An Infection-induced RhoB-Beclin 1-HSP90 Complex Enhances Clearance Of Uropathogenic Escherichia Coli

Objective:Urinary Tract Infection（UTI）is one of the most common bacterial infections in communities and hospitals[1,2].UTI is usually a self-limiting disease,but it has a tendency to relapse.With the increasing resistance of urinary tract pathogens to currently available antibiotics,we need to explore new strategies for the treatment of urinary tract infections.Urinary tract pathogenic Escherichia coli（UPEC）is a gram-negative intracellular pathogen and the main cause of urinary tract infection（UTI）.Therefore,how to resist UPEC infection is currently the main research direction to solve UTI.Host cells can defend against pathogens through a variety of antibacterial pathways,such as cytokine production,phagosome-lysosome fusion,inflammasome activation,autophagy,antigen presentation,and B and T cell immune responses[3-5].Among them,autophagy is a multifunctional cellular homeostasis mechanism,which can target to degrade damaged components or invade pathogens in cells through lysosomal fusion[6,7],thereby maintains a constant internal environment.Due to the diverse types of invading pathogens,the detailed mechanisms of autophagy-mediated bacterial clearance are also diverse.According to reports,UPEC resides in autophagosomes during infection and is affected by autophagy clearance,but its mechanism and the cytokines involved are still unclear.Bacterial infection induces the expression of many host factors,which are responsible for sensing invasive pathogens and initiate antibacterial effects,such as Toll-like receptors（TLRs）on the cell surface,or STING,a cellular factor induced in the cell.We found that RhoB,a member of the small RhoGTPases family,has a rapid and transient up-regulation of its intracellular protein levels after cells being stimulated by LPS or inflammatory cytokines.This process is currently believed to depend on the NF-κB pathway.In the process of autophagy formation,Beclin 1 is the main component of PI3K complex and is mainly responsible for the assembly of various regulatory factors related to autophagy.Many cytokines regulate the level of autophagy by regulating the expression of Beclin 1.However,so far,the roles and underlying mechanisms of RhoB in regulating Beclin 1and bacterial clearance are unknown.Here,using a UPEC infection model,we demonstrate that bacterial infection upregulates RhoB in bladder epithelial cells,which subsequently promotes intracellular bacteria clearance through induction of LC3lipidation and autophagosome formation.RhoB binds with Beclin 1 through its residues at 118 to 140 and the Beclin 1 CCD domain,with RhoB Arg133 being the key binding site.Binding of RhoB to Beclin 1 enhances the HSP90 and Beclin 1 interaction to prevent Beclin 1 degradation,and RhoB also directly interacts with HSP90 to maintain RhoB homeostasis.UPEC infections induce RhoB,Beclin 1 and LC3 levels in the bladder epithelium in vivo;whereas,Beclin 1,LC3 and clearance of UPEC are greatly decreased in RhoB^+/-and RhoB^-/-mice during UPEC infections.We conclude that when stimulated by UPEC infections,host cells promote UPEC clearance through the RhoB-Beclin 1-HSP90 complex.Targeting RhoB may lead to novel treatment strategies for UPEC infections.This paper intends to answer the following three questions:Firstly,what are the effects of RhoB on UPEC clearance in vivo and in vitro models;secondly,what are the effects of RhoB on autophagy and the specific molecular mechanism;lastly,if there are the interactions between RhoB and Beclin 1 and HSP90.Methods:1.In cell experiments,such as UPEC cell infection assay（Infection assay）,immunofluorescent staining（IF）,co-localization,western blotting（WB）and other methods,by constructing plasmid overexpression RhoB or small interfering RNA（si RNA）knocks down RhoB to clarify the effect of RhoB on UPEC clearance in bladder epithelial cells（5637,J82,T24,etc.）.2.In 293 and 5637 cells,we used WB,transmission electron microscope（TEM）,and fluorescent marker plasmid GFP/m Cherry/GFP-m Cherry-LC3 to overexpress autophagy marker protein LC3 and other methods to observe the RhoB pair.The influence of puncta formation and the expression of autophagy marker proteins（LC3,P62,Beclin 1）.3.We used methods such as co-immunoprecipitation（co-IP）,immunofluorescent staining（IF）,and molecular cloning to determine the interaction between RhoB,Beclin1,and HSP90,clarifying the interaction,domains and the key binding site between the three.By using gene mutation method,the function of structural domain and key gene site were verified in cell autophagy and UPEC clearance.4.After establishing a UTI model of RhoB knockout mice,we detected the effect of RhoB on autophagy protein expression and bacterial clearance in vivo by WB,immunohistochemistry（IHC）,IF and infection assay.Result:1.Through cell experiments,such as UPEC infection assay,IF,co-localization,WB and other methods,by constructing plasmid overexpression RhoB or small interfering RNA（si RNA）knocks down RhoB,and it is clear that RhoB promotes UPEC clearance in bladder epithelial cells（5637,J82,T24,etc.）.2.In 293 and 5637 cells,using WB,transmission electron microscope（TEM）,and fluorescent marker plasmid GFP/m Cherry/GFP-m Cherry-LC3 overexpression of autophagy marker protein LC3 and other methods,it is found that RhoB promotes autophagosome formation,and promotes the conversion of the autophagy marker protein LC3-Ⅰto the lipidated form LC3-Ⅱ,promotes the degradation of P62,and upregulation the expression level of Beclin 1.3.By using purified protein or cell lysates,we performed experiments,including co-IP,IF,and molecular cloning and found there are direct interactions between RhoB,Beclin1 and HSP90,and RhoB.Residues 118 to 140 of RhoB interact with CCD domain of Beclin 1,where RhoB residue Arg133 is the key binding site.Deletion of 118-140domain or RhoB R133A mutation resulted in reduced LC3-Ⅱlipidation,but increased amount of intracellular UPEC bacteria.4.In RhoB knockout mouse UTI model,we applied WB,immunohistochemistry（IHC）,IF,and infection assay to show that the levels of autophagy marker protein LC3 and Beclin 1 in RhoB-deficient mice are significantly lower than those of wild-type mice,and the amount of UPEC bacteria in the bladder was significantly increased.Conclutions:1.RhoB restricts intracellular UPEC in bladder epithial cells.2.RhoB induces autophagy through upregulation of Beclin 1.3.RhoB physically interacts with Beclin 1 and prevents its degradation.4.Molecular interactions between RhoB and Beclin 1.5.RhoB-mediated Beclin 1 stabilization is governed by HSP90,where RhoB alsophysically interacts with HSP90.6.RhoB promotes bacterial clearance and autophagy in vivo.