Immunomodulatory Effect Of Omp33-36 From A.baumannii Outer Membrane Vesicles On Macrophages

ObjectiveAcinetobacter baumannii?A.baumannii?is a non-fermentative gram-negative bacilli,an opportunistic pathogenic bacterium widely distributed in the hospital environment,has become an important condition pathogenic bacterium for hospital infection.Acinetobacter baumannii can interact with host cells through outer membrane vesicles?OMVs?to induce the production of inflammatory factors and play a bactericidal and bacteriostatic role.The outer membrane protein Omp33-36 is one of the major toxic proteins that can be released by OMVs.The purpose of this study was to investigate the immunomodulatory effect of the outer membrane protein Omp33-36 from A.baumannii on Raw264.7?a mouse macrophage cell line?.To further clarify the role of outer membrane protein Omp33-36 in the infection of Acinetobacter baumannii.Methods?1?Acinetobacter baumannii ATCC 17978 ^Tand ATCC 19606^T were routinely cultured,and the outer membrane vesicles were isolated by differential centrifugation.The outer membrane vesicles of bacteria were identified by SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining.?2?Serially diluted the isolated outer membrane vesicles,took differentconcentrations of outer membrane vesicles and co-culture with mouse macrophages Raw264.7,the CCK-8 was used to determine the cell survival rate.The concentration of the OMVs from two standard strains with similar effect on cell survival wasdetermined as the stimulation concentration.?3?After co-cultivation with three different stimulating concentrations of outer membrane vesicles and mouse macrophage cell line Raw 264.7 for 24 h,the mRNAs levels of pro-inflammatory factors IL-6,TNF-?and anti-inflammatory factors IL-10,TGF-?were measured.?4?The Rec_Ab system was used to knock out the outer membrane protein Omp33-36 gene of Acinetobacter baumannii ATCC17978^T to construct Acinetobacter baumannii Omp33-36 deletion strain?Omp33::17978.?5?The outer membrane vesicles of Acinetobacter baumannii Omp33-36deletion strain?Omp33::17978 and wild strains were extracted,and the cell survival rate was determined by CCK-8 method to determine the outer membrane vesicle stimulation concentration.After co-cultured with Raw 264.7 for 24 h,the mRNA expression levels of pro-inflammatory factors IL-6,TNF-?,and anti-inflammatory factors IL-10 and TGF-?were determined.?6?The outer membrane vesicles of Acinetobacter baumannii omp33-36deletion strain?omp33::17978 and wild strain were cultured with raw 264.7 for 24hours,Flow cytometry was used to detect CD86 and CD206 related markers of M1and M2 macrophages.?7?The outer membrane protein fragment of Acinetobacter baumannii Omp33-36?20-299aa?without 19aa signal peptide was recombinant expressed and purified.Raw 264.7 was stimulated with two different concentrations of Omp33-36protein,and the mRNA expression levels of pro-inflammatory factor IL-6,TNF-?,anti-inflammatory factor IL-10 and TGF-?were measured.Results?1?There was no significant difference in the ability of Acinetobacter baumannii standard strains ATCC17978^T and ATCC19606^T to produce vesicles,and the SDS-PAGE electrophoresis of OMV proteins was similar.However,compared with the wild strain,the Omp33-36 deleted strain?Omp33::17978 constructed in this study showed a downward trend in the ability to produce outer membrane vesicles.?2?The standard strain of Acinetobacter baumannii had slight cytotoxicity:Acinetobacter baumannii had little effect on the survival of macrophage RAW264.7 at low concentration?<10?g/mL?,even though the high concentration of Acinetobacter baumannii?50?g/mL?had weak cytotoxicity on macrophage RAW264.7.?3?The outer membrane vesicles derived from two different standard strains of Acinetobacter baumannii were co-cultured with the macrophage Raw264.7.Pro-inflammatory factors IL-6,TNF-?mRNA expression levels were significantly higher than the blank control group,but the expression of IL-10 and TGF-?mRNA was different between the two groups.?4?The outer membrane vesicles extracted from Acinetobacter baumannii Omp33-36 deletion strain?Omp33::17978 constructed by Rec_Abb system were co-cultured with Raw 264.7 for 24 h,the mRNA expression levels of pro-inflammatory factors IL-6 and TNF-?were significantly lower than those of the wild strain OMVs treatment group,and the mRNA expression levels of the inflammation inhibitors IL-10 and TGF-?were significantly higher than that of the wild strain OMVs treatment group.When co-cultivated for 24h,the ratio of Raw264.7 to M1-type polarization decreased.?5?After recombinant outer membrane protein fragment of Acinetobacter baumannii omp33-36?20-299aa?were co-cultured with Raw 264.7 for 24 h,the mRNA expression level of the inflammatory factors IL-6,TNF-?and inflammatory inhibitors IL-10 and TGF-?is increased in Raw 264.7,and this change trend is concentration-dependent.The mRNA expression level of pro-inflammatory factor has a more significant increase trend compared with the anti-inflammatory factor.conclusions?1?The outer membrane vesicles of two Acinetobacter baumannii standard strains can stimulate the mouse macrophage cell line Raw 264.7 to express the pro-inflammatory factors IL-6 and TNF-?mRNA at high levels,but the anti-inflammatory factors IL-10,TGF-?mRNA expression levels are regulated differently.?2?The outer membrane protein Omp33-36 from Acinetobacter baumannii OMV plays important roles both in the pro-inflammatory and anti-inflammatory stimulation on mouse macrophage Raw 264.7.?3?The outer membrane protein Omp33-36 from Acinetobacter baumannii OMV can promote the polarization of mouse macrophages Raw 264.7 to M1 type.