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Protocol for HeLa Cells Infection with Escherichia coli Strains Producing Colibactin and Quantification of the Induced DNA-damage
用可产生大肠杆菌毒素的大肠埃希杆菌菌株感染HeLa细胞的实验方案及诱导产生DNA损伤的定量   

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Molecular Microbiology
Mar 2016

Abstract

Strains of Escherichia coli bearing the pks genomic island synthesize the genotoxin colibactin. Exposure of eukaryotic cells to E. coli producing colibactin induces DNA damages, ultimately leading to cell cycle arrest, senescence and death. Here we describe a simple method to demonstrate the genotoxicity of bacteria producing colibactin following a short infection of cultured mammalian cells with pks+ E. coli.

Keywords: Escherichia coli (大肠埃希杆菌), Enterobacteria (肠杆菌), Polyketide (聚酮), Colibactin (大肠杆菌毒素), Genotoxin (基因毒素), DNA damage (DNA损伤), Cell culture (细胞培养), Infection (感染)

Background

Colibactin is a genotoxin discovered in extra-intestinal pathogenic, commensal and probiotic strains of Escherichia coli (Nougayrede et al., 2006). Colibactin is also produced by other Enterobacteriaceae, including Klebsiella pneumonia, Enterobacter aerogenes and Citrobacter koseri (Putze et al., 2009). Colibactin is a polyketide/non-ribosomal peptide hybrid compound, synthesized by a multi-enzymatic machinery, consisting of polyketide and nonribosomal peptide synthases (PKS and NRPS), tailoring and maturation enzymes, and an efflux pump (for a review: Taieb et al., 2016). This synthesis machinery is encoded on a 52 kb genomic locus, the ‘pks’ island. Colibactin induces DNA damages in eukaryotic cells infected with pks+ bacteria. The genotoxic effect induced by colibactin requires a direct contact of live pks+ bacteria with the eukaryotic cells. Indeed, no genotoxic effect is observed with killed bacteria, or with bacterial supernatants or lysates. Thus, to demonstrate the genotoxicity of colibactin producing E. coli, cultured mammalian cells (such as HeLa cells) are infected during 4 h with live pks+ bacteria. The dose of colibactin delivered to the cells varies with the number of infecting bacteria per cell (multiplicity of infection, or MOI). At the end of the 4 h infection, bacterial growth is monitored by optical density measurement (OD600 nm). Then, the cells are washed to remove the bacteria and further incubated three days with antibiotics, to allow the development of the cytopathic phenotype associated with the DNA damage. The cellular DNA damage response results in proliferation (cell cycle) arrest, cell death and senescence (Secher et al., 2013). The microscopic observation of the cell morphology reveals the cellular response to the genotoxic insult, with reduced cell numbers and a striking giant cells phenotype (called megalocytosis) due to the cell cycle arrest and cellular senescence. The genotoxic effect can be quantified by staining the cells with methylene blue, extracting the dye and measuring the optical density at 660 nm (De Rycke et al., 1996).

Materials and Reagents

  1. Tissue culture plate 96 wells flat bottom (P96) (Corning, Falcon®, catalog number: 353072 or equivalent)
  2. Culture flask 250 ml, 75 cm2 (Corning, Falcon®, catalog number: 353136 or equivalent)
  3. Paper towel
  4. HeLa cells (ATCC, catalog number: CCL-2 )
  5. pks+ Escherichia coli strain (stored in LB 20% glycerol at -80 °C). Strains typically used as positive controls in the authors’ laboratory are probiotic strain Nissle 1917 or the commensal mouse strain NC101
  6. Lennox L broth base (LB medium) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12780029 )
  7. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  8. Dulbecco’s modified Eagle medium (DMEM) with 25 mM HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 42430 )
  9. Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H8264 )
  10. Dulbecco’s modified Eagle medium (DMEM), high glucose, GlutaMax Supplement, pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 31966021 )
  11. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 , or equivalent)
  12. Non-essential amino acids solution (NEAA) 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 11140035 )
  13. Gentamicin solution 50 mg/ml (Sigma-Aldrich, catalog number: G1397 )
  14. Paraformaldehyde (PFA) 20% (Electron Microscopy Sciences, catalog number: 15713 )
  15. Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537 )
  16. 10x PBS (Sigma-Aldrich, catalog number: D1408 )
  17. Methylene blue (RAL DIAGNOSTICS, catalog number: 310950 )
  18. Tris-HCl 1 M pH 8.5 (Teknova, catalog number: T1085 )
  19. Hydrochloric acid (HCl) 1 M (Merck, catalog number: 109057 )
  20. HeLa cell culture medium (see Recipes)
  21. Fixation solution (see Recipes)
  22. Methylene blue staining solution (see Recipes)
  23. Methylene blue wash buffer (see Recipes)
  24. Methylene blue extraction solution (see Recipes)

Equipment

  1. Micropipette Research Plus 0.1-10 µl (Dutscher, Eppendorf, catalog number: 035602 )
  2. Micropipette PIPETMAN G 20-200 µl (Dutscher, Gilson, catalog number: 066811 )
  3. Multichannel micropipette 30-300 µl (Dutscher, Finnpipette, catalog number: 050667N )
  4. Incubator for bacterial culture, with shaking (Eppendorf, New BrunswickTM, model: Innova® 42 )
  5. CO2 incubator for cell culture (Forma Scientific)
  6. Colorimeter to measure the absorbance at 600 nm of bacterial cultures (biochrom, model: WPA CO7500 )
  7. Microplate reader for absorbance measurement at 600 and 660 nm (TECAN Infinite Pro)
  8. Vortex (Dutscher, model: Vortex-Genie 2 )
  9. Inverted microscope (Olympus, model: CKX31 )
  10. Biological safety cabinets (Thermo Fisher Scientific, Thermo ScientificTM, model: MSC-AdvantageTM Class II )
  11. Chemical safety hood to handle the fixative

Procedure

  1. Day 1
    1. Seed a 3 ml LB culture from a -80 °C glycerol stock or a colony on an LB agar plate, and grow overnight (16-24 h) at 37 °C with shaking (240 RPM).
    2. Inoculate in a tissue culture 96 wells (P96) microplate, 5 x 103 HeLa cells in each well in 100 μl per well of complete cell culture medium (DMEM 10% FBS 1% NEAA 50 μg/ml gentamicin, see Recipes). Grow for 24 h in a 37 °C 5% CO2 humidified incubator. See the Data analysis section for the number of strains/conditions that can be tested per P96.

  2. Day 2
    1. Bacteria culture
      Inoculate into 9.5 ml pre-warmed (37 °C) DMEM 25 mM HEPES in a tube with 500 μl of the bacterial overnight LB culture. Grow at 37 °C with shaking (240 RPM) to reach OD600 nm = 0.4 to 0.5 (about 2 h). Determine the number of bacteria per ml by measuring the absorbance OD600 nm of 1 ml of the culture. 1 unit of OD600 nm corresponds to 5 x 108 bacteria/ml (typical value for E. coli with a WPA colourwave CO 7500 colorimeter).
    2. HeLa cell washing
      Meanwhile, wash cautiously the HeLa cells 3 times with 100 µl of warm (37 °C) HBSS then add 100 µl per well of DMEM 25 mM HEPES. For row B, add 200 µl instead of 100 µl (see Figure 1).


      Figure 1. Example of plate infection scheme. For dose/effect examination, bacteria are inoculated to HeLa cells at various multiplicity of infection (MOI, i.e., the number of bacteria per cell at the onset of the infection). Typically, bacteria are inoculated in row B with 200 µl per well (preferentially in duplicate or triplicate) then serially diluted (2-fold as shown) to achieve an infectious dose-effect. The final interaction medium volume is 100 µl. Peripheral wells should not be used to avoid edge effects that alter HeLa cell growth and response.

    3. HeLa cells infection
      Inoculate the cells with the bacteria, starting in row B, to achieve the maximum required number of bacteria per cell (multiplicity of infection, MOI). For dose/effect examination, prepare serial two-fold dilutions from row B to G, by transferring between each row 100 µl (see Figure 1). Typically, the highest MOI is 200 in first row B, and the MOI in the last row G is 6.25. Include on the plate a positive (bacteria synthetizing colibactin) and a negative control (bacteria not synthetizing colibactin). The plate is incubated at 37 °C with 5% CO2 for 4 h. Measure the absorbance at 600 nm with a microplate reader to monitor bacterial growth.
    4. Post-infection
      Wash the cells at least three times with 100 µl of pre-warmed (37 °C) HBSS. Check under an inverted microscope whether most of the bacteria are removed. Perform additional washes if required. Then, 100 µl of complete medium (DMEM GlutaMax, 10% FBS, 1% NEAA) supplemented with 200 µg/ml of gentamicin is added to each well. Incubate the P96 at 37 °C with 5% CO2 for 3 days.

  3. Day 3-4
    Check the cells under an inverted microscope every day for bacterial overgrowth, replace with fresh medium supplemented with gentamicin if required.

  4. Day 5
    1. Cell fixation
      The plate is rinsed two times with PBS. Under a chemical safety hood proceed to fixation with 100 µl of fixation solution (PBS 4% formaldehyde, see Recipes) for 20 min. Throw out the fixative in a toxic waste container, and rinse 2 times with PBS then once with methylene blue wash buffer (see Recipes).
    2. Methylene blue coloration
      Stain with 100 µl/well of methylene blue staining solution (see Recipes) for 1 h. Rinse several times with methylene blue wash buffer (see Recipes), tapping the plate between each wash on a paper towel to remove all the liquid. Repeat until no more blue stain is visible on the paper towel. Air dry overnight at room temperature or a few hours at 37 °C.
    3. Microscopic observation
      Observe the cells with an inverted microscope. Control cells should be confluent with a normal cellular morphology. In contrast, cells infected with high MOI of E. coli producing colibactin (MOI around 100) should exhibit a cytopathic phenotype characterized by low numbers of cells, megalocytosis (giant cells) and signs of cell death (apoptotic bodies, cell debris) (see Figure 2). At lower MOI of E. coli producing colibactin (around MOI 25), the phenotype is less marked, and islets of normal cells (that were able to repair the moderate DNA damage and resumed proliferation) should be visible. At MOI < 12 the cells are similar to control cells.


      Figure 2. Megalocytosis phenotype observed three days after a 4 h exposure of HeLa cells to a pks+ E. coli strain at a multiplicity of infection (MOI) of 12 to 100 bacteria per cell. The cells were stained with methylene blue and photographed with a 20x objective. Scale bars = 25 µm.

    4. Genotoxicity quantification
      The methylene blue is extracted in 100 µl per wells of methylene blue extraction solution, 15 min under agitation. Transfer 75 µl to a new plate and measure the absorbance at 660 nm with a microplate reader. Prepare a graph with OD660 nm in function of the MOI and the strain, with control strains as references. The more the strain is genotoxic, the less coloration will be measured, due to cell growth inhibition, senescence (megalocytosis) and cell death.

Data analysis

The cells should be first examined visually under the microscope (Figure 2), then by quantifying the methylene blue (Figure 3). Quantify the stain to measure the protein content in the cell layer, and thus represent the global cell viability inversely correlated to the genotoxicity. We suggest testing each strain at least in technical duplicate wells for each MOI tested. Thus, five independent conditions (strains) with a dose-effect can be tested per plate. If a statistical analysis is required, perform at least three independent experiments to obtain a biological triplicate, and compare the groups by 1-way ANOVA after log-transformation of the data.


Figure 3. Quantification of methylene blue extracted from HeLa cells 3 days after a 4 h exposition to various MOI of pks+ E. coli, the isogenic clbH mutant (that does not produce the genotoxin), or the clbH mutant complemented with pClbH plasmid. The percentage of methylene blue staining relative to control was calculated as the mean optical density in triplicate wells (from one experiment) divided by that in control (untreated) wells. The data can be plotted in function of the multiplicity of infection (MOI: number of infecting bacteria per cultured cell at the onset of infection), or in function of the bacterial growth at the end of the infection (as measured by optical density at 600 nm in the interaction wells). The error bars shown here represent the standard deviation of the mean in the triplicate wells.

Notes

All the HeLa cells washes should be performed with caution to avoid damaging or removing the cells from the monolayer; aspirate and add the medium slowly (about 1-2 sec).

Recipes

  1. HeLa cell culture medium (complete medium)
    500 ml DMEM high glucose with pyruvate and GlutaMax
    55 ml fetal bovine serum
    5.5 ml NEAA
    50 µg/ml gentamicin
  2. Fixation solution, 1x PBS, 4% PFA
    5 ml 20% PFA stock solution
    5 ml 10x PBS
    40 ml distilled H2O
    Aliquot and store at -20 °C
  3. Methylene blue staining solution
    5 g methylene blue
    500 ml 0.01 M Tris pH 8.5 (Methylene blue wash buffer)
    Mix during 3-4 h
    Store at room temperature
  4. Methylene blue wash buffer
    5 ml 1 M Tris pH 8.5
    495 ml distilled H2O
  5. Methylene blue extraction solution
    50 ml 1 M HCl
    450 ml distilled H2O

Acknowledgments

This protocol is based on previously published cultured cell infection method with pks+ E. coli (De Rycke et al., 1996; Nougayrede et al., 2006). This work was supported by the French Institut National du Cancer (grant INCAPLBIO13-123).

References

  1. De Rycke, J., Mazars, P., Nougayrede, J. P., Tasca, C., Boury, M., Herault, F., Valette, A. and Oswald, E. (1996). Mitotic block and delayed lethality in HeLa epithelial cells exposed to Escherichia coli BM2-1 producing cytotoxic necrotizing factor type 1. Infect Immun 64(5): 1694-1705.
  2. Nougayrede, J. P., Homburg, S., Taieb, F., Boury, M., Brzuszkiewicz, E., Gottschalk, G., Buchrieser, C., Hacker, J., Dobrindt, U. and Oswald, E. (2006). Escherichia coli induces DNA double-strand breaks in eukaryotic cells. Science 313(5788): 848-851.
  3. Putze, J., Hennequin, C., Nougayrede, J. P., Zhang, W., Homburg, S., Karch, H., Bringer, M. A., Fayolle, C., Carniel, E., Rabsch, W., Oelschlaeger, T. A., Oswald, E., Forestier, C., Hacker, J. and Dobrindt, U. (2009). Genetic structure and distribution of the colibactin genomic island among members of the family Enterobacteriaceae. Infect Immun 77(11): 4696-4703.
  4. Secher, T., Samba-Louaka, A., Oswald, E. and Nougayrede, J. P. (2013). Escherichia coli producing colibactin triggers premature and transmissible senescence in mammalian cells. PLoS One 8(10): e77157.
  5. Taieb, F., Petit, C., Nougayrede, J. P. and Oswald, E. (2016). The enterobacterial genotoxins: cytolethal distending toxin and colibactin. EcoSal Plus 7(1).

简介

具有pks基因组岛的大肠杆菌菌株合成基因毒素大肠杆菌素。 将真核细胞暴露于产生大肠杆菌的大肠杆菌诱导DNA损伤,最终导致细胞周期停滞,衰老和死亡。 在这里,我们描述了一种简单的方法来证明在用pks +大肠杆菌培养的哺乳动物细胞的短时间感染后,产生大肠杆菌素的细菌的遗传毒性。
【背景】大肠杆菌素是在大肠杆菌的肠外致病,共生和益生菌菌株中发现的基因毒素(Nougayrede等,2006)。大肠杆菌素也由其他肠杆菌科产生,包括肺炎克雷伯杆菌,产气肠杆菌和柠檬酸杆菌(Putze等人,2009)。大肠杆菌素是通过由多酮酶和非核糖体肽合酶(PKS和NRPS),定制和成熟酶以及外排泵组成的多酶机械合成的聚酮化合物/非核糖体肽杂化化合物(综述:Taieb等人。,2016)。该合成机制编码在52kb的基因组,即'pks'岛上。 Colibactin在感染pks +细菌的真核细胞中诱导DNA损伤。由大肠杆菌素诱导的遗传毒性作用需要活的pks +细菌与真核细胞的直接接触。事实上,杀死的细菌或细菌上清液或裂解物没有观察到遗传毒性作用。因此,为了证明产生大肠杆菌的大肠杆菌的基因毒性,培养的哺乳动物细胞(例如HeLa细胞)在4小时内用活的pks +细菌感染。递送至细胞的大肠杆菌素的剂量随每细胞感染细菌的数量而变化(感染的多重性或MOI)。在4h感染结束时,通过光密度测量(OD600nm)监测细菌生长。然后,洗涤​​细胞以除去细菌,并用抗生素进一步培养三天,以允许与DNA损伤相关的细胞病变表型的发展。细胞DNA损伤反应导致增殖(细胞周期)停滞,细胞死亡和衰老(Secher等,2013)。细胞形态的显微镜观察显示细胞对遗传毒性损伤的反应,由于细胞周期阻滞和细胞衰老,细胞数量减少和引人注目的巨细胞表型(称为巨细胞增多症)。可以通过用亚甲蓝染色细胞,提取染料并测量660nm处的光密度来定量遗传毒性作用(De Rycke等,1996)。

关键字:大肠埃希杆菌, 肠杆菌, 聚酮, 大肠杆菌毒素, 基因毒素, DNA损伤, 细胞培养, 感染

材料和试剂

  1. 组织培养板96孔平底(P96)(Corning,Falcon ,目录号:353072或等效物)
  2. 培养瓶250ml,75cm 2(Corning,Falcon ,目录号:353136或等同物)
  3. 纸巾
  4. HeLa细胞(ATCC,目录号:CCL-2)
  5. 大肠杆菌菌株(-80℃下保存在LB 20%甘油中) 在作者实验室中通常用作阳性对照的菌株是益生菌菌株Nissle 1917或共生小鼠株NC101
  6. Lennox L培养基(LB培养基)(Thermo Fisher Scientific,Invitrogen TM,目录号:12780029)
  7. 甘油(Sigma-Aldrich,目录号:G5516)
  8. 具有25mM HEPES(Thermo Fisher Scientific,Gibco TM,目录号:42430)的Dulbecco改良Eagle培养基(DMEM),目录号:42430)
  9. 汉克斯平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:H8264)
  10. Dulbecco改良的Eagle培养基(DMEM),高葡萄糖,GlutaMax Supplement,丙酮酸(Thermo Fisher Scientific,Gibco TM,目录号:31966021)
  11. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106或等同物)
  12. 非必需氨基酸溶液(NEAA)100x(Thermo Fisher Scientific,Gibco TM,目录号:11140035)
  13. 庆大霉素溶液50mg / ml(Sigma-Aldrich,目录号:G1397)
  14. 二甲醛(PFA)20%(电子显微镜科学,目录号:15713)
  15. Dulbecco的磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8537)
  16. 10x PBS(Sigma-Aldrich,目录号:D1408)
  17. 亚甲基蓝(RAL DIAGNOSTICS,目录号:310950)
  18. Tris-HCl 1 M pH 8.5(Teknova,目录号:T1085)
  19. 盐酸(HCl)1 M(Merck,目录号:109057)
  20. HeLa细胞培养基(参见食谱)
  21. 固定溶液(见配方)
  22. 亚麻蓝染色溶液(见食谱)
  23. 亚麻蓝洗涤缓冲液(见配方)
  24. 亚甲基蓝提取液(见配方)

设备

  1. Micropipette Research Plus 0.1-10μl(Dutscher,Eppendorf,目录号:035602)
  2. Micropipette PIPETMAN G 20-200μl(Dutscher,Gilson,目录号:066811)
  3. 多通道微量移液管30-300μl(Dutscher,Finnipipette,目录号:050667N)
  4. 用于细菌培养的培养箱,摇动(Eppendorf,New Brunswick TM,型号:Innova 42)
  5. CO 2细胞培养箱(Forma Scientific)
  6. 比色计测量细菌培养物600nm处的吸光度(biochrom,型号:WPA CO7500)
  7. 用于600和660 nm吸光度测量的微孔板读数器(TECAN Infinite Pro)
  8. 涡旋(Dutscher,型号:Vortex-Genie 2)
  9. 倒置显微镜(Olympus,型号:CKX31)
  10. 生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM ,型号:MSC-Advantage TM II类)
  11. 化学安全罩来处理固定剂

程序

  1. 第1天
    1. 在-80℃甘油储备液或LB琼脂平板上的菌落上种3ml LB培养物,并在37℃振荡(240RPM)下生长过夜(16-24小时)。
    2. 接种在组织培养物中,每孔100μl/孔的完全细胞培养基(DMEM 10%FBS 1%NEAA50μg/ ml)中的96孔(P96)微孔板,5×10 3 HeLa细胞庆大霉素,见食谱)。在37℃5%CO 2加湿培养箱中生长24小时。有关可按P96测试的应变/条件数量,请参见数据分析部分。

  2. 第二天
    1. 细菌培养
      在具有500μl细菌过夜LB培养物的管中接种到9.5ml预温热(37℃)DMEM 25mM HEPES中。在37℃下振荡(240RPM)生长达到OD 600nm = 0.4至0.5(约2小时)。通过测量1ml培养物的吸光度OD 600nm来确定每ml细菌数。 1单位OD 600nm对应于5×10 8细菌/ ml(大肠杆菌的典型值),其具有WPA colourwave CO 7500色度计)。
    2. HeLa细胞洗涤
      同时,用100μl温热(37℃)HBSS小心地洗涤HeLa细胞3次,然后加入每孔100μl的DMEM 25mM HEPES。对于B行,加入200μl而不是100μl(见图1)

      图1.板感染方案示例。 对于剂量/效应检查,细菌以不同的感染复数(MOI,即,感染开始时每个细胞的细菌数)接种到HeLa细胞中。通常,将细菌以每孔200μl(优选一式两份或一式三份)接种在B行中,然后连续稀释(如图所示2倍)以达到感染剂量效应。最终的相互作用介质体积为100μl。不应使用外围孔来避免改变HeLa细胞生长和反应的边缘效应
    3. HeLa细胞感染
      从B列开始接种细菌,以达到每个细胞所需的最大细菌数(感染的多重性,MOI)。对于剂量/效应检查,通过在每行100μl之间转移(参见图1),从行B至G进行连续两倍稀释。通常,第一行B中最高MOI为200,最后一行G中的MOI为6.25。在平板上包括阳性(细菌合成大肠杆菌素)和阴性对照(细菌不合成大肠杆菌素)。将板在37℃下与5%CO 2孵育4小时。使用酶标仪测量600 nm处的吸光度,以监测细菌生长。
    4. 感染后
      用100μl预热(37℃)HBSS将细胞洗涤至少三次。在倒置显微镜下检查大多数细菌是否被去除。如果需要,请执行附加洗涤。然后,向每个孔中加入100μl补充有200μg/ ml庆大霉素的完全培养基(DMEM GlutaMax,10%FBS,1%NEAA)。将P96在37℃下与5%CO 2 孵育3天。

  3. 第3-4天
    每天在倒置显微镜下检查细胞是否有细菌过度生长,如果需要,请用补充有庆大霉素的新鲜培养基替换。

  4. 第5天
    1. 细胞固定
      用PBS冲洗板两次。在化学安全罩下,用100μl固定液(PBS 4%甲醛,参见食谱)固定20分钟。扔出有毒废物容器中的固定剂,然后用PBS冲洗2次,然后用亚甲基蓝洗涤缓冲液一次(参见食谱)。
    2. 亚甲基蓝着色
      用100μl/孔的亚甲蓝染色溶液(参见食谱)染色1小时。用亚甲基蓝洗涤缓冲液冲洗几次(参见食谱),在纸巾上每次洗涤之间敲打板,以清除所有液体。重复一遍,直到纸巾上没有更多的蓝色染色。在室温下空气干燥过夜或在37℃下几小时。
    3. 显微镜观察
      用倒置显微镜观察细胞。对照细胞应与正常细胞形态融合。相比之下,感染了高MOI的E细胞。产生大肠杆菌的大肠杆菌(约100个的MOI)应该显示特征在于细胞数量低,巨细胞(巨细胞)和细胞死亡的迹象(凋亡小体,细胞碎片)的细胞病变表型(参见图2)。在E的较低MOI。大肠杆菌产生大肠杆菌素(围绕MOI 25),表型不太明显,正常细胞的胰岛(能够修复中度DNA损伤并恢复增殖)应该是可见的。在MOI < 12细胞与对照细胞相似

      图2.在HeLa细胞暴露于 p 4小时后3天观察到的巨细胞增生表型 E。大肠杆菌 菌株在感染复数(MOI)为12〜100个细胞/细胞。细胞用亚甲基蓝染色并用20倍物镜拍照。刻度棒=25μm。

    4. 基因毒性定量
      亚甲基蓝在100μl/孔的亚甲蓝萃取溶液中萃取,在搅拌下15分钟。将75μl转移到新板上,用微量酶标仪测量660 nm处的吸光度。在MOI和菌株的功能下制备具有OD <660nm 的图,其中对照菌株作为参考。由于细胞生长抑制,衰老(巨噬细胞增多)和细胞死亡,应变是基因毒性越多,着色越少。

数据分析

首先应在显微镜下观察细胞(图2),然后定量亚甲蓝(图3)。定量染色以测量细胞层中的蛋白质含量,从而代表与遗传毒性反相关的全局细胞活力。我们建议至少在每个测试的MOI的技术重复孔中测试每个菌株。因此,可以每个板测试具有剂量效应的五个独立条件(菌株)。如果需要统计分析,执行至少三次独立实验以获得生物一式三份,并在数据对数转换后通过单因素方差分析进行比较。


图3.在4小时展开后3天从HeLa细胞提取的亚甲基蓝的量化到各种MOI + 大肠杆菌 ,等同性 突变体(不产生基因毒素)或与pClbH质粒互补的 clbH 突变体。 相对于对照的亚甲基蓝染色的百分比计算为一式三份的孔中的平均光密度(来自一个实验)除以对照(未处理)孔中的平均光密度。数据可以以多重感染(MOI:感染发病开始时每培养细胞的感染细菌数)或感染结束时细菌生长的功能(通过光密度600测量) nm)。这里显示的误差条表示三重孔中平均值的标准偏差。

笔记

所有的HeLa细胞洗涤应谨慎进行,以免损伤或从单层中除去细胞;吸出并缓慢加入培养基(约1-2秒)。

食谱

  1. HeLa细胞培养基(完全培养基)
    500毫升DMEM高葡萄糖与丙酮酸和GlutaMax
    55毫升胎牛血清
    5.5 ml NEAA
    50μg/ ml庆大霉素
  2. 固定液,1x PBS,4%PFA
    5 ml 20%PFA储备溶液
    5 ml 10x PBS
    40ml蒸馏的H 2 O O
    等分并储存于-20°C
  3. 亚麻蓝染色液
    5克亚甲蓝
    500ml 0.01M Tris pH 8.5(亚甲基蓝洗涤缓冲液)
    3-4小时内混合
    在室温下存放
  4. 亚麻蓝洗涤缓冲液
    5 ml 1 M Tris pH 8.5
    495ml蒸馏的H 2 O O
  5. 亚甲基蓝提取液
    50ml 1 M HCl
    450ml蒸馏的H 2 O O

致谢

该方案基于以前公布的具有 大肠杆菌的培养细胞感染方法(De Rycke >等人,1996; Nougayrede等人,2006)。这项工作得到法国国家癌症研究所的支持(授予INCAPLBIO13-123)。

参考

  1. De Rycke,J.,Mazars,P.,Nougayrede,JP,Tasca,C.,Boury,M.,Herault,F.,Valette,A.and Oswald,E.(1996)。&lt; a class = ke-insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/8613380“target =”_ blank“>暴露于大肠杆菌的HeLa上皮细胞中的有丝分裂阻滞和延迟致死率产生细胞毒性坏死因子1型的BM2-1。 64(5):1694-1705。
  2. Nougayrede,JP,Homburg,S.,Taieb,F.,Boury,M.,Brzuszkiewicz,E.,Gottschalk,G.,Buchrieser,C.,Hacker,J.,Dobrindt,U.and Oswald,E。(2006 )。大肠杆菌诱导DNA双链断裂在真核细胞中。科学 313(5788):848-851。
  3. Putze,J.,Hennequin,C.,Nougayrede,JP,Zhang,W.,Homburg,S.,Karch,H.,Bringer,MA,Fayolle,C.,Carniel,E.,Rabsch,W.,Oelschlaeger, TA,Oswald,E.,Forestier,C.,Hacker,J.and Dobrindt,U。(2009)。肠杆菌科家族中大肠杆菌素基因组岛的遗传结构和分布。 (11):4696-4703。
  4. Secher,T.,Samba-Louaka,A.,Oswald,E.和Nougayrede,JP(2013)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih。 gov / pubmed / 24116215“target =”_ blank“>产生大肠杆菌的大肠杆菌触发哺乳动物细胞中过早和可传播的衰老。 8(10): e77157。
  5. Taieb,F.,Petit,C.,Nougayrede,JP和Oswald,E.(2016)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/ pubmed / 27419387“target =”_ blank“>肠细菌基因毒素:细胞致死性扩张毒素和大肠杆菌素。 EcoSal Plus 7(1)。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bossuet-Greif, N., Belloy, M., Boury, M., Oswald, E. and Nougayrede, J. (2017). Protocol for HeLa Cells Infection with Escherichia coli Strains Producing Colibactin and Quantification of the Induced DNA-damage. Bio-protocol 7(16): e2520. DOI: 10.21769/BioProtoc.2520.
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