Enhancement Of HlyA-dependent Autophagy Promotes The Excretion Of Pathogenic Escherichia Coli In Urinary Tract

BACKGROUND AND PURPOSEUrinary tract infection(Urinary tract infection,UTI)is the most common bacterial infectious disease in the world.It is an inflammatory reaction caused by pathogenic microorganisms invading the urinary system and reproducing.About 80%of UTI is caused by urinary tract pathogenic Escherichia coli(Urinary Pathogenic E.coli.In the United States,the incidence of UTI is extremely high every year,of which the incidence rate of men is more than 3%.Compared with men,women are the main victims of UTI,with an incidence of more than 10%(of which the incidence has increased to 20%among women over the age of 65).More than 60%of women develop UTI,at least once in their lifetime and 20%of women have frequent relapses.Urinary pathogenic Escherichia coli((Uropathogenic Escherichia coli,UPEC)is the most common pathogen of urinary tract infection.UPEC is caused by invasive factors such as flagella,pili,toxins and so on.A kind of exotoxin secreted by UPEC:?-hemolysin(?-hemolysin,HlyA))is an efficient hemolytic toxin,a typical member of the RTX(repeats-in-toxin)family.HlyA is related to urinary tract infection and septicemia.After synthesizing protoxin ProHlyA in vivo,it is acylated by two internal lysine(Lys-563 and Lys-689).Acylation makes the combination of toxin and cell membrane irreversible,triggering a series of hemolytic reactions.As a degradation pathway,the main physiological function of autophagy in various organisms is to adapt to nutritional hunger by producing amino acids.When stimulation occurs,the process of autophagy will transfer intracellular substances to lysosomes through double membrane organelles,called autophagosomes.Lysosomes degrade autophagosomes to produce amino acids to compensate for various stimuli.In addition to increased autophagy during starvation,basic autophagy is also important for the intracellular energy homeostasis of resting cells.It is important to note that,LC3 protein can be used as a marker of autophagy.In addition to these basic functions,autophagy is also involved in intracellular bacteria,antigen presentation,tumor inhibition,and cell death.More and more studies have shown that cell autophagy is involved in the process of bacterial infection and involves the mechanism of bacterial excretion.When pathogenic urinary Escherichia coli(UPEC)infects bladder epithelial cell(BECs),they can be used as targets for autophagy,but because of their ability to neutralize lysosome pH,they avoid being degraded by autophagosomes into lysosomes.This change was detected by mucin Trp channel 3(TRPML3),the transient receptor potential cationic channel of lysosomes.Then,the activation of TRPML3 initiates the exocytosis of lysosomes spontaneously,resulting in the excretion of bacteria encapsulated by exosomes.In addition,some of the bacteria are degraded by lysosomes,while others continue to be stored in cells in the form of QIR.Urinary tract infection has always been a difficult problem for doctors and patients all over the world.at present,it is the focus and focus of scholars at home and abroad to study the pathogenesis of urinary tract infection and how to effectively prevent and treat it.It can be predicted that if urinary tract infection can be effectively prevented and treated,it will produce huge social and economic benefits.METHOD1.Using "urinary tract infection","Urinary tract pathogenic Escherichia coli","urinary tract infection","urinary tract infection" and "urinary tract pathogenic Escherichia coli" as search words,Pubmed,China knowledge Network and Wanfang database were searched systematically.To collect the studies on urinary tract infection and the typing of pathogenic bacteria published at home and abroad from January 2017 to December 2018(including our hospital),and count the patients who were diagnosed with urinary tract infection and cultured pathogenic bacteria in the middle clean urine,collect and sort out the proportion of all kinds of bacteria in positive strains.2.The mouse model of urinary tract infection was established by UPEC,and the urine of infected mice was collected,and the bacterial load of bladder was detected by bacterial coating plate method to verify the success of the model.3.At the tissue and cellular level,Western-Blot technique was used to verify the interaction between UPEC infection and autophagy.4.The mouse model of urinary tract infection was established with HDM strain lacking in HlyA.The success of the model was verified by the same method,and the relationship between HDM strain lacking HlyA and autophagy was verified at tissue and cell level.5.Bacterial expulsion test was used to verify whether the excretion of bacteria was related to autophagy,and the relationship between bacterial excretion and autophagy was verified again by comparing the cell autophagy inhibitor with the UPEC infection group without inhibitor.6.MTT method was used to detect and compare the cell activity of the experimental group with autophagy inhibitor and UPEC infection group without autophagy inhibitor to verify whether the bacterial excretion was released by the cleavage of infected cells.RESULT1.A total of 5217 positive strains were collected,of which Escherichia coli accounted for the most,accounting for 65.08%,followed by Enterococcus faecium with a total of 467 strains,accounting for 8.95%.There were 312 strains of Klebsiella pneumoniae and 153strains of fungi,accounting for 7.00%,5.98%,2.93%and 10.06%,respectively.2.The mouse model of urinary tract infection caused by UPEC infection was established.24 hours after infection,the urine was collected and smeared with bacteria.It was found that the bacterial load of bladder in the infection group was much higher than that in the control group.(p<0.05)3.The tissue protein was extracted from the infected mouse bladder tissue.The cell protein was extracted after UPEC infection of bladder epithelial cells(BECs,5637 line)in vitro.The Maker protein LC3 of autophagy was detected by Western-Blot method.The expression of LC3 in vivo and in vitro was higher than that in normal group.(P<0.05)4.The same method was used to infect HDM strain,and the results showed that there was no difference in LC3 expression between HDM infection group and normal group.(p>0.05)5.The results of bacterial expulsion test showed that the amount of bacteria excreted in the UPEC infection group was significantly higher than that in the bacterial excretion in the UPEC infection group with autophagy inhibitor.(p<0.05)6.Using MTT method to detect the cell activity of UPEC infection group without inhibitor and infection group with inhibitor,it was found that there was no difference in cell activity between the two groups.(p>0.05)7.CONCLUSION1.At present,patients with urinary tract infections in China are mainly caused by Escherichia coli.2.UPEC depends on its invasion factor HlyA to promote the process of autophagy.3.The promotion of autophagy can increase the excretion of bacteria by host cells,but this process does not lead to the release of bacteria caused by cell understanding.