Mechanism Of Autophagy In Translocation Of Hypervirulent Klebsiella Pneumoniae

ObjectiveKlebsiella Pneumoniae is a gram-negative enterobacter bacteria that can cause life-threatening infections and sepsis.Hypervirulent K.pneumoniae(hvKp)can lead to liver abscesses and other metastatic infections.It is believed that Kp destroys the integrity of the intestinal barrier and enters the systemic circulation from the intestinal tract and causes metastatic infection.Autophagy is essential for host resistance to bacterial infection,intestinal barrier integrity and mucosal immune response.We aim to explore the mechanism of autophagy in intestinal hvKp translocation infection.MethodsFifty C57BL/6J mice were divided into two groups:gavage group(GA)(n=40)and control group(CO)(n=10).In GA group,hvKp was administered continuously for5 days,200μL/d,colony number was 10~9CUF/m L,and the migration infection model of highlyvirulent Klebsiella Pneumoniae was established.Control group was given aseptic saline 200μL/d by intragastric administration.After the experiment,the mice were anesthetized with phenobarbital sodium and euthanized with cervical dislocation under anesthesia.Peripheral venous blood of mice was collected to detect Bacterial Translocation(BT)by 16S r DNA sequencing.Hematoxylin-eosin staining was used to evaluate intestinal and liver morphology.The ultrastructural changes of intestinal and liver tissues were observed by electron microscope.We measured the levels of Superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione peroxidase(GP_X)to evaluate the oxidative stress status.Translocationwas detected by in situ hybridization.Western blot was used to measure the expression of tight junction protein and autophagy protein.The expression of autophagy protein and TJ was observed by immunofluorescence.The genes that may be involved in hvKp translocation infection were evaluated by reverse transcription assay.Results:1.At the end of the experiment,2 mice died in the GA group and no mice died in the CO group,with a mortality rate of 4%.No bacteria were detected in peripheral blood of CO group,but 18 of 38 mice in the GA group were detected in peripheral blood.The mice in the gavage group were divided into two groups:intestinal BT(+)group(n=18)and intestinal BT(-)group(n=20),and the BT rate was 37.5%.Klebsiella Pneumoniae was detected in 8 mice in 18 intestinal BT(+)group,which were divided into liver BT(+)group(n=8)and liver BT(-)group(n=10),and the BT rate was44.44%.2.The damage degree of intestinal BT(+)intestinal tissue was significantly higher than that of CO group.The degree of liver tissue damage in BT(+)group was significantly higher than that in CO group.3.The activities of SOD and GPx in intestinal BT(+)group were lower than those in CO group.Compared with BT(+)group,MDA level in CO group was obviously decreased,SOD and GPx levels were increased.There were also significant differences in the above three indexes between the intestinal BT(-)group and the intestinal BT(+)group.MDA concentration in liver BT(+)group was obviously increased.There were evident differences in SOD and GPx activities and MDA content between liver BT(-)group and liver BT(+)group.4.After 5 days of intestinal feeding,displaced Kp was detected in the lamina propria of the intestinal mucosa.5.Compared with CO group,LC3-II and Beclin-1 in GA group were significantly increased.The expressions of LC3-II and Beclin-1 in intestinal BT(-)group were obviously higher than those in intestinal BT(+)group.There are autophagosomes in the intestinal mucosa.These results indicated that intestinal mucosal autophagy was activated after hvKp continuous gavage.6.Compared with CO group,BT(+)significantly decreased the expression of ZO-1and Claudin-2.It means that the intestinal mucosal barrier is broken.Compared with intestinal BT(-)group,the expressions of ZO-1 and Claudin-2 in intestinal BT(+)group were also significantly decreased.7.Compared with CO group,LC3-II and Beclin-1 in liver BT(-)and liver BT(+)groups were significantly increased.The expressions of LC3-II and Beclin-1 in liver BT(-)group were evidently higher than those in liver BT(+)group.Autophagosomes are seen in the liver tissue.LC3-II and Beclin-1 were accumulated in hepatocyte cytoplasm in liver BT(-)group and liver BT(+)group,indicating that autophagy was activated in hepatocytes.8.Eukaryotic reference transcriptional analysis showed that the expression of autophagy genes HMOX-1 and ADRB2 was up-regulated in the BT(+)group in the liver,which may be involved in the pathogenesis of KLA.Conclusion1.hvKp can activate intestinal mucosal autophagy and reduce the damage to intestinal mucosal barrier function by downregulating oxidative stress level.2.Increasing the level of autophagy in hepatocytes can reduce the incidence of hvKLA,and the autophagy genes HMOX-1 and ADRB2 may be involved in the pathogenesis of KLA.