs?or NOD-like receptors?NLRs?,which recognize specific pathogen-associated molecular patterns?PAMP?.For example,lipopolysaccharide?LPS?or peptidoglycan?PG?of Gram-negative bacteria.At present,many studies have confirmed the connection between TLRs or NLRs and autophagy,and these works provide a missing link between pathogen recognition and autophagy initiation.For example,LPS participates in autophagy in phagocytic cells through TLR4;single-stranded RNA induces autophagy against Mycobacteria by TLR7 activation;cytosolic receptors NOD1 and NOD2 recruited ATG16L to Shigella flexneri to initiate autophagy.In addition to recognizing the above receptors,integrin family receptors are also important host cell surface receptors,which are distributed in almost all cells.In addition to mutual recognition with the above receptors,integrins are also important receptors for pathogen recognition,which is distributed on almost all cell surfaces.It plays an important role in the adhesion of pathogens and invasion of host cells.In our study,we found that autophagy induced by GAS infection in Hep2cells has nothing to do with the well-reported Toll-like receptors,but closely related to integrin family receptors.Integrin?5?1 initiates autophagy by recognizing each other with the structural protein FbaA of GAS.And we have conducted an in-depth discussion of its mechanism.Regarding pathogens,the molecular link between integrin receptors and autophagy has rarely been reported.We will further demonstrate whether integrin receptors can be used as a general event to initiate autophagy and protect host cells when host cells are infected by pathogenic bacteria.Our research provides new insights into the interaction pattern between pathogen and host.And it provides a theoretical basis for discovering new antibacterial strategies and developing drugs for autophagy adjuvant therapy.Part one Respiratory epithelial cells clear group A Streptococcus by autophagyObjective:In vivo and in vitro experiments were conducted to determine the changes of bacterial viability and autophagic activity in M1 GAS infected respiratory epithelial cells.The relationship between M1 GAS and autophagy were explored by molecular biology techniques.Methods:1 M1 GAS?MOI=100?infected Hep2 cells?without antibiotics in the medium?,and after 2 hours,extracellular M1 GAS was killed using a medium containing 10 times the concentration of antibiotics.The cells were cultured continuously,and at 5 h,10 h,and 25 h after infection with M1 GAS,the cells were disrupted and live bacteria was performed on blood agar plates.Count the number of live bacteria in the cells at different time points.2 Effect of autophagy on intracellular M1 GAS.2.1 An interfering plasmid of sh-RNA against Atg5 was constructed,and the plasmid of sh-Atg5 was transferred into Hep2 cells by lentivirus.The expression of Atg5 protein was detected by Western blot,and to screen out the Atg5 interfered Hep2 stable cell lines.2.2 M1 GAS was used to infect Hep2 cells and hep2-sh-atg5 cells.After 2h,the extracellular M1 GAS was killed with a culture medium containing 10times the concentration of antibiotics.The cells were further cultured,and after 6h of infection with M1 GAS,the cells were broken and cultured with live bacteria on blood agar plates.Count the number of live bacteria of Hep2cells and Hep2-sh-Atg5 cells at the same time point.3 Effect of M1 GAS on autophagy formation in Hep2 cells.3.1 Hep2 cells were infected with M1 GAS.After infection for 0h,2h,4h,and 6h,cells were harvested and whole cell proteins were extracted.The level of expression of the expression of LC3 protein?the marker protein of autophagy?was detected by Western blot.The time course of M1 GAS-induced autophagy was determined.3.2 The plasmid of p BABE-puro-EGFP-LC3 was transfected into Hep2 cells using the transfection reagent Lipofectamine 2000.After transfection for 24 h,Hep2 cells were infected with M1 GAS for 6 h,the distribution of EGFP-LC3in the cells was observed by Confocal Laser Scanning Microscope?CLSM?.3.3 Hep2 cells were infected with M1 GAS for 6 hours,and the cells were fixed with 2.5%glutaraldehyde.The structure of autophagosomes were observed by Transmission Electron Microscopy?TEM?.4 In vivo experiments demonstrated the interaction of M1 GAS with autophagy.4.1 The mice were nasally drips with M1 GAS?3×10 8 CFU/50?l?,and after 24 hours,the lung tissues of the mice were taken?the whole process was maintained in a sterile environment?to detect the expression of LC3 protein and the survival of GAS in the mice lung.4.2 The expression of Atg5 protein in the mice lung were detected by Western blot to determine whether the Atg5?KO?-deficient mice were successfully constructed.WT healthy mice and Atg5?KO?-deficient mice were infected with M1 GAS for 24 hours.Then,the lung tissue was taken to compare the number of live bacteria in WT healthy mice and Atg5?KO?deficient mice.5 Heat-inactivated M1 GAS induces autophagy.5.1 The M1 GAS were heat inactivated at 70°C for 1 h.Then,Hep2 cells were infected?MOI=100?with heat-inactivated M1 GAS.At the 0h,2h,4h and 6h time points of infection,cells were harvested and whole cell proteins were extracted to detect the expression of LC3.5.2 The plasmid of p BABE-puro-EGFP-LC3 were transfected into Hep2 cells using the transfection reagent Lipofectamine 2000 for 24h.The cells were infected with heat-inactivated M1 GAS.After 6 hours,the distribution of EGFP-LC3 in the cells was observed by CLSM.6 The effect of M1 GAS strain with defective M protein or FbaA protein on autophagy.6.1 Hep2 cells were infected with WT M1 GAS,FbaA-M1 GAS and M-M1GAS for 6 h,respectively.Cells were harvested and whole cell proteins were extracted to detect the expression of LC3.6.2 The plasmid of p BABE-puro-EGFP-LC3 were transfected into Hep2 cells using the transfection reagent Lipofectamine 2000 for 24h.Hep2 cells were infected with WT M1 GAS,FbaA-M1 GAS and M-M1 GAS,respectively.After 6 hours,the distribution of EGFP-LC3 in the cells was observed by CLSM.6.3 Hep2 cells were infected with WT M1 GAS,FbaA-M1 GAS and M-M1GAS,respectively.After 6 hours,count the number of three bacteria in the cell on blood agar plate.7 The effect of FbaA and M of M1 GAS on autophagy.7.1 Hep2 cells were stimulated with different concentrations?0?g/ml,10?g/ml,20?g/ml,30?g/ml,40?g/ml,50?g/ml?of FbaA protein and M protein.The optimal stimulation concentration was determined by detecting the expression level of the LC3 protein.7.2 Hep2 cells were stimulated with FbaA protein and M protein at the optimal concentration?30?g/ml?,and the changes of LC3 protein and P62 protein were detected by Western blot.7.3 The cells were pretreated with PBS,FbaA protein,FbaA protein+3-MA?autophagy inhibitor 3-methyladenosine,10 m M?for 30 min.Then,Hep2 cells were infected with M1 GAS for 6h,and the cells were harvested.Intracellular live bacterial culture was performed on blood agar plates.The number of bacteria was counted after 24 h.8 FbaA protein was expressed in segments to determine the effective fragments for inducing autophagy.8.1 According to previous studies in our laboratory,functional fragments of FbaA protein were identified and purified.The four segments:A?aa#37-110?,B?aa#68-161?,C?aa#104-277?,D?aa#160-324?.8.2 The above four-stage FbaA protein fragment were used to stimulate Hep2cells,and the cells were collected at 0h,2h,4h,and 6h,respectively.The change of LC3 protein was detected by Western blot.8.3 Hep2 cells?containing p BABE-puro-EGFP-LC3 plasmid?were stimulated with four FbaA protein fragments.The distribution of EGFP-LC3 was observed using CLSM.Results:1.After the Hep-2 cells were infected with M1 GAS,the cells were lysed at different time points.The results showed that with the extension of time,the number of live bacteria in the cells gradually decreased,and the presence of M1 GAS could hardly be detected after 25h.2.The number of live bacteria in Hep2-sh-Atg5 cells with M1 GAS infection were much higher than Hep2 cells?P<0.05?.3.After M1 GAS infection of Hep2 cells,LC3?protein began to high expression in 4 h?P<0.05?.The distribution of EGFP-LC3 protein from uniform distribution in the cytoplasm to point-like aggregation distribution by TEM.A large number of vesicles?autophagosomes?with a bilayer membrane structure were formed in the cytoplasm,containing M1 GAS by CLSM.4.After M1 GAS was intranasally infected with mice,the lungs were homogenized.The results of Western blot showed that the LC3II protein was significantly increased compared with the PBS group.The amount of live bacteria in the lungs of Atg5-deficient mice were significantly higher than WT healthy mice?P<0.05?.5.Heat-inactivated M1 GAS stimulation Hep2 cells,LC3?protein began to high expression.The distribution of EGFP-LC3 protein from uniform distribution in the cytoplasm to point-like aggregation distribution by TEM.6.WT M1 GAS and M-M1 GAS induce higher levels of autophagy than FbaA-M1 GAS.The specific realization:high expression of LC3,point-like aggregation.7.The results of FbaA protein-stimulated cells showed that LC3?expression was increased and P62 was decreased.8.Among the FbaA protein fragments,only the B?aa#68-161?fragment caused high expression of LC3?a nd punctate aggregation.Summary:1.M1 GAS was cleared after entering Hep2 cells for 9 hours.2.M1 GAS can induce autophagy in Hep2 cells,and autophagy is beneficial to clear the M1 GAS.3.The FbaA is an effective protein for inducing autophagy.Part Two FbaA protein initiates autophagy by Fn-mediated binding to nismObjective:To understand which molecules of the host cell initiate autophagy and the signaling pathways involved in this the process.Methods:1.FbaA protein stimulates Hep2 cells,TLR2 knockdown cells and TLR4knockdown cells.Expression of LC3?,TLR2 and TLR4 were detected.2.The binding of FbaA or FbaA+Fn to integrin?5?1 was detected by Pull down experiment.3.Hep2 cells were stimulated with low concentration?10?g/ml?and high concentration?40?g/ml?of FbaA protein to detect changes in the expression of Fn protein and integrin?5?1 receptor protein.4.Comparison of Fn?different doses?and FbaA+Fn stimulated cells,causing changes of LC3?.5.FbaA protein stimulates Hep2 cells,Fn or Integrin?5 or Integrin?1knockdown cells.Expression of Fn,Integrin?5,Integrin?1,LC3?were detected.6.The rapamycin pretreated cells?si-Control cells,si-Fn cells,si-Integrin?5 cells,si-Integrin?1 cells?.Then,M1 GAS infected these four cells.Count the number of live bacteria in the corresponding cells.7.The expression of m TOR,p-m TOR,ULK1,p-ULK1,Beclin-1 and p-Beclin-1 protein were detected by FbaA protein stimulated cells for 6h.8. The expression of m TOR,p-m TOR,ULK1,p-ULK1,Beclin-1 and p-Beclin-1 protein were detected by FbaA protein stimulated cells?si-Control cells,si-Fn cells,si-Integrin?5 cells,si-Integrin?1 cells?for 6h.9. The interaction relationship among Vps34,beclin-1 and Rab7 were detected by Co-IP under different cell conditions?Control group,FbaA group,si-integrin 5+FbaA group,si-integrin 1+FbaA group?.10.The interaction relationship among Vps34,beclin-1 and Rab7 were detected by Co-IP under different cell conditions?Control group,sh-Beclin-1group?.Results:1.FbaA protein stimulates Hep2 cells.Experiment result shows that TLR2 and TLR4 protein expression did not change.Knockdown of TLR2 or TLR4 did not cause LC3?protein changes.2.The Pull down experiment showed that the binding of FbaA to integrin?5?1 was more obvious in the present of Fn.3.FbaA protein-stimulated cells do not cause changes expression of the Fn protein and integrin?5?1 receptor protein.4.Individual and different doses of Fn protein did not cause changes in the expression of LC3II protein;however,when FbaA protein was added together,we found that LC3II protein expression was elevated and dose-dependent with the concentration of Fn.5.When Fn protein or Integrin?5 protein or Integrin?1 protein was knocked down alone,the expression of LC3II protein was not changed compared with the control group;the expression of LC3II was increased in FbaA protein-stimulated group compared with the group without FbaA protein stimulation;LC3II expression was lower in the knockdown group than the unknocked group?while adding FbaA protein stimulation?.6.The results showed that the number of intracellular bacteria in the si-Fn group,si-Integrin?5 group and si-Integrin?1 group was higher than that in the si-Control group?P<0.05?.The number of intracellular bacteria in the Rapamycin-treated group was lower than that in the corresponding group?P<0.05?.7.After FbaA protein stimulated Hep2 cells,the results of Western blot showed that the expression of m TOR,ULK1 and Beclin-1 did not change with time;However,the expression of p-m TOR and p-ULK1 began to decrease from 2h until 6h;The expression of p-Beclin-1 began to increase at 2h until6h.8.Western blot results showed that knockdown of Fn protein or Integrin?5 protein or Integrin?1 protein alone did not affect the expression changes of m TOR,p-m TOR,ULK1,p-ULK1,Beclin-1,and p-Beclin-1 proteins.When FbaA protein was added,the expression of p-m TOR,p-ULK1 was decreased,and the expression of p-Beclin-1 was increased in the si-Control group compared with the si-Fn group or the si-Integrin?5 group or the si-Integrin?1group.9. The results of Co-IP showed that when FbaA protein stimulated normal Hep2 cells,Vps34,Beclin-1 and Rab7 were immunoprecipitated;However,when Fn protein or Integrin?5 protein or Integrin?1 protein was knocked down alone,Vps34,Beclin-1,Rab7 were not immunoprecipitated.10. Rab7 and Vps34 were not immunoprecipitated in the stable cell line of Hep2-sh-Beclin-1.Summary:1.FbaA protein induces autophagy,which is independent of TLR2 and TLR4 receptors.2.FbaA protein binding to Fn protein initiates autophagy via the integrin?5?1 receptor.3.FbaA protein activates Vps34 through the intracellular m TOR-ULK1-Beclin-1 signaling pathway,and then recruits Rab7 to initiate autop-hagy formation.Part three The effect of autophagy on group A streptococcus infection and its molecular mechanism were studied in animalsObjective:To explore the mechanism of M1 GAS activating autophagy and the effect of autophagy in vivo by using healthy mice and genetically deficient mice.Methods:1.Healthy mice were infected with WT M1 GAS and FbaA-M1 GAS,respectively.The expression of LC3II protein and the number of live bacteria were detected in the lung.2.Air pouch model of infection:After anesthesizing with Pelltobarbitalum Natricum,mices were injected subcutaneously with 1 ml of air to form an air pouch and then inoculated with 0.5 ml PBS?containing3×108 GAS?.After 48h,mices Skin lesion area were measured by Image J software.3.The sg RNA-Control,sg RNA-Integrin?5 and sg RNA-Integrin?1(1×1011 vg/mice)were constructed with AAV6 by CRISPR-Cas9.And the vectors were delivered to mice lung using intranasal.After 3 weeks,the mice lungs were taked for Western blot verification.4.WT mice,sg RNA-Integrin?5 knockdown mice and sg RNA-Integrin?1knockout mice were infected with M1 GAS?3×108 CFU/50?l?.After 24 h,the expression of LC3II protein and the number of live bacteria were detected in the lung.5.The pulmonary inflammation of WT mice with WT M1 GAS and FbaA-M1 GAS infection were observated.And pulmonary inflammation were observated causing by WT M1 GAS infection in Atg5?KO?mice,sg RNA-Integrin?5 knockdown mice and sg RNA-Integrin?1 knockdown mice.Results:1.Western blot results showed that compared with WT M1 GAS-infected healthy mice,FbaA-M1 GAS-infected mice caused lower expression of LC3II protein?P<0.05?,but the survival of live bacteria were higher?P<0.05?.2.FbaA-M1 GAS infected mice caused a larger area of skin damage.3.Western blot results showed that sg RNA-Integrin?5 knockdown mice and sg RNA-Integrin?1 knockout mice were successfully induced,and the lung Integrin?5 protein or Integrin?1 protein was almost undetectable.4.Compared with WT mice,M1 GAS-infected sg RNA-Integrin?5knockdown mice and sg RNA-Integrin?1 knockout mice induced lower expression of LC3II protein,but the number of live bacteria was higher than of WT mice.5.H&E staining of mouse lungs showed that mice infected with WT M1GAS or FbaA-M1 GAS caused severe lung inflammation compared with the PBS group,which was characterized by lymphocytic infiltration and thickening of the alveolar septum.Compared with WT mice infection group,the Atg5?KO?mice,sg RNA-Integrin?5 knockout mice and sg RNA-Integrin?1 knockout mice showed more obvious inflammatory cell invasion,severe thickening of alveolar septum,massive hemorrhage in alveoli,and larger area of inflammatory lesions.Summary:1.M1 GAS induces autophagy in the lungs of mice,and FbaA protein is the main effector protein.2.M1 GAS initiates autophagy through the mouse lung integrin?5?1receptor.Conclusions:1.The clearance of M1 GAS in epithelial cells is associated with autophagy induction by FbaA of M1 GAS,and the 68-161 amino acid sequence of FbaA protein is autophagy induction peptide.2.FbaA protein initiates autophagy by Fn-mediated binding to the epithelial cell integrin?5?1 receptor.3.Intracellular signal of autophagy induced by FbaA protein:activation of Vps34 through m TOR-ULK1-Beclin-1 signaling pathway,thereby recruiting Rab7 to initiate autophagy.4.Autophagy induced by FbaA of M1 GAS interaction with integrin?5?1 protein is beneficial to the lung of mice clear invading M1 GAS.5.M1 GAS infection can cause more severe inflammatory lesions in the lungs of mice after knocking out autophagy-related proteins. |
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