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Analysis of the Virulence of Uropathogenic Escherichia coli Strain CFT073 in the Murine Urinary Tract
小鼠泌尿系统中CFT073致病性大肠杆菌的毒力分析

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Abstract

This urinary tract infection model was used to monitor the efficacy of a new virulence factor of the uropathogenic Escherichia coli strain CFT073 in vivo. The new virulence factor which we designated TIR-containing protein C (TcpC) blocks Toll-like receptor signaling and the NLRP3 inflammasome signaling cascade by interacting with key components of both pattern recognition receptor systems (Cirl et al., 2008; Waldhuber et al., 2016). We infected wild type and knock-out mice with wildtype CFT073 and a mutant CFT073 strain lacking tcpC. This protocol describes how the mice were infected, how CFT073 was prepared and how the infection was monitored. The protocol was derived from our previously published work and allowed us to demonstrate that TcpC is a powerful virulence factor by increasing the bacterial burden of CFT073 in the urine and kidneys. Moreover, TcpC was responsible for the development of kidney abscesses since infection of mice with wildtype but not tcpC-deficient CFT073 mutants caused this complication.

Keywords: Uropathogenic Escherichia coli(致病性大肠杆菌), Toll-like receptor(Toll-like 受体), Inflammasome(炎症体), TIR-containing protein C(TIR-containing 蛋白C.), Urinary tract infection(泌尿系统感染)

Background

Urinary tract infections (UTIs) are some of the most common bacterial infections worldwide (Dielubanza and Schaeffer, 2011) and are predominantly caused by uropathogenic Escherichia coli (UPEC) (Zhang and Foxman, 2003). There is a high rate of recurrent infections (Dielubanza and Schaeffer, 2011) and also an increase in the emergence of antibiotic resistant E. coli strains (Eurosurveillance editorial, 2015). Therefore the understanding of host and bacterial factors in the pathophysiology of urinary tract infections is of high relevance in order to develop new therapeutic agents.
   The murine UTI model system is the primarily used animal model system to study the pathogenicity of UPEC isolates and bacterium-host interactions and to identify underlying molecular mechanism. Besides the murine UTI model system, other animal model systems like porcine, avian, zebra fish and nematodes exist, which have been demonstrated to be useful for investigating UTIs. However, these models are associated with one or several limitations and disadvantages such as no possibility for genetic modification, the lack of a vertebrate-like immune system and/or urinary tract system or high costs. In addition to animal model systems cell culture based systems with primary immune cells or immortalized urinary tract tissue-derived cells are available. In vitro culture methods can be used to analyze UPEC interactions with host cells but they, of course, cannot reflect the complexity of the host environment involving a number of different cell types, tissue architecture and host defense mechanisms. Mice have much in common with humans including conserved immunological factors and a similar urinary tract system. Further, the availability of a variety of genetically distinct mouse strains to assess the impact of the genetic background makes the murine mouse model very accessible to study host-pathogen interactions in order to develop therapeutic agents.

Materials and Reagents

  1. Microcentrifuge tubes (1.5 ml for urine collection and 2.0 ml for tissue homogenization)
  2. 1 ml syringes (BD, catalog number: 309628 )
  3. 27 G needles (BD, catalog number: 305136 )
  4. ELISA plates (Nunc MaxiSorp® flat-bottom 96 well plate) (Sigma-Aldrich, catalog number: M9410 )
  5. Unfrosted or frosted glass slides (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10149870 )
  6. poly-L-lysine-coated glass slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: P4981 )
  7. Coverslips 60 x 24 mm
  8. Mice
    Only female mice were used for the UTI infection model because the urethra in male mice is extremely difficult to catheterize due to its anatomy. The following strain of mice at an age of 8 to 16 weeks was used:
    C57BL/6
    The following knock-out mice were successfully used in the model to study the relevance of a number of host-defense genes but these mice are optional and not required to generate the model:
    Tlr4−/−
    Myd88−/−
    Trif−/−
    TrifLps2/Lps2
    Il-1β−/−
    Irf3−/−
    Note: All knock-out mice were at least 8x backcrossed to C57BL/6 mice. All mice were bred and housed in specific pathogen free conditions. Mice were only used upon permission of the local animal experimental ethics committee.
  9. Bacteria
    1. UPEC strain CFT073, provided by ATCC (ATCC, catalog number: 700928 )
    2. tcpC-deficient CFT073 tcpC::kan, generated in the lab using the method described by Datsenko/Wanner (Datsenko and Wanner, 2000).
    3. Complemented mutant strain CFT073 tcpC::kan+pTcpC, the plasmid was generated as described (Cirl et al., 2008).
    Note: Bacterial strains used to inoculate the animals were maintained on LB agar plates containing appropriate antibiotics. The strains were grown overnight on tryptic soy agar. Bacteria were harvested in phosphate-buffered saline and bacterial cell density (OD597 = 1 corresponded to 1 x 109 bacteria/ml) was subsequently adjusted on the basis of a standard curve of absorbance at 597 nm to a concentration of 1010 bacteria per ml by dilution in phosphate-buffered saline.
  10. Ice
  11. Dry ice
  12. Ethanol (70%)
  13. Isoflurane (Abbott Laboratories, Forene®, catalog number: 506949 )
  14. Ketamine (Ketaminol®, catalog number: 511519 )
  15. Xylazine (Rompun®, catalog number: 0 22545 )
  16. 0.9% NaCl
  17. Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D8662 )
  18. Gentamicin
  19. Tissue-Tek® O.C.T. compound (SAKURA FINETEK, catalog number: 4583 )
  20. Tissue-Tek® Cryomold (SAKURA FINETEK, catalog number: 4565 )
  21. Isopentane (Sigma-Aldrich, catalog number: PHR1661 )
  22. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  23. Normal goat serum (Agilent Technologies, catalog number: X0907 )
  24. Rat monoclonal antibody NIMP-R14 (1: 200) (Abcam, catalog number: ab2557 )
  25. Antiserum to a synthetic peptide within the PapG adhesin (1:200), produced in the laboratory
  26. Goat anti-rat immunoglobulins (1:200), conjugated with Alexa 488 dye (A488; 495ext/519em nm) (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11006 )
  27. Goat anti-rabbit immunoglobulins (1:200), conjugated with Alexa 568 dye (A568; 578ext/603em nm) (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11011 )
  28. Anti-NLRP3 antibody (Santa Cruz Biotechnology, catalog number: sc-66846 )
  29. Anti-IL-1β antibody (Abcam, catalog number: ab9722 )
  30. Anti-ASC antibody (Abcam, catalog number: ab155970 )
  31. DAPI (Sigma-Aldrich, catalog number: D9542 )
  32. VECTASHIELD mounting medium (Vector Laboratories, catalog number: H-1000 )
  33. MIP-2 quantification kit (R&D Systems, catalog number: MM200 )
  34. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  35. Tryptic soy agar (TSA) (BD, DifcoTM, catalog number: 236950 )
  36. LB agar (BD, catalog number: 244520 )
  37. Columbia 5% blood agar (BD, catalog number: 221263 )
  38. Sucrose (Sigma-Aldrich, catalog number: 84100 )
  39. Ketamine/Xylazine solution (see Recipes)
  40. Media for cultivation of bacteria (see Recipes)
    Tryptic soy agar plates, LB, Columbia 5% blood agar
  41. Sucrose solution (see Recipes)
  42. 4% paraformaldehyde (see Recipes)

Equipment

  1. Burker chamber
  2. ELISA washer
  3. Drop glass jar for gas anesthesia
  4. Biosafety hood in biosafety level 2 facility
  5. Curved forceps, scissors
  6. Soft polyethylene catheter (0.61 mm outer diameter; Clay Adams) (BD, catalog number: 427401 )
  7. Stomacher 80 homogenizer (Seward medical, catalog number: 0080/000/EU )
  8. Cryostat section were made with a Microtome blade C-35 (FEATHER Safety Razor, model: C-35)
  9. Fluorescence microscopy (Olympus, model: AX60 , equipped with filter sets [excitation/emission] 495ext/519em nm and 578ext/603em nm)
  10. AxioCam ERc 5s camera (Zeiss, model: AxioCam ERc 5s)
  11. Labsystems Multiskan PLUS reader (Analytical Instruments, Golden Valley, USA)

Procedure

  1. Infection model (direct instillation of bacteria into the bladder)
    The infection protocol of mice was originally described by Hagberg et al. (1983) and was further modified by Cirl et al. (2008), Yadav et al. (2010) and Waldhuber et al. (2016).
    1. To sample the urine the mouse will be placed to sit normally on the metal mesh of the cage. Upon lifting up the tale and hind legs, the mouse will hold on to the cage firmly with the front legs thus stretching the belly and exposing the underside. Mouse bladder is now emptied by gentle manual compression with index finger in the abdominal region (Figure 1) and urine is collected in sterile microcentrifuge tubes (kept on ice). Collected urine can be used to quantify colony-forming units (CFU) on TSA plates, for examination of inflammatory cells (e.g., neutrophils) or for cytokine analysis by ELISA. Alternatively, urine can be collected after anesthesia.


      Figure 1. Urine collection in mice. Place the mouse on a cage metal mesh and hold tail to lift hind legs. Most mice will urinate naturally and urine can be collected in a sterile microcentrifuge tube. Sometimes, using index finger, gentle pressure on abdominal area can be applied to encourage mice to urinate.

    2. The animals are anesthetized using either isoflourane in a drop glass jar or by intraperitoneal injection of ketamine-xylazine cocktail (Xylazin: 6-8 mg/kg body weight, Ketamin: 90-120 mg/kg body weight (normal body weight of 8-16 week old mice: 19-23 g) in 0.9% NaCl; diluted to a final volume of 100-200 µl/mouse for i.p. administration)
    3. If not done before anesthesia the bladder is now emptied by gentle compression of the abdomen. A drop of urine is caught directly at the urethral orifice with a calibrated loop (taking up 10 µl of fluid) and spread on blood agar plate to ensure that the mice are uninfected.
    4. Immediately thereafter, a soft polyethylene catheter (0.61 mm outer diameter; Clay Adams) adapted to a 0.4 by 20-mm gauge needle on a 1 ml tuberculin syringe (supplier BD) is transurethrally inserted into the bladder.
      Note: By gently grabbing and pulling out the clitoral fold of the mouse, the urethra is straightened out and allows for fast and atraumatic catheterization. We use a forceps to pull the tissue prior to inserting the catheter with the mouse on the back (Figure 2).


      Figure 2. Murine urinary tract infection model. Image showing the position of the mouse during transurethral catheter insertion.

    5. We used the high dose model in most experiments and instilled 0.1 ml of the bacterial suspension containing 2 x 108 or 1 x 109 CFU/ml. In a few experiments we lowered the bacterial amount to 2 x 105/mouse (low dose model) to study the effects of IL-1β deficiency on bacterial burden.
    6. After injection, the catheter is immediately withdrawn and no further manipulations are performed. Control mice were injected with sterile PBS.
    7. The model can then be used to study bladder and/or kidney infections (Figure 3).


      Figure 3. Ascending urinary tract infection in mice. Mice were infected with E. coli CFT073 by intravesical inoculation and sacrificed after 7 days. A. Uninfected control mice show healthy kidney with smooth and round contours, whereas kidney from infected mice display severe abscess (arrows) formation (B).

  2. Monitoring infection
    1. The establishment and persistence of bacteria in the mouse urinary tracts is monitored by sequential urine cultures until sacrifice.
    2. Urine is collected into sterile tubes through gentle pressure on the mouse’s abdomen (5 h, 24 h and up to 7 days) and neutrophils are quantified by light microscopy using a Burker chamber.
    3. The urine samples and serial dilutions (starting dilution 1:100, then 1:1,000, 1:10,000 in PBS) are quantitatively cultured (100 μl) on tryptic soy agar (TSA) plates. LB or Columbia 5% blood agar plates are good alternatives. TSA plates do not contain antibiotics, since there is no antibiotic which would select between the experimental strain and any possible contamination. The experimental strain and possibly contaminating bacteria are differentiated by colony morphology. In addition sham infected controls may also reveal possible contaminations, but these occur only rarely.
    4. After sacrifice by cervical dislocation, bladders and kidneys are removed under sterile conditions, placed into a plastic bag containing 5 ml PBS (pH 7.2) and homogenized in a Stomacher 80 homogenizer (100 rotations/min, 5 min; the instrument was cleaned with 70% alcohol between samples).
    5. Samples are placed on ice and analyzed in serial dilutions plated on TSA plates. The number of bacteria is expressed as the number of CFU per entire tissue.
    6. The number of intracellular bacteria is determined by killing extracellular bacteria with gentamicin (200 μg/ml) and subsequent washing and homogenization of the remaining organ (more information in Notes).
    7. Subsequently, blood agar and TSA plates are incubated at 37 °C overnight and visually scored for bacterial colonies.

  3. Post infection analysis
    1. Histology
      1. Kidneys and bladders are fixed in 1 ml of freshly prepared 4% PFA immediately after sacrifice and incubated overnight at 4 °C.
      2. Subsequently, the fixed tissues are incubated in 15% sucrose (4 °C/24 h) and washed in 25% ice-cold sucrose (4 °C/24 h). Tissues are then embedded in O.C.T. compound and frozen in isopentane at -40 °C and stored at -80 °C for further use. Cryostat sections (thickness 5 μm) are made with Microtome blade C-35, and mounted onto poly-L-lysine-coated glass slides and stained.
      3. The kidney sections from different groups of mice are double-stained for analysis by immunohistochemistry.
    2. Immunohistochemistry
      1. Briefly, tissue sections are dried at 37 °C for 15 min, washed in PBS-0.2% Triton X-100 (pH 7.2) (2 x,10 min/RT) and incubated (30 min/RT) with PBS-0.2% Triton X-100 plus 5% goat normal decomplemented serum.
      2. Sections are subsequently incubated with a NIMP-R14 rat mAb (1:200) to detect neutrophils and a rabbit antiserum to the synthetic peptide CRPSAQSLEIKHGDL within the PapG adhesin (1:200) (produced in the lab) to detect UPECs for 2-3 h at RT. Negative controls consisted of only normal goat serum (1:200). Antibodies are diluted in PBS-0.2% Triton X-100 plus 5% goat normal serum.
      3. The kidney sections (thickness 5 μm) are washed in PBS (3 x, 5 min) by submersion in a washing chamber and incubated (1 h/RT) with secondary goat anti-rat immunoglobulins (1:200), conjugated with Alexa 488 (A488; 495ext/519em nm), and secondary goat anti-rabbit immunoglobulins (1:200), conjugated with Alexa 568 (A568; 578ext/603em nm) as fluorochrome.
      4. After washing in PBS (2 x, 5 min), specimens are counterstained (3 min/RT) with DAPI (0.05 µM, 1 ml) to stain cell nuclei.
      5. Sections are washed again in PBS (10 min) and mounted with VECTASHIELD mounting medium and kept in the dark (4 °C).
      6. Sections are analyzed by fluorescence microscopy (Carl Zeiss) with an AxioCam ERc 5s camera at 200x magnification.
      7. Alternative targets analyzed by immunohistochemistry included: E. coli, NLRP3, IL-1β, and ASC.
    3. Cytokine measurements
      1. Urine samples are collected at 0.6 h, 24 h and up to 7 days and stored at -20 °C.
      2. MIP-2 in urine is quantified by ELISA using the Mouse MIP-2 quantification kit according to the manufacturer's instructions. Urine is diluted five-fold in sample buffer.
      3. The ELISA plates are read at 450 nm in a Labsystems Multiskan PLUS reader.

Data analysis

Parameters such as bacterial load in urine, urine cytokine content, number of polymorphonuclear leucocytes in urine were depicted as mean plus/minus standard deviation from at least 3 individual mice. The statistical difference of two groups or more were compared by Mann-Whitney rank sum test (Waldhuber et al., 2016), Student’s t-test (Yadav et al., 2010) or Fisher’s exact test (Cirl et al., 2008). Multiple groups were analyzed by 1-way ANOVA, post hoc Bonferroni’s multiple comparisons test (Cirl et al., 2008; Waldhuber et al., 2016).

Notes

Note: Intracellular detection of CFT073.

  1. Mice were infected transurethrally with CFT073.
  2. The bladder was removed micro-surgically at day 3 or 7 post infection.
  3. It was cut open by inserting a small scissor at the trigonum and it was further cut to the apex.
  4. If desired, adhering bacteria and residual urine was removed by flushing gently with 500 µl of PBS.
  5. The remaining cut open bladder was submersed in gentamicin solution (200 μg/ml, 15 min) to kill extracellular bacteria.
  6. Afterwards, the bladder was rinsed in PBS twice and the bladder was transferred into a microcentrifuge cup (2 ml, Eppendorf) containing PBS/Tween (0.3%, v/v) and a steel ball (4 mm stainless).
  7. The sample was stored on ice (critical to avoid overheating during homogenization) until homogenization was performed with a Retsch MM200 tissue homogenizer for 30 sec. with a frequency of 30/sec. Samples were placed back onto ice and analyzed in serial dilutions.
  8. The prepared material was plated on blood agar plates and individual colonies were counted.

Recipes

  1. Ketamine/Xylazine solution
    Xylazin: 6-8 mg/kg body weight
    Ketamin: 90-120 mg/kg body weight in 0.9% NaCl
    Diluted to a final volume of 100-200 µl/mouse for i.p. administration
  2. Media for cultivation of bacteria
    1. Tryptic soy agar plates
      1. Add 40 powder in 1 L distilled water
      2. Heat to boiling while stirring to dissolve powder
      3. Autoclave for 15 min at 121 °C to sterilize
      4. Allow to cool to about 50 °C. Pour into petri dishes and allow to solidify (5 mm thickness)
    2. LB
      1. Add 35 g powder in 1 L distilled water
      2. Heat to boiling while stirring to dissolve powder
      3. Autoclave for 15 min at 121 °C to sterilize
      4. Allow to cool to about 50 °C. Pour into petri dishes and allow to solidify (5 mm thickness)
    3. Columbia 5% blood agar
      1. These agar plates are available ready to use from BD (see Materials and Reagents)
  3. Sucrose solution
    15% and 25% sucrose solution
  4. 4% paraformaldehyde (500 ml)
    1. Take 400 ml of PBS in a glass beaker and keep on a stir plate inside a ventilated hood (paraformaldehyde is toxic)
    2. Heat the solution while stirring to approximately 60 °C
    3. Add 20 g of paraformaldehyde powder to the solution
    4. To dissolve paraformaldehyde powder, slowly add 1 N NaOH dropwise until the solution becomes clear. Cool down the solution and filter and adjust the volume to 500 ml with PBS
    5. Adjust the pH to 6.9 with diluted HCl
    6. Aliquot the solution and freeze at -20 °C and use within 2 months

Acknowledgments

TM was supported by the Deutsche Forschungsgemeinschaft (DFG) grant MI471/6-1. The protocol was initially developed by Hagberg et al. (1983) and modified in the publications of Cirl et al. (2008), Yadav et al. (2010) and Waldhuber et al. (2016). Authors declare that there are no conflicts of interest or competing interests that may impact the design and implementation of their protocol.

References

  1. Cirl, C., Wieser, A., Yadav, M., Duerr, S., Schubert, S., Fischer, H., Stappert, D., Wantia, N., Rodriguez, N., Wagner, H., Svanborg, C. and Miethke, T. (2008). Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins. Nat Med 14(4): 399-406.
  2. Datsenko, K. A. and Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97(12): 6640-6645.
  3. Dielubanza, E. J. and Schaeffer, A. J. (2011). Urinary tract infections in women. Med Clin North Am 95(1): 27-41.
  4. Eurosurveillance editorial, t. (2015). ECDC publishes 2014 surveillance data on antimicrobial resistance and antimicrobial consumption in Europe. Euro surveillance 20(46).
  5. Hagberg, L., Engberg, I., Freter, R., Lam, J., Olling, S. and Svanborg Eden, C. (1983). Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin. Infect Immun 40(1): 273-283.
  6. Waldhuber, A., Puthia, M., Wieser, A., Cirl, C., Durr, S., Neumann-Pfeifer, S., Albrecht, S., Rommler, F., Muller, T., Zheng, Y., Schubert, S., Gross, O., Svanborg, C. and Miethke, T. (2016). Uropathogenic Escherichia coli strain CFT073 disrupts NLRP3 inflammasome activation. J Clin Invest 126(7): 2425-2436.
  7. Yadav, M., Zhang, J., Fischer, H., Huang, W., Lutay, N., Cirl, C., Lum, J., Miethke, T. and Svanborg, C. (2010). Inhibition of TIR domain signaling by TcpC: MyD88-dependent and independent effects on Escherichia coli virulence. PLoS Pathog 6(9): e1001120.
  8. Zhang, L. and Foxman, B. (2003). Molecular epidemiology of Escherichia coli mediated urinary tract infections. Front Biosci 8: e235-244.

简介

该尿路感染模型被用于监测新生的致病性大肠杆菌菌株CFT073在体内的功效。我们指定含TIR的蛋白C(TcpC)的新的毒力因子通过与模式识别受体系统的关键组分相互作用来阻断Toll样受体信号传导和NLRP3炎性信号级联反应(Cirl等人)。 ,2008; Waldhuber等人,2016)。我们用野生型CFT073和缺乏tcpC的突变体CFT073菌株感染野生型和敲除小鼠。该协议描述了小鼠如何感染,如何制备CFT073以及如何监测感染。该方案源于我们以前发表的工作,并允许我们证明TcpC是一种强大的毒力因子,通过增加CFT073在尿液和肾脏中的细菌负担。此外,TcpC负责肾脓肿的发展,因为感染具有野生型但不是tcpC的缺乏CFT073突变体的小鼠引起这种并发症。

背景 尿路感染(UTIs)是全世界最常见的细菌感染(Dielubanza和Schaeffer,2011),主要是由欧洲病原大肠杆菌(UPEC)引起的(Zhang和Foxman,2003)。复发性感染率高(Dielubanza和Schaeffer,2011),抗生素抗性E的出现也有所增加。大肠杆菌菌株(Eurosurveillance editorial,2015)。因此,为了开发新的治疗剂,对宿主和细菌因子对尿路感染病理生理学的了解具有很高的相关性。
 鼠类UTI模型系统是主要使用的动物模型系统,用于研究UPEC分离株和细菌 - 宿主相互作用的致病性,并确定潜在的分子机制。除了鼠类UTI模型系统,存在其他动物模型系统,如猪,禽,斑马鱼和线虫,已被证明可用于调查UTI。然而,这些模型与一个或多个限制和缺点相关联,例如不存在遗传修饰的可能性,缺乏脊椎动物样免疫系统和/或尿道系统或高成本。除了具有初级免疫细胞或永生化尿道组织来源细胞的基于细胞培养的系统的动物模型系统是可用的。文化方法可用于分析UPEC与宿主细胞的相互作用,但当然,它们不能反映涉及多种不同细胞类型,组织结构和宿主防御机制的宿主环境的复杂性。小鼠与人类有很多共同点,包括保守的免疫因素和类似的泌尿系统。此外,各种遗传上不同的小鼠品系的可用性以评估遗传背景的影响使得鼠类小鼠模型非常容易研究宿主 - 病原体的相互作用以开发治疗剂。

关键字:致病性大肠杆菌, Toll-like 受体, 炎症体, TIR-containing 蛋白C., 泌尿系统感染

材料和试剂

  1. 微量离心管(1.5ml用于尿液收集,2.0ml用于组织匀浆)
  2. 1 ml注射器(BD,目录号:309628)
  3. 27 G针(BD,目录号:305136)
  4. ELISA板(Nunc MaxiSorp平底96孔板)(Sigma-Aldrich,目录号:M9410)
  5. 未磨砂或磨砂玻璃片(Thermo Fisher Scientific,Fisher Scientific,目录号:10149870)
  6. 聚-L-赖氨酸包被的玻片(Thermo Fisher Scientific,Thermo Scientific TM,目录号:P4981)
  7. 盖片60 x 24毫米
  8. 小鼠
    只有雌性小鼠被用于UTI感染模型,因为雄性小鼠的尿道由于其解剖学而非常难以导管。使用下列8至16周龄的小鼠:
    C57BL/6
    模型中成功地使用了以下敲除小鼠来研究许多宿主防御基因的相关性,但是这些小鼠是可选的,不需要产生模型:
    Tlr4 -/-
    Myd88 -/-





    Irf3 -/-
    注意:所有敲除小鼠与C57BL/6小鼠回交至少8x回交。将所有小鼠繁殖并饲养在特定的无病原体条件下。小鼠仅在当地动物实验伦理委员会允许的情况下使用。
  9. 细菌
    1. UPEC菌株CFT073,由ATCC(ATCC,目录号:700928)提供
    2. 使用Datsenko/Wanner(Datsenko和Wanner,2000)描述的方法在实验室中生成的tcpC -deficient CFT073 tcpC :: kan 。
    3. 补体突变菌株CFT073 > pTcpC,如所述(Cirl等人,2008)产生质粒。
    注意:用于接种动物的细菌菌株保持在含有适当抗生素的LB琼脂平板上。将菌株在胰蛋白酶大豆琼脂上生长过夜。在磷酸盐缓冲盐水中收获细菌,随后根据标准物质调节细菌细胞密度(OD 597 = 1对应于1×10 9细菌/ml)通过在磷酸盐缓冲盐水中稀释,在597nm处的吸光度曲线的浓度为每10毫升10 10细菌。

  10. 干冰
  11. 乙醇(70%)
  12. 异氟烷(Abbott Laboratories,Forene ,目录号:506949)
  13. 氯胺酮(Ketaminol ®,目录号:511519)
  14. 赛拉嗪(Rompun ®,目录号:022545)
  15. 0.9%NaCl
  16. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8662)
  17. 庆大霉素
  18. Tissue-Tek ® O.C.T.化合物(SAKURA FINETEK,目录号:4583)
  19. Tissue-Tek ® Cryomold(SAKURA FINETEK,目录号:4565)
  20. 异戊烷(Sigma-Aldrich,目录号:PHR1661)
  21. Triton X-100(Sigma-Aldrich,目录号:X100)
  22. 正常山羊血清(Agilent Technologies,目录号:X0907)
  23. 大鼠单克隆抗体NIMP-R14(1:200)(Abcam,目录号:ab2557)
  24. 在实验室中生产的PapG粘附素(1:200)中的合成肽的抗血清
  25. 与Alexa 488染料(A488; 495ext/519em nm)缀合的山羊抗大鼠免疫球蛋白(1:200)(Thermo Fisher Scientific,Invitrogen,Sup。,目录号:A-11006)
  26. 与Alexa 568染料(A568; 578ext/603em nm)缀合的山羊抗兔免疫球蛋白(1:200)(Thermo Fisher Scientific,Invitrogen,目录号:A-11011)
  27. 抗NLRP3抗体(Santa Cruz Biotechnology,目录号:sc-66846)
  28. 抗IL-1β抗体(Abcam,目录号:ab9722)
  29. 抗ASC抗体(Abcam,目录号:ab155970)
  30. DAPI(Sigma-Aldrich,目录号:D9542)
  31. VECTASHIELD安装介质(Vector Laboratories,目录号:H-1000)
  32. MIP-2量化试剂盒(R& D Systems,目录号:MM200)
  33. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  34. 胰蛋白酶大豆琼脂(TSA)(BD,Difco TM,目录号:236950)
  35. LB琼脂(BD,目录号:244520)
  36. 哥伦比亚5%血琼脂(BD,目录号:221263)
  37. 蔗糖(Sigma-Aldrich,目录号:84100)
  38. 氯胺酮/赛拉嗪溶液(见配方)
  39. 培养细菌培养基(见食谱)
    胰蛋白酶大豆琼脂平板,LB,哥伦比亚5%血琼脂
  40. 蔗糖溶液(参见食谱)
  41. 4%多聚甲醛(见食谱)

设备

  1. Burker室
  2. ELISA洗衣机
  3. 滴玻璃瓶气体麻醉
  4. 生物安全二级设施生物安全罩
  5. 弯曲镊子,剪刀
  6. 软聚乙烯导管(外径0.61 mm;粘土亚当斯)(BD,目录号:427401)
  7. Stomacher 80均质机(Seward medical,目录号:0080/000/EU)
  8. 冷冻机切片用切片刀C-35(FEATHER安全剃刀,型号:C-35)制成
  9. 荧光显微镜(Olympus,型号:AX60,配有滤光片组[激发/发射] 495ext/519em nm和578ext/603em nm)
  10. AxioCam ERc 5s相机(Zeiss,型号:AxioCam ERc 5s)
  11. Labsystems Multiskan PLUS阅读器(Analytical Instruments,Golden Valley,USA)

程序

  1. 感染模型(直接滴入细菌进膀胱)
    小鼠的感染方案最初由Hagberg等人描述。 (1983),并被Cirl等人进一步修改。 (2008),Yadav等人。 (2010)和Waldhuber等人。 (2016)。
    1. 为了取样尿液,将鼠标放置在笼子的金属网上。提起故事和后腿后,鼠标将牢牢地握在笼子上,使前腿伸展腹部并露出下侧。小鼠膀胱现在通过用食指在腹部区域进行温和的手动压缩(图1)清空,并将尿液收集在无菌微量离心管中(保存在冰上)。收集的尿液可用于定量TSA平板上的集落形成单位(CFU),用于检查炎性细胞(例如嗜中性粒细胞)或通过ELISA进行细胞因子分析。也可以在麻醉后收集尿液

      图1.小鼠中的尿液收集。将鼠标放在网状金属网上,并抓住尾巴抬起后腿。大多数小鼠自然排尿,尿液可以收集在无菌微量离心管中。有时使用食指,可以应用腹部轻微的压力来鼓励小鼠排尿。

    2. 使用异氟烷在滴玻璃罐中麻醉动物,或通过腹膜内注射氯胺酮 - 赛拉嗪混合物(Xylazin:6-8mg/kg体重,Ketamin:90-120mg/kg体重(正常体重8- 16周龄小鼠:19-23g)在0.9%NaCl中;稀释至终体积为100-200μl/小鼠以进行ip给药)
    3. 如果在麻醉之前没有完成,膀胱现在通过轻轻的腹部压缩而排空。一滴尿直接在尿道口处用校准回路(摄取10μl流体)直接捕获并铺展在血琼脂平板上,以确保小鼠未感染。
    4. 此后,立即将适用于1毫升结核菌素注射器(供应商BD)上的0.4×20毫米规格针的软质聚乙烯导管(0.61mm外径; Clay Adams)经尿道插入膀胱。
      注意:通过轻轻地抓住并拉出鼠标的阴蒂部分,尿道被拉直并允许进行快速和无创性的导管插入。在使用鼠标将导管插入背部之前,我们使用镊子拉动组织(图2)。


      图2.小鼠尿路感染模型 显示经尿道导管插入期间小鼠位置的图像。

    5. 我们在大多数实验中使用高剂量模型,并灌注0.1ml含有2×10 8或1×10 9 cfu/ml的细菌悬浮液。在一些实验中,我们将细菌数量降至2×10 5 /小鼠(低剂量模型),以研究IL-1β缺乏对细菌负担的影响。
    6. 注射后,立即取出导管,不进行进一步的操作。对照小鼠注射无菌PBS
    7. 然后该模型可用于研究膀胱和/或肾脏感染(图3)

      图3.小鼠上升的尿路感染小鼠用E感染。大肠杆菌CFT073通过膀胱内接种并在7天后处死。 A.未感染的对照小鼠显示具有光滑圆形轮廓的健康肾脏,而来自感染小鼠的肾脏显示严重的脓肿(箭头)形成(B)。

  2. 监测感染
    1. 通过连续的尿培养物监测小鼠尿道中细菌的形成和持续性,直至处死。
    2. 通过小鼠腹部(5小时,24小时和最多7天)的温和压力将尿液收集到无菌管中,并通过使用Burker腔的光学显微镜来定量中性粒细胞。
    3. 在胰蛋白酶大豆琼脂(TSA)板上定量培养尿液样品和连续稀释液(起始稀释1:100,然后在PBS中1:100,000,1:100,000)。 LB或哥伦比亚5%血琼脂平板是很好的选择。 TSA板不含抗生素,因为没有抗生素可以在实验菌株和任何可能的污染物之间进行选择。实验菌株和可能的污染细菌通过菌落形态分化。此外,假冒感染的对照也可能揭示可能的污染,但这些只是很少发生。
    4. 经无菌条件处死后,将膀胱和肾脏置于无菌条件下,放入含有5ml PBS(pH 7.2)的塑料袋中,并在Stomacher 80均质机中匀浆(100转/分钟,5分钟;仪器用70样品之间的酒精%)。
    5. 将样品置于冰上,并在TSA平板上连续稀释分析。细菌数量表示为每个整个组织的CFU数。
    6. 通过用庆大霉素(200μg/ml)杀死细胞外细菌并随后洗涤和匀浆剩余的器官来确定细胞内细菌的数量(Notes中的更多信息)。
    7. 随后,将血琼脂和TSA平板在37℃下孵育过夜,目视评价细菌菌落
  3. 感染后分析
    1. 组织学
      1. 肾脏和膀胱在处死后立即固定在1ml新鲜制备的4%PFA中,并在4℃温育过夜。
      2. 随后,将固定的组织在15%蔗糖(4℃/24小时)中孵育,并在25%冰冷的蔗糖(4℃/24小时)中洗涤。然后将组织嵌入O.C.T.复合并在-40℃的异戊烷中冷冻并储存在-80℃下进一步使用。冷冻切片(厚度5μm)用切片刀C-35制成,并安装在聚-L-赖氨酸涂层的玻璃片上并染色。
      3. 来自不同组小鼠的肾切片经双重染色,通过免疫组化进行分析
    2. 免疫组织化学
      1. 简言之,将组织切片在37℃下干燥15分钟,在PBS-0.2%Triton X-100(pH7.2)(2×10分钟/RT)中洗涤,并用PBS-0.2% Triton X-100加5%山羊正常实施的血清
      2. 随后将切片与NIMP-R14大鼠单克隆抗体(1:200)一起孵育,以检测PapG粘附素(1:200)(实验室中产生)中的合成肽CRPSAQSLEIKHGDL的嗜中性粒细胞和兔抗血清,以检测2-3 h。阴性对照仅由正常山羊血清(1:200)组成。抗体在PBS-0.2%Triton X-100加5%山羊正常血清中稀释
      3. 将肾切片(厚度5μm)用PBS洗涤(3×5分钟),浸泡在洗涤室中,与第二山羊抗大鼠免疫球蛋白(1:200)孵育(1小时/室温),与Alexa 488 (A488; 495ext/519emnm)和与Alexa 568(A568; 578ext/603emnm)缀合的荧光染料的次级山羊抗兔免疫球蛋白(1:200)。
      4. 在PBS(2×5分钟)洗涤后,用DAPI(0.05μM,1ml)将样品重新染色(3分钟/RT)以染色细胞核。
      5. 切片再次在PBS(10分钟)中洗涤,并用VECTASHIELD安装培养基安装并保持在黑暗中(4℃)。
      6. 通过荧光显微镜(Carl Zeiss)用200x放大倍率的AxioCam ERc 5s照相机分析切片。
      7. 通过免疫组织化学分析的替代目标包括:大肠杆菌,NLRP3,IL-1β和ASC。
    3. 细胞因子测量
      1. 在0.6小时,24小时和7天内收集尿样,并储存在-20℃
      2. 通过使用Mouse MIP-2定量试剂盒根据制造商的说明书通过ELISA定量尿中的MIP-2。尿液在样品缓冲液中稀释5倍
      3. 在Labsystems Multiskan PLUS读数器中,在450nm读取ELISA板。

数据分析

尿中的细菌负荷,尿细胞因子含量,尿中多形核白细胞数等参数,表示为至少3只单独小鼠的平均加/减标准差。通过Mann-Whitney秩和检验(Waldhuber等人,2016),Student's -test(Yadav等)比较了两组以上的统计学差异al。,2010)或Fisher精确检验(Cirl等人,2008)。多组分析通过单因素方差分析,事件Bonferroni的多重比较检验(Cirl等人,2008; Waldhuber等人,2016)分析。

笔记

注意:CFT073的细胞内检测

  1. 用CFT073经尿道感染小鼠。
  2. 感染后第3或7天,手术切除膀胱
  3. 通过在三角形中插入小剪刀将其切开,并进一步切割成顶点。
  4. 如果需要,用500μlPBS轻轻冲洗,除去附着的细菌和残留的尿液。
  5. 将剩余的切开的膀胱浸入庆大霉素溶液(200μg/ml,15分钟)中以杀死细胞外细菌。
  6. 然后将膀胱在PBS中冲洗两次,将膀胱转移到含有PBS /吐温(0.3%,v/v)和钢球(4mm不锈钢)的微量离心杯(2ml,Eppendorf)中。
  7. 将样品储存在冰上(在匀浆期间避免过热至关重要),直到用Retsch MM200组织匀浆器进行均化30秒。频率为30 /秒。将样品放回冰上并以连续稀释度进行分析。
  8. 将制备的材料铺在血琼脂平板上,并计数个体菌落。

食谱

  1. 氯胺酮/赛拉嗪溶液
    Xylazin:6-8 mg/kg体重
    氯胺酮:在0.9%NaCl中90-120mg/kg体重
    稀释至100-200μl/小鼠的终体积。管理
  2. 培养细菌的培养基
    1. 胰蛋白酶大豆琼脂板
      1. 在1升蒸馏水中加入40粒粉末
      2. 加热至沸腾,同时搅拌溶解粉末
      3. 在121℃高压灭菌15分钟以消毒
      4. 允许冷却至约50℃。倒入培养皿中,使其固化(厚度为5mm)
      1. 在1升蒸馏水中加入35克粉末
      2. 加热至沸腾,同时搅拌溶解粉末
      3. 在121℃高压灭菌15分钟以消毒
      4. 允许冷却至约50℃。倒入培养皿中,使其固化(厚度为5mm)
    2. 哥伦比亚5%血琼脂
      1. 这些琼脂板可从BD(见材料和试剂)可以使用
  3. 蔗糖溶液
    15%和25%蔗糖溶液
  4. 4%多聚甲醛(500 ml)
    1. 在玻璃烧杯中取400毫升PBS,并在通风罩内保持搅拌板(多聚甲醛有毒)
    2. 加热溶液,同时搅拌至约60°C
    3. 向溶液中加入20克多聚甲醛粉末
    4. 为了溶解多聚甲醛粉末,缓慢加入1N NaOH直至溶液变澄清。冷却溶液并过滤,并用PBS将体积调节至500ml
    5. 用稀盐酸调节pH至6.9
    6. 将溶液等分,并在-20°C下冷冻并在2个月内使用

致谢

TM由德意志民主共和国(DFG)授权MI471/6-1提供支持。该协议最初是由Hagberg等人开发的。 (1983),并在Cirl等人的出版物中进行了修改。 (2008),Yadav等人。 (2010)和Waldhuber等人。 (2016)。作者声明,没有可能影响其协议的设计和实施的利益冲突或竞争利益。

参考文献

  1. Cirl,C.,Wieser,A.,Yadav,M.,Duerr,S.,Schubert,S.,Fischer,H.,Stappert,D.,Wantia,N.,Rodriguez,N.,Wagner, Svanborg,C。和Miethke,T。(2008)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/18327267"target ="_ blank"通过独特的细菌Toll /白细胞介素-1受体结构域的蛋白质家族颠覆Toll样受体信号传导。 Nat Med 14(4):399-406。 >
  2. Datsenko,KA和Wanner,BL(2000)。  One - 使用PCR产物使大肠杆菌K-12中的染色体基因失活。 Proc Natl Acad Sci USA 97(12):6640-6645。 />
  3. Dielubanza,EJ和Schaeffer,AJ(2011)。尿液妇女的道路感染。 Med Clin North Am 95(1):27-41。
  4. 欧巡监察社,t。 (2015)。 ECDC发布2014年抗生素耐药性监测数据和欧洲的抗菌消费。欧洲监管 20(46)。
  5. Hagberg,L.,Engberg,I.,Freter,R.,Lam,J.,Olling,S。和Svanborg Eden,C.(1983)。  由源自人类大肠埃希氏大肠杆菌引起的小鼠升高,无阻塞的尿路感染。感染免疫 40(1):273-283。
  6. Waldhuber,A.,Puthia,M.,Wieser,A.,Cirl,C.,Durr,S.,Neumann-Pfeifer,S.,Albrecht,S.,Rommler,F.,Muller,T.,Zheng,Y 。,Schubert,S.,Gross,O.,Svanborg,C.和Miethke,T。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih E.gov/pubmed/27214553"target ="_ blank"> Uropathogenic大肠杆菌菌株CFT073破坏了NLRP3炎性细胞活化。 J Clin Invest 126(7):2425 -2436。
  7. Yadav,M.,Zhang,J.,Fischer,H.,Huang,W.,Lutay,N.,Cirl,C.,Lum,J.,Miethke,T。和Svanborg,C.(2010) TcpC抑制TIR域信令:MyD88依赖和独立效应在大肠杆菌毒力上。 PLoS Pathog 6(9):e1001120。
  8. Zhang,L.和Foxman,B.(2003)。大肠杆菌介导的尿路感染的分子流行病学。前面Biosci 8:e235-244。
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引用:Waldhuber, A., Puthia, M., Wieser, A., Svanborg, C. and Miethke, T. (2017). Analysis of the Virulence of Uropathogenic Escherichia coli Strain CFT073 in the Murine Urinary Tract. Bio-protocol 7(3): e2129. DOI: 10.21769/BioProtoc.2129.
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