Mechanism Of Staphylococcus Aureus Against Autophagy Clearance By Inhibiting Autophagy In Bovine Mammary Epithelial Cells

Bovine mastitis is an inflammatory reaction of mammary gland parenchyma under the stimulation of various pathogenic bacteria and physical and chemical factors.It is mainly characterized by the physical and chemical properties of milk and pathological changes in mammary tissue,resulting in reduced milk yield and quality.There are problems such as the high cost of treatment and widespread epidemics,which seriously affect the welfare and health of sick bovines,and even cause public health problems.The most common pathogen of infectious mastitis is Staphylococcus aureus(S.aureus).The remarkable success of S.aureus as a facultative pathogen is facilitated by its vast arsenal of virulence factors and an ability to acquire antibiotic resistance readily.These factors enable it to survive and reproduce in the mammary gland.Persistent intramammary infection generally presents long-term increases in somatic cell counts and repeated clinical cases,which makes S.aureus mastitis more difficult to eliminate from dairy herds.Previous studies have demonstrated that S.aureus can invade bovine host cells and thus evade immune defenses.Autophagy is regulated by a series of signaling pathways and more than 30 key regulators(autophagy-related genes(Atgs)).Xenophagy acts as a "cell guard" to clear intracellular pathogens involved in homeostasis.Autophagy is a process that occurs in three steps as follows: formation of autophagosomes,formation of autolysosomes by fusion between autophagosomes and lysosomes,and finally,degradation of the cargo within the lysosomes.The complete autophagy flux starts from the autophagosomes that form the double-membrane structure.The key step for autophagy to finally produce biological effects is the formation of autolysosomes by fusion between autophagosomes and lysosomes.Lysosomes are monolayer coated vesicles containing a variety of acidic hydrolases,which can eventually degrade a variety of pathogens in autolysosomes.Unfortunately,studies have verified that although S.aureus can invade cells and induce autophagy,the activation of signaling pathways,the integrity of the induced autophagic flux and its intracellular survival after S.aureus invades bovine mammary epithelial cells has not been studied.Therefore,after the invasion of mammary epithelial cells by S.aureus,the signaling pathways that induce autophagy after S.aureus invades cells were identified by analyzing autophagosome maturation,autophagosome and lysosome fusion and autophagolysosome The activation of the three stages of body degradation,and then find the key factors that interfere with the survival of S.aureus in the cell,and finally clarify the autophagy marker protein LC3 as the core to screen the Clumping factor A(Clf A)of S.aureus to interfere with autophagosomes Maturation and mechanisms that cause autophagosome and lysosome fusion barriers to escape autophagic degradation.The main research contents include the following three parts:1.Autophagy of Bovine mammary epithelial cell induced by intracellular Staphylococcus aureusThe purpose of this study was to investigate the signaling pathway activated by S.aureus invasion of bovine mammary epithelial cells and its effect of autophagy on intracellular S.aureus survival..In the current study,autophagy caused by S.aureus was detected,and the correlation between autophagy and intracellular S.aureus survival was assessed.First,a model of intracellular S.aureus infection was established.Then,the autophagy of MAC-T cells was evaluated by confocal microscopy and western blot.Moreover,the activation of the PI3K-Aktm TOR and Erk1/2 signaling pathways was determined by western blot.Finally,the relationship between intracellular bacteria and autophagy was analyzed by using autophagy regulators(3-methyladenine(3-MA),rapamycin(Rapa)and chloroquine(CQ)).The results showed that S.aureus could indeed invade bovine mammary epithelial cells,and has no effect on cell viability within 8 h of persistent infection of cells.S.aureus caused obvious induction of autophagosome formation,transformation of LC3I/II,and degradation of p62/SQSTM1 in MAC-T cells;furthermore,the PI3K-Akt-m TOR and Erk1/2 signaling pathways were activated.The number of intracellular S.aureus increased significantly with autophagy activation by rapamycin,whereas the number decreased when the autophagy flux was inhibited by chloroquine.Therefore,this study indicated that intracellular S.aureus can induce autophagy and utilize it to survive in bovine mammary epithelial cells.And the integrity of autophagic flow is beneficial to the intracellular survival of S.aureus.2.S.aureus avoids autophagy clearance of bovine mammary epithelial cells by inhibiting autophagy fluxThis chapter aimed to assess the ability of S.aureus to evade autophagic degradation and survive by exploring different stages of autophagic flux.Firstly,the intracellular infection model was constructed,then the bacteria in autophagosome was detected by the transmission electron microscopy.Autophagosome generation was detected by western blot and confocal microscopy.Moreover,the autophagy flux induced by the S.aureus was evaluated by autophagy marker protein immunoblot analysis and fluorescent labeling LC3 method.In addition,In addition,the changes of acridine orange and lysosome red fluorescent probes were observed by confocal microscopy and the changes of LAMP2,CTSD and CTSL expressions were detected by western blotting to verify the level of lysosomal acidity and degradation function.Results showed that,after infected by S.aureus,a double-layer membrane structure around the S.aureus was observed in MAC-T,indicating that autophagy occurred.The change of autophagy marker protein and fluorescent labeling autophagosome also confirmed this.However,along with the time prolonging,western blot results showed that the expression of autophagy marker protein LC3 was continuously increased and the degradation of p62 protein was blocked.Confocal microscopy observed that the yellow fluorescence of transiently transfected doublelabeled LC3 persisted.The autophagy flux was significantly inhibited,leading to obvious autophagosome accumulation.At the same time,the red fluorescence of the acridine orange and red lysosomal probes is attenuated.Tthe lysosomal alkalization and degradation ability of MAC-T were also impaired.Collectively,these results indicate that S.aureus can escape autophagic degradation by inhibiting autophagy flux and damaging lysosomal function after invading MAC-T.3.Autophagy is disturbed by Clf A of S.aureusAutophagy reduces the pathogen burden,and thus,pathogens are prone to develop different strategies for overcoming autophagy.Intracellular LC3 accumulation and blocking of autophagy flux take place in Staphylococcus aureus,but no study has systemically investigated host-selective targeting and the regulation of S.aureus-mediated autophagy.In this study,we first found that by using an in-silico interaction estimation method,MAP1LC3/LC3 can bind to clumping factor A(Clf A).Subsequently,the potential interactions between Clf A and LC3 were verified by performing in vitro Glutathione-S-transferase pull-down assay and also observed by in vivo laser confocal microscopy in vivo.The isogenic clf A deletion strain ATCC25923?clf A(ATCC?clf A)and wild strain ATCC25923(ATCC)were constructed to ascertain whether S.aureus was used Clf A for modulating autophagy to increase its intracellular survival.Transcriptomics and proteomics analyses of ATCC-and ATCC?clf A-infected cells confirmed that Clf A interfered with autophagyrelated cellular biological processes such as activation of the PI3K/Akt/m TOR signaling pathway,autophagosome maturation/fusion,and lysosome fusion via LC3 binding.ATCC?clf A-infected cells showed unobstructed autophagy flux,increased p62 decomposition,LC3-II turnover,and apparent punctate structure of LC3.In addition,ATCC?clf A invasion increased the expression of Rab7,Tecpr1,VAMP8,Vti1 b,and other fusion-related proteins,and its survival in cells was significantly lower than that of the wild-type strain during persistent infection.Overall,based on these findings,S.aureus utilized Clf A for damaging autophagy of host cells,thus,improving its intracellular survival.In summary,after S.aureus invades cells,it first activates the Akt/m TOR and Erk1/2signaling pathways to induce autophagy,and then promotes the formation of autophagosomes through the targeted binding of Clf A and the autophagy protein LC3,which in turn blocks the formation of autophagosomes.Fusion of autophagosomes and lysosomes and disruption of lysosomal degradation capacity ultimately helped S.aureus escape autophagic degradation and survive intracellularly.This study provides novel insights into the impact of Clf A on S.aureus infection and will help to further elucidate the relationship between S.aureus and the host autophagy.In addition,understanding the autophagy pathway in Clf A-associated immunity is potentially important for the prevention or treatment of S.aureus infection.