| Acinetobacter baumannii (A. baumannii) is a gram-negative, opportunistic pathogen. A.baumannii infection could lead to a wide range of infectious disease. A. baumannii can moreeasily affects immunodeficient patients suffering from other severe diseases, such as trauma,post operation, coma and some chronic diseases, and who require hospitalization. Mortalityrates involved in A. baumannii infection range from19to54%. Now, A. baumannii becomesincreasingly important also because of its strong survival ability in the medical environmentand the wide spread of multiple-resistant strain. A. baumannii is regarded as an extracellularbacteria before, but recently, several studies have showed that A. baumannii could attach toand invade several mammalian cell lines and had the ability to survive in infected host cells.Thus, whether host innate immune adopted the same mechanism defence against intracellularA. baumannii as that they deal with other traditional intracellular pathogenic bacteria is anintriguing topic.Autophagy is a programmed cell death process and an important conservativeevolutionary process. Autophagy plays a very important role on turning the intracellularmaterial over and survival in starvation in eukaryotic cell. Autophagy could remove someredundant or aged organelles to maintain the metabolic balance and renewal of someorganelles. In this process some damaged proteins or organelles are packaged with a doublemembrane structure called autophagic vacuoles packages and then form autophagosomes.Autophagosomes could fuse with lysosomes and these proteins or organelles degradeeventually in autophagolysosomes.Autophagy was found to be related with several intracellular microorganisms infection.There were no reportes about whether A. baumannii could induce autophagy formation andwhat is the effect of autophagy on A. baumannii infection. Our research team worked on theautophagic response of mammalian cells to A. baumannii.The objection of our research is to investigate whether A. baumannii could inducemammalian cells to form autophagy and on this basis we investigated the initial stage (which proteins involved), the pathway and the effect on the infection of this autophagic response.And the crucial factor of A. baumannii was studied too.We used use A. baumannii to infect HeLa cells which had been transfected withLC3-fluorescence protein expression plasmid and also used the autophagic inducer to pretreatthe HeLa cells and inhibitor to pretreated the HeLa cells before infection. Then LC3punctawere observed with the confocal microscope. The autophagosomes of the A. baumanniiinfected HeLa cells were observed with transmission electron microscope to confirm theformation of autophagy. LC3expression level was detected by western blotting and the ratioof LC3-II/LC3-I was used to present the level of autophagy. By immunofluorescence staining,the colocalization between the intracellular bacteria and sensing proteins was detected toinvestigate which proteins were involved in the initial stage of this autophagic response. Weknocked down some sensing protein with RNA interference technology in HeLa cells andthen these cells were infected with A. baumannii. By immunofluorescence staining, thecolocalization between the intracellular bacteria and sensing proteins was detected toinvestigate the effect of this protein on the sensing stage. To know whether the autophagy wasa complete one or not, the colocalization among intracellular bacteria, LC3puncta andacidified lysosome or LAMP1were observed by immunofluorescence staining and p62expression level were detected by western blotting. To investigate the signal pathways of theautophagy, LC3puncta were observed with confocal microscopy and LC3expression levelwere detected by western blotting by using RNA interference and signal pathway specificinhibitor. We observed LC3puncta with the confocal microscope and detected LC3expression level by western blotting in A. baumannii-infected or phospholipase Dmutant-infected HeLa cells. To know the role of phospholipase D on the autophagy inducedby A. baumannii, prokaryotic expression of phospholipase D and immuno fluorescenttechnique were used. Finally, we used A. baumannii to infect macrophages using autophagicinducer or inhibitor by counting the colony forming unit on LB agarose plate and observationwith confocal microscopy.The results were as follows:（1） We observed A. baumannii infected HeLa cells with confocal microscopy anddetected LC3expression level by western blotting and found A. baumannii could induce HeLa cells autophagy. The autophagy formation was confirmed by observation ofautophagosome with transmission electron microscopy. This autophagy was demonstratedthat it was a complete one with immunofluorescence staining and western blotting.（2）A. baumannii was first recognized by ubiquitin E3ligase LRSAM1responsible forubiquitination, the ubiquitinated coat of bacteria is bound by ubiquitin interacting autophagyadaptor proteins NDP52and p62/SQSTM1, resulting in recruitment of autophagic machinery,knockdown experiments of septins sept2and sept9indicated that the p62/SQSTM1andNDP52target intracytosolic A. baumannii to autophagy pathways also dependent uponcytoskeleton component. The autophagy induced by A. baumannii was mediated by differentautophagy pathways including Beclin-1/Atg7/Atg8pathway, the MEK/ERK pathway, and theAMPK-mTOR-Ulk1pathway.（3） Gentamicin protection assay corroborated that the inhibition or induction ofautophagy significantly increased or reduced the production of A. baumannii progeny.Together, in this experiment, we verified A. baumannii infection can induce autophagicresponse and revealed its specific autophagy pathways, especially, corrborated the mechanismof recognization and clear of intracellular A. baumannii by host cells.The innovation of this study is to investigate the autophagy induced by A. baumannii inmammalian cell and the effect of autophagy on A. baumannii infection. We also investigated themechanismofoccurrenceandthesignalpathwaysandthecrucialfactorofA.baumanniitoautophagy. |
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