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Intracellular Macrophage Infections with E. coli under Nitrosative Stress

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Molecular Microbiology
Mar 2012



Escherichia coli (E. coli) produces disseminated infections of the urinary tract, blood, and central nervous system where it encounters professional phagocytes such as macrophages, which utilize reactive nitrogen intermediates (RNI) to arrest bacteria. In vitro, extraintestinal pathogenic E. coli (ExPEC) can survive within bone marrow-derived macrophages for greater than 24 h post-infection within a LAMP1+ vesicular compartment, and ExPEC strains, in particular, are better adapted to intracellular macrophage survival than commensal strains (Bokil et al., 2011). This protocol details an intracellular murine macrophage-like cell infection, including modulation of the host nitrosative stress response, to model this host-pathogen interaction in vitro. To accomplish this, RAW 264.7 murine macrophage-like cells are pre-incubated with either L-arginine, an NO precursor, or IFNγ to yield a high nitric oxide (NO) physiological state, or L-NAME, an inducible NO synthase (iNOS)-specific inhibitor, to yield a low NO physiological state. This protocol has been successfully utilized to assess the contribution of a novel ExPEC regulator to intracellular survival and the nitrosative stress response during macrophage infections (Bateman and Seed, 2012), but can be adapted for use with a variety of E. coli strains or isogenic deletions.

Materials and Reagents

  1. E. coli isolate UTI89 (Note 1) (or other ExPEC strain)
  2. Luria-Bertani broth (LB) culture medium
  3. RAW 264.7 mouse macrophage-like cells (Note 2)
  4. DMEM [high glucose (HG), 4,500 mg/L]
  5. Fetal bovine serum (FBS) (Sigma-Aldrich)
  6. 1x Phosphate buffered saline (PBS)
  7. Gentamicin (50 mg/ml stock)
  8. L-arginine
  9. L-NAME (Cayman Chemical Company)
  10. IFNγ, recombinant mouse (EMD Millipore, catalog number: IF005 )
  11. 1% Triton-X 100 (see Recipes)
  12. RAW 264.7 media (DMEM + 10% FBS) (see Recipes)
  13. 1 M L-arginine stock solution (see Recipes)
  14. 1M L-NAME stock Solution (see Recipes)


  1. 24 well tissue culture trays
  2. Table-top swinging bucket centrifuge with microtray adaptors
  3. 37 °C incubator with aeration (for liquid bacterial cultures)
  4. 37 °C incubator with 5% CO2 [for macrophage cultures (Note 3)]
  5. Spectrophotometer
  6. 0.2 micron filter


  1. Day 1
    1. Start 5 ml overnight (16-18 h) culture of E. coli isolate UTI89 (or other ExPEC strain) in LB at 37 °C with aeration.
    2. Seed RAW 264.7 cells into 24 well trays (Note 4) at a density of 1 x 105 cells/well and allow to adhere and grow to confluence for 36-42 h prior to infection. For general maintenance, RAW 264.7 murine macrophages are routinely grown in DMEM (HG) + 10% FBS and sub-cultured 1:4 approximately every 3 days using a cell scraper to detach adherent cells for sub-culture.

  2. Day 2
    1. Back-dilute bacterial culture 1:100 into 10 ml fresh LB media, and grow statically at 37 °C for 18-24 h to induce expression of type 1 pili adherence factor. This can be accomplished in an Erlenmeyer flask or sterile petri dish.
    2. To stimulate NO expression and the production of cytokines, pre-treat several wells of RAW 264.7 cells with 1 ng/ml IFNγ for 16-18 h (overnight) prior to infection (see diagram of 24-well tray below).

  3. Day 3
    1. 1 mM L-arginine is included in the media of a second group of RAW 264.7 cells for 1 h to pre-load cells for nitric oxide production upon stimulation with E. coli infection. L-arginine is a precursor to NO.
    2. A third group of RAW 264.7 cells is pre-treated with 1 mM of the iNOS-specific inhibitor L-NAME for 1 h prior to infection to inhibit NO production. L-arginine and L-NAME are subsequently included in the media for the duration of the experiments.
    3. Finally, a fourth group of RAW 264.7 cells is left untreated (naive) to serve as an un-stimulated control.
    4. Infect cells with E. coli by adding 10 μl of an OD600= 0.8 bacterial suspension (~1 x 107 colony forming units (CFU)7 or multiplicity of infection (MOI) of 10) in PBS to confluent cell monolayers (~1 x 106 cells/well) for all 4 treatment groups.
    5. Centrifuge plates in a swinging bucket clinical centrifuge with microtray adaptors at low speed to bring bacteria in contact with the cell monolayer (5 min at 1,000 rpm) and then incubated for 1 h at 37 °C with 5% CO2.
    6. Determine initial 1 h adherence/invasion for the various treatment groups by following steps m through p). This number will be used to normalize CFU/well at 24 h post-infection to account for any difference in initial adherence/invasion among the various treatment groups or bacterial strains (Note 6).
    7. For the remainder of the treatment groups, wash infected monolayers 3 times with 0.5 ml PBS (avoid touching the monolayer with pipette tips or directly applying the wash onto the monolayer as this may disturb the integrity of the monolayer. This step is to remove the majority of extracellular bacteria).
    8. Incubate for the remainder of the 24 h experiment using a step-down gentamicin protocol (100 μg/ml gentamicin in DMEM HG + 10% FBS for 2 h then exchange for media with 50 μg/ml gentamicin for 21 h) (Note 7).

  4. Day 4
    1. Wash cells 3 times with 0.5 ml PBS.
    2. Remove last wash and add 1 ml PBS + 0.1% Triton-X. Pipet vigorously to lyse cells. Minor foaming is acceptable.
    3. Determine intracellular bacterial burden by plating serial dilutions in LB (including appropriate antibiotics depending on bacterial strain) and incubating at 37 °C overnight.

  5. Day 5
    1. Count bacterial CFU on plates.
    2. Intracellular burden at 24 h post-infection for various treatment groups and/or bacterial strains should be normalized to the corresponding 1 h adherence/invasion counts. Bacterial burden for each strain and/or group can be plotted using your favorite graphing program (Excel, GraphPad, etc.). Unpaired, two-tailed Student’s t-tests can be used to determine statistical significance, which is defined as attaining p-values ≤ 0.05.

Treatment Groups and Controls


  1. UTI89 is a prototypic ExPEC cystitis isolate that was obtained from an adult patient with cystitis and has been well described in the literature (Mulvey, 1998). Other ExPEC isolates can be used as well.
  2. RAW 264.7 cells were purchased from the ATCC.
  3. Note that you will need to put bacteria-infected macrophages into your tissue culture incubator if you do not separate CO2-injected incubators for microbiology and sterile tissue culture work. If you have only a single incubator that is considered sterile, you may place your infected macrophage culture trays inside a plastic food container without the lid. Over the top, place an unfolded dry autoclaved paper towel and use a rubber band to seal it over the plastic food contained. Wipe the outside of the plastic contained with 70% ethanol and place the whole container in the tissue culture incubator. The paper towel will allow gas exchange of the cultures while preventing aerosols from escaping.
  4. The number of trays needed will depend on several factors. There are 4 treatment groups: untreated, L-NAME (inhibition of NO), L-arginine (high NO), and IFNγ (activation of macrophages leading to high NO and cytokine production). One set will be used to determine initial 1 h adherence/invasion and one set will be used for 24 h (or another time-point) intracellular infections. The diagram of treatment groups and controls is meant as a starting point only. Additional technical and biological replicates should be included to determine experimental variation.
  5. Prior to starting infections, researchers should determine bacterial CFU in a PBS suspension by serial dilution plating. This will give you an estimate for the amount of bacterial suspension to add per well. Serial dilutions of the bacterial inoculation should also be performed at every infection to determine the exact MOI for ach experiment. In parallel, use a hemocytometer to count cells in a confluent monolayer at the time of infection to give an accurate MOI.
  6. This is an important step to determine the intracellular survival after different treatments or in different strains. If initial adherence/invasion rates are different, subsequent intracellular survival rates at 24 h post-infection will be skewed. Proceed to step (m) for initial 1 h adherence/invasion. Continue through all steps for 24 h infections.
  7. IFNγ is not included in the media after the initiation of infections; however, L-arginine and L-NAME are included in the media for the duration of the experiments.


  1. RAW 264.7 media (DMEM + 10% FBS)
    450 ml DMEM (HG)
    50 ml FBS
    Filter sterilize through 0.2 micron filter unit. Make as much as need based on the number of bacterial strains tested and replicates.
  2. 1 M L-arginine stock solution
    0.871 g L-arginine
    5 ml dI H2O
    Resuspend and filter sterilize through 0.2 micron filter unit. Use at a working concentration of 1 mM or 1,000-fold dilution.
  3. 1 M L-NAME stock solution
    1.35 g L-NAME
    5 ml diH2O
    Resuspend and filter sterilize through 0.2 micron filter unit. Use at a working concentration of 1 mM or 1,000-fold dilution.
  4. 1% Triton-X 100
    1 ml Triton-X 100
    99 ml diH2O
    Mix well. Use 1 ml per well to resuspend and lyse RAW 264.7 cells with intracellular E. coli infection.


This protocol is adapted from and has been successfully utilized in Bateman and Seed (2012).


  1. Bateman, S. L. and Seed, P. C. (2012). Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli. Mol Microbiol 83(5): 908-925.
  2. Bokil, N. J., Totsika, M., Carey, A. J., Stacey, K. J., Hancock, V., Saunders, B. M., Ravasi, T., Ulett, G. C., Schembri, M. A. and Sweet, M. J. (2011). Intramacrophage survival of uropathogenic Escherichia coli: differences between diverse clinical isolates and between mouse and human macrophages. Immunobiology 216(11): 1164-1171.
  3. Mulvey, M. A., Lopez-Boado, Y. S., Wilson, C. L., Roth, R., Parks, W. C., Heuser, J. and Hultgren, S. J. (1998). Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science 282(5393): 1494-1497.


大肠杆菌(大肠杆菌)产生尿道,血液和中枢神经系统的扩散感染,其中它遇到专业吞噬细胞,例如巨噬细胞,其利用反应性氮中间体RNI)以阻止细菌。 体外,肠外致病性。大肠杆菌(ExPEC)可以在LAMP1 +囊泡区室内在感染后在骨髓来源的巨噬细胞中存活超过24小时,并且ExPEC菌株特别地比共生菌株更适合细胞内巨噬细胞存活(Bokil& em>等人,2011)。该协议详细描述了细胞内鼠类巨噬细胞样细胞感染,包括调节宿主亚硝基化应激反应,在体外模拟这种宿主 - 病原体相互作用。为了实现这一点,将RAW 264.7鼠巨噬细胞样细胞与L-精氨酸,NO前体或IFNγ预温育以产生高一氧化氮(NO)生理状态或L-NAME,诱导型NO合酶(iNOS )特异性抑制剂,产生低NO生理状态。该方案已经成功地用于评估新型ExPEC调节剂对巨噬细胞感染期间的细胞内存活和亚硝基化应激反应的贡献(Bateman和Seed,2012),但是可以适用于多种E。大肠杆菌菌株或同基因缺失。


  1. E。 大肠杆菌分离UTI89(注1)(或其他ExPEC株)
  2. Luria-Bertani肉汤(LB)培养基
  3. RAW 264.7小鼠巨噬细胞样细胞(注2)
  4. DMEM [高葡萄糖(HG),4,500mg/L]
  5. 胎牛血清(FBS)(Sigma-Aldrich)
  6. 1x磷酸盐缓冲盐水(PBS)
  7. 庆大霉素(50mg/ml母液)
  8. L-精氨酸
  9. L-NAME(Cayman Chemical Company)
  10. IFNγ,重组小鼠(EMD Millipore,目录号:IF005)
  11. 1%Triton-X 100(参见配方)
  12. RAW 264.7培养基(DMEM + 10%FBS)(参见配方)
  13. 1 M L-精氨酸储备液(见配方)
  14. 1M L-NAME储备溶液(见配方)


  1. 24孔组织培养皿
  2. 带微型板适配器的台式摇摆式离心机
  3. 37°C通气培养箱(用于液体细菌培养)
  4. 37℃具有5%CO 2的培养箱[用于巨噬细胞培养(注3)]
  5. 分光光度计
  6. 0.2微米过滤器


  1. 第1天
    1. 开始5ml过夜(16-18小时)的E培养。 大肠杆菌在37℃,通气的LB中分离UTI89(或其他ExPEC菌株)。
    2. 将种子RAW 264.7细胞以1×10 5个细胞/孔的密度接种到24孔盘(注释4)中,并允许在感染前粘附并生长至汇合36-42小时。 对于一般维持,RAW 264.7鼠巨噬细胞常规地在DMEM(HG)+ 10%FBS中生长,并且使用细胞刮刀大约每3天亚培养1:4以分离粘附细胞用于亚培养。

  2. 第2天
    1. 反向稀释细菌培养1:100到10ml新鲜LB培养基,并在37℃静态生长18-24 h诱导1型菌毛粘附因子的表达。 这可以在锥形瓶或无菌培养皿中完成
    2. 为了刺激NO表达和细胞因子的产生,在感染之前用1ng/mlIFNγ预处理几个孔的RAW 264.7细胞16-18小时(过夜)(参见下面的24孔板图)。 >

  3. 第3天
    1. 1mM L-精氨酸包含在第二组RAW 264.7细胞的培养基中1小时,以在用E刺激时预加载细胞用于一氧化氮产生。 大肠杆菌感染。 L-精氨酸是NO的前体
    2. 第三组RAW 264.7细胞用1mM的iNOS特异性抑制剂L-NAME预处理1小时,然后感染以抑制NO产生。 在实验期间,L-精氨酸和L-NAME随后包括在培养基中
    3. 最后,第四组RAW 264.7细胞未处理(幼稚的)以用作未刺激的对照。
    4. 用 E感染细胞。通过加入10μl的OD 600 = 0.8细菌悬浮液(〜1×10 7个集落形成单位(CFU) 7 >或多重感染(MOI)为10)在PBS中用于所有4个治疗组的铺满的细胞单层(〜1×10 6个细胞/孔)。
    5. 在具有微型板接头的摇摆式临床离心机中在低速下离心板以使细菌与细胞单层接触(在1,000rpm下5分钟),然后在37℃下用5%CO 2孵育1小时, sub>。
    6. 通过以下步骤m至p)确定各种治疗组的初始1小时粘附/侵袭。该数量将用于在感染后24小时使CFU /孔正常化,以解释各种治疗组或细菌菌株之间初始粘附/侵袭的任何差异(注6)。
    7. 对于剩余的治疗组,用0.5ml PBS洗涤感染的单层3次(避免用移液管吸头接触单层,或直接将洗涤施加到单层上,因为这可能干扰单层的完整性)。该步骤是除去多数的细胞外细菌)
    8. 使用降低的庆大霉素方案(100μg/ml庆大霉素在DMEM HG + 10%FBS中培养2小时,然后用含有50μg/ml庆大霉素的培养基更换21小时)孵育24小时实验的剩余部分(注释 7)。

  4. 第4天
    1. 用0.5ml PBS洗涤细胞3次。
    2. 取出最后一次洗涤,加入1ml PBS + 0.1%Triton-X。 吸管剧烈裂解细胞。 少量起泡是可接受的。
    3. 通过在LB中接种连续稀释液(包括取决于细菌菌株的合适的抗生素)并在37℃下孵育过夜来确定细胞内细菌负荷。

  5. 第5天
    1. 计数板上的细菌性CFU
    2. 各种治疗组和/或细菌菌株的感染后24小时的细胞内负荷应该归一化为相应的1小时粘附/侵袭计数。 可以使用您最喜欢的图形化程序(Excel,GraphPad,等)绘制每个菌株和/或组的细菌负荷。 不成对的双尾学生t检验可用于确定统计学显着性,其定义为达到p值≤0.05。



  1. UTI89是从膀胱炎的成年患者获得的原型ExPEC膀胱炎分离株,并且已经在文献中充分描述(Mulvey,1998)。也可以使用其他ExPEC分离物
  2. RAW 264.7细胞购自ATCC。
  3. 注意,如果你不分离CO 2 sub注射孵化器微生物学和无菌组织培养工作,你将需要把细菌感染的巨噬细胞进入你的组织培养孵化器。如果你只有一个被认为是无菌的孵化器,你可以把你感染的巨噬细胞培养托盘放在没有盖子的塑料食品容器内。在顶部,放置一个展开的干燥的高压灭菌的纸巾,并使用橡胶带密封它包含的塑料食品。擦拭含有70%乙醇的塑料的外部,并将整个容器在组织培养孵化器。纸巾将允许文化的气体交换,同时防止气溶胶逃逸
  4. 所需的托盘数量将取决于几个因素。有4个治疗组:未治疗的,L-NAME(抑制NO),L-精氨酸(高NO)和IFNγ(巨噬细胞的活化导致高NO和细胞因子 生产)。一组将用于确定初始1小时的粘附/侵袭,一组将用于24小时(或另一时间点)细胞内感染。治疗组和对照图仅作为起点。应包括额外的技术和生物学重复以确定实验变化
  5. 在开始感染之前,研究人员应通过连续稀释法测定PBS悬浮液中的细菌CFU。这将给您估计每孔添加的细菌悬浮液的量。还应在每次感染进行细菌接种的系列稀释以确定ach实验的确切MOI。同时,使用血细胞计数器在感染时计数汇合单层细胞以给出准确的MOI。
  6. 这是确定不同处理后或在不同菌株中的细胞内存活的重要步骤。如果初始粘附/侵袭率不同,则在感染后24小时的后续细胞内存活率将偏斜。对于初始1小时的粘附/侵入,进行步骤(m)。继续通过所有步骤24小时感染。
  7. 在感染开始后,IFNγ不包括在培养基中;然而,在实验期间,L-精氨酸和L-NAME包括在培养基中


  1. RAW 264.7培养基(DMEM + 10%FBS)
    450ml DMEM(HG)
    50ml FBS
    通过0.2微米过滤器过滤灭菌。 根据测试和复制的细菌菌株数量,根据需要进行制备。
  2. 1μML-精氨酸储备液
    0.871g L-精氨酸 5ml dI H 2 O·
    重悬和过滤灭菌通过0.2微米过滤单元。 在1 mM或1,000倍稀释的工作浓度下使用。
  3. 1 M L-NAME储备液
    1.35 g L-NAME
    5ml二H 2 O 2 / 重悬和过滤灭菌通过0.2微米过滤单元。 在1 mM或1,000倍稀释的工作浓度下使用。
  4. 1%Triton-X 100
    1ml Triton-X 100
    99ml diH 2 O x/v 混合好。 使用1毫升/孔重悬,并用细胞内的E裂解RAW 264.7细胞。 大肠杆菌感染




  1. Bateman,S.L.和Seed,P.C。(2012)。 亚硝化应激反应和细胞内巨噬细胞存活的表观遗传调节 肠外致病性大肠杆菌 。 Mol Microbiol 83(5):908-925。
  2. Bokil,N.J.,Totsika,M.,Carey,A.J.,Stacey,K.J.,Hancock,V.,Saunders,B.M.,Ravasi,T.,Ulett,G.C.,Schembri,M.A.and Sweet,M.J。(2011)。 uropathogenic大肠杆菌的巨噬细胞存活::不同临床分离物之间的差异 小鼠和人巨噬细胞。 Immunobiology 216(11):1164-1171。
  3. Mulvey,M.A.,Lopez-Boado,Y.S.,Wilson,C.L.,Roth,R.,Parks,W.C.,Heuser,J.and Hultgren,S.J。(1998)。 通过1型piliated uropathogenic大肠杆菌诱导和逃避宿主防御 。 Science 282(5393):1494-1497。
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引用:Bateman, S. L. and Seed, P. (2012). Intracellular Macrophage Infections with E. coli under Nitrosative Stress. Bio-protocol 2(20): e275. DOI: 10.21769/BioProtoc.275.