阅读说明：本技术 靶向菌毛黏附素YadC的宿主受体ANXA2用于改善急性下*** (The host receptor ANXA2 of targeting pili adhesin YadC is for improving emergency lower urinary tract infection ) 是由 王荃 李晓 裴庚 于 2019-10-10 设计创作，主要内容包括：本发明涉及靶向尿路致病性大肠杆菌的菌毛黏附素(YadC)的宿主受体——膜联蛋白A2(annexin A2,ANXA2)的治疗药物在改善急性下尿路感染中的应用。通过细胞生物学实验和动物实验证明了靶向菌毛黏附素YadC的宿主受体ANXA2的细胞内钙离子螯合剂BAPTA-AM可有效阻止尿路致病性大肠杆菌对膀胱上皮细胞的黏附与侵袭,并在小鼠体内显著降低尿道致病性大肠杆菌在膀胱中的定植,具有改善尿路致病性大肠杆菌造成的急性下尿路感染的效果。(The present invention relates to the host receptors of the pili adhesin (YadC) of targeting urinary tract enteropathogenic E. Coli --- and the therapeutic agent of Annexin A2 (annexin A2, ANXA2) is improving the application in emergency lower urinary tract infection.Urinary tract enteropathogenic E. Coli sticking and invade to Urothelial Cell can be effectively prevented by the intracellular calcium chelating agent BAPTA-AM that Cell Biology Experiment and zoopery demonstrate the host receptor ANXA2 of targeting pili adhesin YadC, and field planting of the avian pathogenic Escherichia coli in bladder is significantly reduced in Mice Body, have the effect of emergency lower urinary tract infection caused by improving urinary tract enteropathogenic E. Coli.)

1. the therapeutic agent of the host receptor ANXA2 of the pili adhesin YadC based on urinary tract enteropathogenic E. Coli changes in preparation Application in kind emergency lower urinary tract infection drug；The therapeutic agent refers to the host receptor of targeting pili adhesin YadC The intracellular calcium chelating agent BAPTA-AM of ANXA2, chemical name are as follows: 1,2- bis- (2- amino-benzene oxygen)-ethane-N, N, N ', N '-tetraacethyl).

2. application described in claim 1, wherein the emergency lower urinary tract infection refers to: avian pathogenic Escherichia coli is made At emergency lower urinary tract infection.

3. a kind of host receptor of the pili adhesin YadC containing described in claim 1 based on urinary tract enteropathogenic E. Coli The composition of the therapeutic agent of ANXA2, it is characterised in that add pharmaceutically acceptable pharmaceutic adjuvant and be prepared into hard capsule, flexible glue Capsule, tablet, granule, powder, suspension or syrup, wherein the tablet includes: dispersible tablet, lozenge, chewable tablets or effervescent tablet.

4. the host receptor ANXA2's of the pili adhesin YadC described in claim 1 based on urinary tract enteropathogenic E. Coli The composition of therapeutic agent is being used to prepare the application improved in emergency lower urinary tract infection drug.

Technical field

The present invention relates to the treatments of the host receptor ANXA2 of the pili adhesin YadC based on urinary tract enteropathogenic E. Coli Drug is improving the application in emergency lower urinary tract infection, and including the determination of fimbrial antigen type YadC, YadC is on bladder The identification of the receptor ANXA2 of endothelial cell surface, and the intracellular calcium chelating agent BAPTA-AM of targeting host receptor ANXA2 Selection.

Background technique

Urinary tract infection (urinary tract infections, UTIs) is one of most common bacterial infection disease, Urinary tract infection caused by pathogenetic bacteria will lead to acute simplex cystitis, acute simplex pyelonephritis, complexity urethra sense The clinical common urinary system infection contamination disease such as dye, repeated relapsing urinary tract infection.It is reported that about 40% women and 12% Male can at least undergo primary Symptomatic urinary tract infection in life, wherein 10% women can after infection 6-12 months In recurrent infection again.Statistical data shows that the whole world has more than more than 1,000 ten thousand urinary system infection contamination case every year.Urinary system Infection is also important one of nosocomial infection, accounts for United States Hospital infection and bacteremic first place.Urinary system caused by pathogenetic bacteria Togetherness dye also brings huge social economical burden, only by taking the statistical data in the U.S. as an example, spends in its treatment aspect every year Expense reach as many as 3,500,000,000.Therefore, prevent and control the infection of urinary system pathogenetic bacteria to be global urgent problem to be solved.

The pathogenetic bacteria for causing urinary system infection contamination is mainly gramnegative bacterium, including Escherichia coli, proteus, Providence Salmonella, pseudomonas aeruginosa etc..The Escherichia coli of urinary tract infections can be caused, and to be named as urinary tract pathogenic big Enterobacteria (UropathogenicEscherichia coli, UPEC).Urinary system infection contamination caused by UPEC accounts for pure 80% or more of infection.The living environment of urinary system infection contamination bacterium in human body is more special, and pathogenetic bacteria is established thin to host The antibacterial material etc. that the infection of born of the same parents is generated firstly the need of the scouring force for resisting urine in urethra, urine and urothelial cell, this The urinary system pathogenic bacteria of pyelonephritis is caused to need to go upward to kidney along urethra outside, therefore urinary system pathogenetic bacteria usually has There is surface antigen abundant, and fimbrial antigen is to meet bacterium effectively to adhere to and the key effect factor of systemic infection.

Urinary tract infections usually uses antibiotic treatment；However after antibiotic treatment in several weeks, it can still be found in the urinary tract UPEC bacterial strain, and multi-drug resistant bacterial strain is also increasing year by year, and has highlighted the importance of exploitation replacement therapy strategy.Many researchs will It focuses on the anti-adhesive treatment for the pili adhesin for being responsible for UPEC field planting.Some anti-adhesives have been developed at present Agent, such as the mannoside for 1 type pili and the spherical tetrose for P pili are treated as the non-antibiotic for treating UTI Method.The receptor analogs that sweet dew carbohydrates and their derivative (mannoside) is the adhesin FimH of 1 type pili are proved, can be blocked Combination and field planting of the UPEC in bladder, to weaken the toxicity of UPEC.Most of UPEC bacterial strains can express in course of infection A plurality of types of pili.Therefore, other pili adhesins important in UPEC is pathogenic are identified and are directed to the pili adhesin Anti-adhesive treatment (including a variety of drugs) will be more effective.

Summary of the invention

It is an object of the invention to establish a kind of method for improving emergency lower urinary tract infection, i.e. targeting pili adhesin YadC Host receptor ANXA2 intracellular calcium chelating agent BAPTA-AM.Embodiment disclosed by the invention meets this mesh 's.

To achieve the above object, the invention discloses following technology contents:

The therapeutic agent of the host receptor ANXA2 of pili adhesin YadC based on urinary tract enteropathogenic E. Coli improves in preparation Application in emergency lower urinary tract infection drug；The therapeutic agent refers to the host receptor of targeting pili adhesin YadC The intracellular calcium chelating agent BAPTA-AM of ANXA2.The BAPTA-AM is referred to: 1,2- bis- (2- amino-benzene oxygens)- Ethane-N, N, N ', N '-tetraacethyl).The emergency lower urinary tract infection refers to: anxious caused by avian pathogenic Escherichia coli Property lower urinary tract infection.

The present invention further discloses the host receptors of the pili adhesin YadC based on urinary tract enteropathogenic E. Coli The composition of the therapeutic agent of ANXA2, it is characterised in that add pharmaceutically acceptable pharmaceutic adjuvant and be prepared into hard capsule, flexible glue Capsule, tablet, granule, powder, suspension or syrup, wherein the tablet includes: dispersible tablet, lozenge, chewable tablets or effervescent tablet.

Composition can be configured to tablet, dispersible tablet, sugar-coat agent, granule, dry powder doses, solution or capsule.To prepare mouth Lactose can be used for medication compositions or starch does carrier, gelatin, sodium carboxymethylcellulose, methylcellulose, polyvinyl pyrrole Alkanone etc. is suitable adhesive.Starch or microcrystalline cellulose can be selected as disintegrating agent, often with talcum powder, santocedl, firmly Glycerol, calcium stearate or magnesium, polyethylene glycol-4000, polyethylene glycol-6000, sodium pyrosulfite etc.；As suitable anti- Adhesive and lubricant.For example, tablet can be prepared by compacting wet granular.Active constituent and carrier and selectivity with one Part disintegration additive composition mixture, the aqueous solution of the mixture and adhesive, alcohol or aqueous alcohol solution are suitable It is granulated in equipment, other disintegrating agents, lubricant and antiplastering aid is then added by this mixture tabletting in dry particle.

Host receptor the invention discloses the pili adhesin YadC of UPEC on Urothelial Cell surface --- film connection Albumin A 2(annexin A2, ANXA2).Annexin A2 is distributed widely in various cells, including endothelial cell, monocyte And epithelial cell, and many biochemical processes, such as cell Proliferation are participated in, endocytosis, autophagy and film transport.ANXA2 is with calcium dependent Mode is reversibly bound on negatively charged membrane phospholipid, and is mainly present on cell membrane with stable different tetramer, The heterologous tetramer is by ANXA2 and p11(S100A10) two molecular compositions, it is formed ANXA2/p11 compound (A2t). S100A10 is EF hand-type Ca^{2 +}The member of binding protein S100 family, intracellular S100A10 participate in several including ANXA2 Kind transport of the albumen to plasma membrane.Helical form N-terminal in ANXA2 occurs for the combination of the two, and in the compound, ANXA2 can be with Protect S100A10 from by quick ubiquitination and degradation, and S100A10 can then increase the Ca of ANXA2^{2 +}Sensibility and its knot Close the ability of film and F- actin.ANXA2 is accredited as the potential receptor of pseudomonas aeruginosa and virus, it was reported that ANXA2 It is related with bacterium and virus infection epithelial cell with A2t.But have not been reported effect of the ANXA2 in UPEC infection.Mass spectrum and Co-IP experiment confirms that the YadC albumen of purification can be with the ANXA2 of endogenous cellular ANXA2 albumen and purification Albumen has direct interaction.

The invention discloses the intracellular calcium chelating agent BAPTA-AM(of targeting ANXA2 is commercially available) it refers to: (1,2- Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl Ester), bis- (the 2- amino-benzene oxygen)-ethane-N, N, N ' of 1,2-, N '-tetraacethyl) improving the work in emergency lower urinary tract infection With.

The chemical structure of BAPTA-AM is as follows:

BAPTA-AM is a kind of cytoplasm calcium chelating agent of cell-permeable, does not damage to cell, is widely used in studying With Ca^{2 +}The physiological function of related cytochrome.Research finds BAPTA-AM:

1) there is cytoprotection, be mainly reflected in the effect of protection nerve cell, antioxidation and inhibition Apoptosis；

2) liver protection effect, BAPTA-AM have obtained national patent to the treatment research of the disease characterized by a large amount of cell deaths (patent No. ZL200310106055.2, Song Biwei etc.)；

3) cardiovascular and respiratory system is also had a major impact, is mainly reflected in vasodilator effect, treatment drug induccd ventricle is fine It quivers, alveolar type II cells is promoted to secrete surface reactive material.

BAPTA-AM and its derivative are expected to become the serious conditions such as apoplexy, liver failure, Respiratory Distress Syndrome(RDS) Important salvage drug.But application of the BAPTA-AM in the emergency lower urinary tract infection caused by UPEC does not have been reported that also.Due to ANXA2 With Ca^{2 +}The mode combination membrane phospholipid of dependence, and there are some researches prove BAPTA-AM inhibition can be with ANXA2 and S100A10 albumen Interaction, reduce A2t albumen composition formation, therefore we be used as hinder ANXA2 function inhibitor.In vitro It can significantly reduce UPEC sticking and invade to Urothelial Cell using the BAPTA-AM of targeting ANXA2 as the result is shown；In vivo As a result also demonstrating can significantly reduce bacterium after giving BAPTA-AM bladder Local treatment before UPEC infects C57BL/6J mouse In the field planting of bladder, improve emergency lower urinary tract infection.

Embodiment of the present invention is related to improving urgency with the intracellular calcium chelating agent BAPTA-AM of targeting ANXA2 The method of property lower urinary tract infection.

Detailed description of the invention

Fig. 1 YadC promotes that CFT073's is pathogenic in vivo and in vitro；

A: wild strain (CFT073), deletion mycopremna (ΔyadC), covering bacterial strain (ΔyadC p-yadC) 5637 cells of infection, Stick bacterium amount by coated plate counting statistics each group；

B: wild strain (CFT073), deletion mycopremna (ΔyadC), covering bacterial strain (ΔyadC p-yadC) 5637 cells of infection, Pass through the invasion bacterium amount of coated plate counting statistics each group；

C: wild strain (CFT073), deletion mycopremna (ΔyadC), covering bacterial strain (ΔyadC p-yadC) infection C57BL/6J After mouse 12 hours, bacterium amount is colonized by the bladder of coated plate counting statistics each group；

D: wild strain (CFT073), deletion mycopremna (ΔyadC), covering bacterial strain (ΔyadC p-yadC) infection C57BL/6J it is small After mouse 24 hours, bacterium amount is colonized by the bladder of coated plate counting statistics each group；

Fig. 2 ANXA2 albumen and YadC albumen have direct interaction；

A:Far-western blotting and LC-MS/MS mass-spectrometric technique identifies YadC in the interaction of 5637 cell surfaces Albumen is ANXA2, and arrow meaning is the band of Mass Spectrometric Identification；

B: co-immunoprecipitation (co-immunoprecipitation, co-IP) technology detection purifying FLAG-YadC albumen with The interaction of endogenous ANXA2 albumen；

The interaction of the ANXA2 albumen of the FLAG-YadC albumen and purifying of c:co-IP technology detection purifying；

Effect of the BAPTA-AM of Fig. 3 receptor targeted ANXA2 in UPEC course of infection；

A: figure column is successively defined as 1-4 from left to right；1,3 be respectively wild strain (CFT073), deletion mycopremna (ΔyadC),

Infected in the presence of DMSO count after 5637 cells stick bacterium amount；2,4 be respectively wild strain (CFT073), missing Bacterial strain (ΔyadC) infected in the presence of 5 μm of BAPTA-AM count after 5637 cells stick bacterium amount；

B: figure column is successively defined as 1-4 from left to right；1,3 be respectively wild strain (CFT073), deletion mycopremna (ΔyadC), The invasion bacterium amount counted after 5637 cells is infected in the presence of DMSO；2,4 be respectively wild strain (CFT073), deletion mycopremna (ΔyadC) the invasion bacterium amount counted after 5637 cells is infected in the presence of 5 μm of BAPTA-AM；

C:C57BL/6J female mice per urethra injects 2 mg/kg BAPTA-AM or DMSO processing to bladder, takes bladder group after 24 hours It knits and carries out H&E dyeing；

D:C57BL/6J female mice per urethra injects 2 mg/kg BAPTA-AM or DMSO processing to bladder, connects after 1 hour through urethra Kind 1 × 10⁸CFU CFT073 puts to death mouse and bladder body is taken to be homogenized after 24 hours, the bladder of counting is colonized bacterium amount；It will Figure column is successively defined as 1-4 from left to right；1,3 be respectively wild strain (CFT073), deletion mycopremna (ΔyadC), deposit in DMSO Bladder under is colonized bacterium amount；2,4 be respectively wild strain (CFT073), deletion mycopremna (ΔyadC) in 2 mg/kg BAPTA- Bladder in the presence of AM is colonized bacterium amount.

Embodiment is as follows:

1) 5 μm of BAPTA-AM pre-process human bladder epithelium 5637(ATCC HTB-9) cell 1 is after h, with CFT073(ATCC 700928) andyadCDeletion mycopremna (this experimental construction, following construction methods) infects 5637 cells, each by coated plate counting statistics Group is sticked and invasion bacterium amount；

2) 1 h gives mouse 2 mg/kg BAPTA-AM bladder Local treatment before UPEC per urethra infecting mouse, after 24 h Dead mouse takes bladder to carry out tissue homogenate, passes through the field planting bacterium amount of coated plate counting statistics each group.