Role Of Capsular In Raw 264.7 Infected With Acinetobacter Baumannii

Objective: Acinetobacter baumannii is a common clinically resistant bacteria.The infection caused by multiple/pan-resistant A.baumannii has become an important public health issue of global concern,but it is rare to study the mechanism of A.baumannii virulence.The capsule is an important virulence factor of bacteria,which can protect bacteria and resist the phagocytosis of host immune cells.However,the virulence mechanism of A.baumannii capsule has not been studied yet.In this study,we aimed to investigate the mechanism of autophagy and apoptosis of Raw 264.7 cells induced by A.baumannii capsular cells by interacting with different capsular thicknesses of A.baumannii and purified capsular polysaccharides.Methods: 1.Design primers based on the specific sequence of A.baumannii and other Acinetobacter 16 S rRNA genes,and establish a multiplex PCR method to identify A.baumannii and other Acinetobacter.2.Using Congo red dye to stain A.baumannii,observe the capsule thickness of A.baumannii under oil microscope,and establish a quick and simple method to identify the capsule thickness of A.baumannii.Two A.baumannii strains with significant difference in capsule thickness,namely thick capsular strain(Ab-H strain)and thin capsular strain(Ab-B strain),were selected for subsequent study.3.Ab-H strain and Ab-B strain were cultured on the blood agar,and the strain was collected on the blood plate after being cultured at 37 ° C for 24 h.The bacterial lysate was added to the bacteria,and combined with ultrasonic lysis of the bacteria,the supernatant was collected and the supernatant was added to a solution of 1% hexadecyl trimethyl ammonium bromide(CTAB)for the first time to precipitate the capsular polysaccharide,and the precipitate was 1 mol/L CaCl2 solution was redissolved,and the capsular polysaccharide was re-precipitated with 80% ethanol.The precipitate was finally washed with absolute ethanol and then the A.baumannii capsular polysaccharide extract.The extracted capsular polysaccharide was determined by phenol-concentrated sulfuric acid method,the endotoxin content was detected by sputum reagent method,and the capsular polysaccharide component was analyzed by GC-MS method.4.Ab-H strain and Ab-B strain and different concentrations of purified capsular polysaccharide were co-cultured with Raw 264.7 cells for 1h,3h and 6h,respectively.qRT-PCR was used to detect the expression levels of autophagy-related genes Atg3,Atg4 B,Atg5,Atg7,Atg12,Atg16L1,UNL1,and GABARAPL1.The autophagy-associated proteins LC3,Atg12,Beclin-1 and Cleaved Caspase-3 of Raw 264.7 cells were detected by Western blotting.The apoptosis rate of Raw 264.7 cell was detected by AnnexinV-FITC/PI.5.Ab-H strain and Ab-B strain and different concentrations of purified capsular were co-cultured with Raw 264.7 cells for 1 h,3h and 6h,respectively.qRT-PCR was used to detect the expression levels of TLR2,TLR4,NLRP3 and IL-1? in Raw 264.7 cells.The expression of reactive oxygen species(ROS)was detected by flow cytometry.The expression levels of phosphorylated proteins of PI3 K,Akt and mTOR were detected by Western blotting.To explore the mechanism of autophagy and apoptosis induced by capsular act to Raw 264.7 cells.Results: 1.By multiplex PCR amplification,A.baumannii can obtain two bands,425 bp and 208 bp,while other Acinetobacter species only have 208 bp After gene sequencing analysis,the 425 bp band was a specific band of A.baumannii,and the 208 bp band was a specific band of other Acinetobacter,which was consistent with expectations.In hospital,the VITEK 2 Compact automatic bacterial identification system identified 98% of the calcium acetate-A.baumannii complex as A.baumannii.2.After dyed with Congo red dye,the bacteria are blue,the background is red,and the bacterial capsule is not colored,which is easy to be recognized,under the oil microscope.Among the tested strains,the bacterial capsule thickness was not the same,and two strains with significant difference in capsule thickness,namely thick capsular strain(Ab-H)and thin capsular strain(Ab-B),were selected for subsequent study.3.The contents of capsular polysaccharides of Ab-H strain and Ab-B strain were 127.593 ?g/mL and 30.047 ?g/mL,and the endotoxin content was 0.01 EU/mL by Limulus Amebocyte Lysate.The elements of two capsules polysaccharide are the same were analyzed by LC-MS.4.Ab-H,Ab-B and purified capsular polysaccharide can induce autophagy and apoptosis of Raw 264.7 cells.After A.baumannii and Raw 264.7 cells were co-cultured,the expression of LC3 II and Beclin-1 protein continued to increase,and the increase in Ab-H group was higher than that in Ab-B group(p<0.01).The apoptotic trend is the same as that of autophagy;purified capsules can also induce autophagy and apoptosis in Raw 264.7 cells,and the induced autophagy and apoptosis intensity are directly proportional to the concentration of capsular polysaccharide.5.A.baumannii and capsular all can induce the expression of reactive oxygen species in Raw 264.7 cells.The expression of reactive oxygen species in Ab-H group was higher than that in Ab-B group(p<0.01).The capsular induced expression of reactive oxygen species in Raw 264.7 cells is proportional to the concentration.6.Both A.baumannii and capsular can inhibit the phosphorylation of PI3 K,Akt and mTOR proteins in Raw 264.7 cells.The expression levels of phosphorylation proteins pPI3 K,pAkt and pmTOR in Ab-B group were higher than those in Ab-H group.(p<0.01);capsular induced a negative correlation between the expression levels of phosphorylation proteins pPI3 K,pAkt and pmTOR.Conclusion: Both A.baumannii and its capsule can induce autophagy and apoptosis in Raw 264.7 cells.The capsular is an important virulence factor for inducing autophagy and apoptosis of Raw 264.7 cells,and its inducing ability is positively correlated with its concentration.Autophagy and apoptosis induced by A.baumannii in Raw 264.7 cells may be caused by capsular-induced Raw 264.7 cells producing a large amount of reactive oxygen species and inhibiting the PI3K-Akt-mTOR signaling pathway,which ultimately causes autophagy and apoptosis of Raw 264.7 cells.