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Dec 2013

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Gentamicin Protection Assay to Determine the Number of Intracellular Bacteria during Infection of Human TC7 Intestinal Epithelial Cells by Shigella flexneri
庆大霉素保护试验确定福氏志贺菌侵染人TC7肠上皮细胞的胞内寄生数目   

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Abstract

Shigella flexneri is an intracellular bacterial pathogen that gains access to the gut epithelium using a specialized Type III Secretion System (T3SS). Various determinants mediating this invasive infection have been experimentally verified using the classical gentamicin protection assay presented here. In this assay epithelial cell lines are infected by bacteria in vitro and the extracellular bacteria are killed by gentamicin. The internalized bacteria, which are protected from the bactericidal action of gentamicin, are recovered by lysing the epithelial cells and enumerated by determining the colonies formed on solid medium. Various techniques based on light microscopy, such as immunofluorescence and bacteria expressing fluorescent proteins, are also used for studying intracellular bacteria. However, these techniques are not only labor intensive and require sophisticated equipment, but mostly are also not quantitative. Despite being an easy quantitative method to study invasiveness of bacteria, the gentamicin protection assay cannot distinguish between the survival and multiplication of the internalized bacteria over longer incubation periods. To alleviate the complications created by multiplication and dissemination of internalized bacteria, complementary assays like plaque formation assays are required. This protocol presents an easy and cost-effective method to determine the invasiveness and the capacity to establish an infection of Shigella under different conditions.

Keywords: Shigella (志贺氏菌), Intracellular pathogen (胞内致病菌), Invasion (侵染), Type III Secretion System (iii型分泌系统), Intestinal epithelial cells (肠上皮细胞)

Background

Shigella infects about 160 million people leading to about 600,000 deaths every year (Reference 8). The clinical manifestations of Shigella infection, or shigellosis, arise only after the bacterium enters the epithelium, where it multiplies and spreads to adjacent cells causing cell death and tissue necrosis. Thus, the entry into the epithelial cells is a critical step in the infectious life cycle of Shigella (Ashida et al., 2015). Most of the determinants mediating this step have been ascribed to a large virulence plasmid that encodes a T3SS responsible for injecting bacterial proteins into the host cell via a needle-like projection (Puhar and Sansonetti, 2014).

The gentamicin protection assay is a classical method that is used to assess the invasiveness of Shigella and has led to the identification of a number of mediators of invasion by mutational analysis and comparisons with the wild-type bacteria. In a typical experiment, the bacteria are allowed to infect the intestinal cells in a synchronized manner followed by removal of external bacteria by gentamicin treatment. The invasiveness is assessed by determining the number of surviving bacteria (protected from gentamicin being intracellular) from the lysates of infected cells. A lower number of surviving bacteria with respect to the wild-type indicates a defect in invasion or intracellular survival. If short infection times are used after the gentamicin treatment (typically 1h), the gentamicin protection assay allows to compare the bacterial capacity to enter host cells only. However, if longer infection times are used after gentamicin treatment (2 h or more), the assay will also reflect the bacterial capacity to survive and multiply within the target cell besides the capacity to invade. Hence, if the gentamicin protection assay is employed to study the capacity to survive and multiply by allowing long infection times, these experiments should always be complemented with tests carried out at short infection times to ensure correct interpretation of the results. The gentamicin protection assay, however, is not suitable to assess the extent of intercellular spread of bacteria throughout the monolayer, which is conveniently determined by a plaque formation assay. Although the invasiveness of bacterial pathogens can also be studied by fluorescence microscopy-based techniques such as immunofluorescence and FACS; it requires the use of costly reagents like labeled probes, special reporter strains and sophisticated equipment. The gentamicin protection assay is not only relatively easy to perform and cheap, it is fairly rapid and less labor intensive making it amenable to higher throughput.

The protocol presented here reprises the classical gentamicin protection assay, which has been optimized for use with Shigella flexneri and TC7 intestinal epithelial cells but can be modified to be used with other intracellular pathogens and other host cell types.

Materials and Reagents

  1. Conical flask (Duran, catalog number: 2121628)
  2. Microfuge tubes (Eppendorf, Safe-Lock tubes, catalog numbers: 0030120086, 0030120094)
  3. Centrifuge tubes (Sarstedt, catalog numbers: 62.554.502, 62.547.254)
  4. Culture tubes (TPP, catalog number: 91016)
  5. Pipette tips (VWR, catalog numbers: 89041-404, 89041-412, 89041-400)
  6. Serological pipettes (VWR, catalog numbers: 612-3702, 612-3700, 612-3698)
  7. 6-well tissue culture test plates (TPP, catalog number: 92406)
  8. TC7 cells (human cell line, a derivative of Caco-2 colon adenocarcinoma cells) (Chantret et al., 1994) 
  9. Shigella flexneri M90T (Sansonetti et al., 1982)
  10. Dulbecco's Modified Eagle Medium (DMEM) (Gibco, catalog number: 21885-025)
  11. Penicillin-Streptomycin solution (10,000 U/ml) (Gibco, catalog number: 15140122)
  12. Minimum Essential Medium Non-Essential Amino Acids solution (100x) (Gibco, catalog number: 11140-035)
  13. Dulbecco’s Phosphate Buffered Saline (PBS) solution (Gibco, catalog number: 14190-144)
  14. Fetal Bovine Serum (Gibco, catalog number: 10500056)
  15. HEPES 1 M solution (Gibco, catalog number: 15630-080)
  16. Trypsin-EDTA (0.05%), phenol red (Gibco, catalog number: 25300-054)
  17. Trypan Blue Stain (0.4%) (Thermo Scientific, catalog number: T10282)
  18. Agarose (VWR Life Science, catalog number: 35-1020)
  19. Tryptic Soy Broth (TSB) ready to use powder (Merck, catalog number: 105459)
  20. Tryptic Soy Agar (TSA) ready to use powder (Merck, catalog number: 105458)
  21. Ethanol (VWR chemicals, catalog number: 20821.558)
  22. Congo red (Sigma, catalog number: C6277) 
  23. Sodium deoxycholate (Sigma, catalog number: 30970)
  24. Gentamicin sulfate (Sigma, catalog number: G1264)
  25. Sterile disposable petriplates (Sigma, catalog number: P5606-400EA)
  26. Congo red solution (see Recipes)
  27. Growth medium (see Recipes)
  28. Infection buffer (see Recipes)
  29. Sodium Deoxycholate solution (see Recipes)
  30. Gentamicin solution (see Recipes)

Equipment

  1. Biosafety cabinet (Thermo Scientific, HERAsafe KS 18)
  2. Spectrophotometer (Amersham, Utrospec 2100 pro)
  3. Pipette controller (VWR, Accurpette)
  4. CO2 Incubator (Thermo Scientific, Heracell VIOS 160i)
  5. Benchtop centrifuge (VWR, Micro Star 17R)
  6. Centrifuge (Eppendorf, 5810R with Rotor A-4-81 for plates)
  7. Water bath (Grant, JBA 12)
  8. Pipettes (Eppendorf, Research plus)
  9. Inverted microscope (Motic, AE2000 Binocular)
  10. Cell counter (Thermo Scientific, Countess II FL)
  11. Cell counter slides (Thermo Scientific, Countess Cell Counting Chamber Slides, C10228)
  12. Orbital shaker (Edmund Bühler, Swip SM25)
  13. Vacuum pump (VWR, Mini diaphragm vacuum pump VP 86)

Software

  1. Prism 7 (GraphPad, https://www.graphpad.com)

Procedure

Note: The experiment should be carried out in a biosafety level 2 lab.


  1. Preparation of bacteria
    Refer to Procedure A of the Protocol–Plaque Assay to Determine Invasion and Intercellular Dissemination of Shigella flexneri in TC7 human Intestinal Epithelial Cells (Sharma and Puhar, 2019).

  2. Preparation of TC7 cells
    Refer to Procedure B of the Protocol–Plaque Assay to Determine Invasion and Intercellular Dissemination of Shigella flexneri in TC7 human Intestinal Epithelial Cells (Sharma and Puhar, 2019).

  3. Infection and cell lysis
    Day 3
    1. On Day 3, when the bacteria reach OD600nm = 0.3-0.4, pipette 1-1.5 ml culture into a microcentrifuge tube.
    2. Spin the tube at 3,000 x g for 5 min. Aspirate the supernatant. Add an equal volume of PBS at room temperature and resuspend the pellet by tapping the microcentrifuge tube or by gentle vortexing.
    3. Repeat Step C2 at least twice and finally resuspend the pellet in infection buffer (see Recipes) at room temperature. Record the OD600nm of the bacterial suspension.
      The recommended multiplicity of infection (MOI) for this experiment is 5. Since there are 1 x 106 cells in each well, the number of bacteria required per well is 5 x 106.
      The number of Shigella cells at OD600nm = 1 is 0.5 x 109/ml; hence at OD = z, the number of bacterial cells = z x 0.5 x 109. Calculate the volume of bacterial suspension required for infecting each well using the following formula:

      Volume of bacterial suspension required (in ml) = 5 x 106/z x 0.5 x 109

    4. Aspirate the medium from the cells and add 2 ml of sterile PBS at room temperature to each well.
    5. Swirl the plate gently and carefully aspirate all the liquid. After at least 2 washings, add 2 ml of infection buffer (see Recipes) at room temperature to each well.
    6. Add the desired volume of bacterial suspension (typically about 10 μl, calculated in Step C3) to each well of the 6-well plate. Centrifuge the plate at 180 x g for 10 min at room temperature.
    7. Incubate the plate at 37 °C, 10% CO2, for the desired infection time, typically 1 h. When Shigella comes in contact with the cells at the incubation temperature of 37 °C (35 °C and above) the secretion of effector proteins through the T3SS is triggered which mediates the bacterial invasion.
    8. After incubation, wash the wells with sterile PBS three times as in Steps C4 and C5.
    9. Add 2 ml of gentamicin solution (see Recipes) to each well and incubate the plate for one more hour at 37 °C and 10% CO2. If intracellular bacterial survival and multiplication is assessed, this incubation time can be extended.
    10. After incubation with gentamicin, wash the wells thrice with PBS (as in Steps C4 and C5).
    11. Aspirate the PBS from the wells and add 1 ml of sodium deoxycholate solution (see Recipes) to lyse the TC7 cells (the bacteria resist deoxycholate).
    12. Using a 1 ml pipette, scrape the cells off the surface and pipette up and down to lyse the cells effectively and homogenize the lysate.

  4. Plating
    1. Prepare log serial dilutions of this lysate in sterile PBS as follows.
    2. Dispense 450 μl of sterile PBS in 1.5 ml microcentrifuge tubes.
    3. Add 50 μl of cell lysate to one tube and mark it 10-1 dilution (as in Figure 1). Vortex the tube for a few seconds and transfer 50 μl of suspension from this tube to the next tube containing 450 μl of sterile PBS. Mark this new tube as 10-2 dilution. Repeat this procedure to obtain further dilutions of the lysate (Figure 1).
    4. For shorter infection time points, a lower dilution will provide significant results. For example, if infection time is 1 h, 10-1 dilution is enough; but if the infection time is increased to 2 h, 10-2 dilution would be more appropriate owing to bacterial multiplication. Similarly, consider higher dilutions for even longer infection times.


      Figure 1. Schematic representation of logarithmic serial dilution of the cell lysate

    5. Mark sectors on the agar plates (on the bottom of the plate) as in Figure 2.
    6. Put a 10 μl drop of diluted suspension (10-1/10-2/10-3/10-4 as labeled on the plate in Figure 2) on one of the sectors on the TSA plate. Let the drop dry.
    7. Put at least three drops of each dilution (R1, R2 and R3 in Figure 2; representing technical replicates). 
    8. On another plate, mark sectors in a similar way (on the bottom of the plate) and put drops of suspension prepared from the lysate of a duplicate well. This plate serves as another technical replicate.
    9. Incubate the plates overnight at 37 °C.
    10. Count the colonies in the drop. Only consider the dilutions in which the number of colonies is within the countable range (3-30 colonies).
    11. In parallel, make log serial dilutions of the starting bacterial suspension and spot 10 μl drops on TSA plates in a similar way as described above. 
    12. Incubate the plates overnight at 37 °C, along with the other plates, and count the colonies in the drop similar to Step D10. This colony count of the inoculum serves as a verification of the calculation performed in Step C3.


      Figure 2. Sectors on a TSA plate for putting drops of up to four different dilutions in triplicate

Data analysis

  1. From the raw colony count, determine the CFU/ml using the following formula:

    CFU/ml = No. of colonies x dilution factor x 100

    For example, if 15 colonies were counted in 10-2 dilution, CFU/ml would be calculated using the aforementioned formula as follows:

    CFU/ml = 15 (No. of colonies) x 100 (dilution factor, from 10-2) x 100 = 150,000 or 1.5 x 105

  2. Using Prism software plot the number of bacteria expressed in CFU/ml against time for every strain to be analyzed and perform statistical analysis using the same software (Figure 3). For comparing only two sample values at a fixed time point, use Student’s t-test. However, for studying the statistical significance of multiple samples across multiple time points, use two-way ANOVA with Tukey’s post-hoc test.


    Figure 3. An example plot of the number of intracellular bacteria in CFU/ml at different infection times for two different strains. Each dot represents an independent value obtained from a replicate.

Notes

  1. To prepare glycerol stocks of Shigella flexneri, dilute the overnight culture 200 times in fresh TSB and incubate at 37 °C with 200 rpm shaking. Monitor the OD600 of the culture periodically. At OD600 = 0.6, withdraw 1 ml of culture in a 2 ml cryovial and add sterile glycerol to a concentration of 15-50% (v/v). Mix gently and store the tube at -80 °C. The frozen stocks are stable for years at -80 °C, however, multiple freeze-thaw cycles may reduce the shelf life.
  2. Carefully select only the red colonies on Congo red agar for the experiment. The off-white colonies have lost the virulence plasmid and are invasion-deficient. If plates are not used immediately after overnight culture, but kept at 4 °C (which is possible for up to 3 weeks), white colonies need to be marked because they will turn red due to non-specific binding of Congo red. Red staining of invasive colonies only develops during growth above 35 °C.
  3. Congo red is able to induce secretion via the Type III Secretion System (T3SS) in Shigella. The dye binds to the bacterial colonies that have an active T3SS making them appear red in color. It is a simple and quick screening method to differentiate virulent Shigella colonies from the avirulent ones (due to curing of the virulence plasmid). Unfortunately, the mechanism of action of Congo red is not understood.
  4. The TC7 cells are cultured and maintained in presence of Penicillin and Streptomycin (100 U/ml). However, infections are carried out in infection buffer that do not contain any antibiotics (which may inhibit the bacterial growth). This also highlights the importance of washing the cell monolayers before starting the infections.
  5. The 6-well plates are centrifuged after addition of bacteria to aid binding, as Shigella lacks adhesion factors.
  6. Keep all the buffers and reagents that are required at initiation of invasion and thereafter, warmed up to 37 °C before using. The secretion of T3SS effectors mediating invasion is temperature dependent. It is active above 35 °C, but inactive at room temperature. In order to synchronize the invasion, it is important to keep the bacteria in buffers at room temperature before invasion.
  7. The medium containing gentamicin should be prepared on the day of use.
  8. Even during longer incubations, gentamicin won’t be able to penetrate the cells and kill the intracellular bacteria. The intracellular bacteria remain ‘protected’ all the times.
  9. Deoxycholate is a detergent and therefore TC7 cell lysates should be made with little pipetting to avoid bubbles.
  10. Use a dedicated incubator for infections.
  11. When using the pipette controller, gently dispense the solutions with gravity (“G” setting in Accurpipette) or at the lowest flow speed into the 6-well plate to avoid detaching the cell monolayer.
  12. Be careful while swirling the plate, washing the cells and during centrifugation, because the confluent cell monolayer detaches easily.
  13. The TC7 cells can survive for about 6 h in a medium devoid of serum. Therefore, additional serum is not required for infection times up to about 6 h. However, for longer incubation times, serum may be added after gentamicin treatment.
  14. For further reading and details on basic methods that have not been detailed in this manuscript (like bacterial inoculation, pouring the plates, etc.) the reader is suggested to go through the following books: A Laboratory Manual (Cappuccino and Welsh, 2017) and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (Freshney, 2010).

Recipes

  1. Congo red solution
    1% (w/v) Congo red in water
    Weigh 1 g of Congo red dye and dissolve it in 70 ml of water. Bring the volume of the solution to 100 ml and filter sterilize the solution, using 0.2 μm membrane filters. The Congo red solution is stable for a long time at room temperature
  2. Growth medium
    DMEM
    1x amino acid solution
    100 U/ml Pen-Strep solution
    10% heat inactivated FBS
    To 500 ml DMEM, add 5 ml of amino acid solution, 5 ml of Pen-Strep solution and 50 ml of FBS (inactivated by incubation at 55 °C for 30 min)
    Note: The growth medium must be stored at 4 °C and can be used for 3-4 weeks.
  3. Infection buffer
    DMEM (serum free)
    20 mM HEPES, pH 7.4
    To 50 ml of serum free DMEM, add 1 ml of 1 M sterile HEPES pH 7.4
    Note: Infection buffer is stable at 4 °C for months.
  4. Sodium Deoxycholate solution
    0.5% sodium deoxycholate in PBS
    To 40 ml of PBS, add 200 mg of sodium deoxycholate and filter-sterilize
    Note: The solution is stable at room temperature for several months.
  5. Gentamicin solution
    50 μg/ml gentamicin in infection buffer
    To 15 ml of infection buffer, add 25 μl of gentamicin from a stock solution of 30 mg/ml (prepared in water)
    Note: The stock solution of gentamicin must be stored at -20 °C in small aliquots. Although the frozen stock solution is stable for several months, repeated freeze-thaw cycles may render the antibiotic inefficient. The working gentamicin solution should not be stored and always be freshly prepared.

Acknowledgments

We thank members of the Puhar lab for critical reading. A.S. is the recipient of a stipend from The MIMS Excellence by Choice Postdoctoral Programme under the patronage of Emmanuelle Charpentier. The programme is financed by the Kempe Foundations and the Knut and Alice Wallenberg Foundation. A.P. acknowledges generous funding from MIMS, UCMR, Umeå University and the Wallenberg Academy Fellow programme. This protocol was used in Puhar et al. (2013).

Competing interests

The authors declare no conflict of interest.

References

  1. Ashida, H., Mimuro, H. and Sasakawa, C. (2015). Shigella manipulates host immune responses by delivering effector proteins with specific roles. Front Immunol 6: 219.
  2. Cappuccino, J. G. and Welsh, C.T (2017). Microbiology: A Laboratory Manual, 11th Edition. ISBN: 978-0134298672.
  3. Chantret, I., Rodolosse, A., Barbat, A., Dussaulx, E., Brot-Laroche, E., Zweibaum, A. and Rousset, M. (1994). Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line Caco-2: evidence for glucose-dependent negative regulation. J Cell Sci 107 (Pt 1): 213-225.
  4. Freshney, R. I. (2010). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. Sixth Edition. ISBN: 9780470528129.
  5. Puhar, A. and Sansonetti, P. J. (2014). Type III secretion system. Curr Biol 24(17): R784-791.
  6. Puhar, A., Tronchere, H., Payrastre, B., Nhieu, G. T. and Sansonetti, P. J. (2013). A Shigella effector dampens inflammation by regulating epithelial release of danger signal ATP through production of the lipid mediator PtdIns5P. Immunity 39(6): 1121-1131.
  7. Sansonetti, P. J., Kopecko, D. J. and Formal, S. B. (1982). Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect Immun 35(3): 852-860.
  8. Shigellosis–Chapter 3–2018 Yellow Book | Travelers’ Health | CDC. Available at: https://wwwnc.cdc.gov/travel/yellowbook/2018/infectious-diseases-related-to-travel/shigellosis.

简介

福氏志贺菌是一种细胞内细菌病原体,使用专门的III型分泌系统(T3SS)进入肠道上皮细胞。使用本文提出的经典庆大霉素保护测定已经实验验证了介导这种侵入性感染的各种决定因素。在该测定中,上皮细胞系被细菌体外感染,并且细胞外细菌被庆大霉素杀死。通过裂解上皮细胞回收保护免受庆大霉素杀菌作用的内化细菌,并通过测定在固体培养基上形成的菌落进行计数。基于光学显微镜的各种技术,例如免疫荧光和表达荧光蛋白的细菌,也用于研究细胞内细菌。然而,这些技术不仅劳动强度大,而且需要复杂的设备,但大多数也不是定量的。尽管是一种简单的定量方法来研究细菌的侵袭性,但庆大霉素保护试验无法区分内化细菌在较长的潜伏期内的存活和繁殖。为了减轻由内化细菌的增殖和传播所产生的并发症,需要诸如噬斑形成测定的互补测定。该方案提供了一种简单且经济有效的方法来确定在不同条件下建立志贺氏菌感染的侵袭性和能力。
【背景】志贺氏菌每年感染约1.6亿人,导致约600,000人死亡(参考文献8)。 志贺氏菌感染或志贺菌病的临床表现仅在细菌进入上皮细胞后出现,其中它会繁殖并扩散到邻近的细胞,导致细胞死亡和组织坏死。因此,进入上皮细胞是志贺氏菌的传染性生命周期中的关键步骤(Ashida 等人,,2015)。介导该步骤的大多数决定簇归因于编码T3SS的大毒力质粒,所述T3SS负责通过针状投影将细菌蛋白质注入宿主细胞(Puhar和Sansonetti,2014)。

庆大霉素保护测定是用于评估志贺氏菌的侵袭性的经典方法,并且已经通过突变分析和与野生型细菌的比较鉴定了许多入侵介质。在典型的实验中,允许细菌以同步方式感染肠细胞,然后通过庆大霉素处理除去外部细菌。通过从感染细胞的裂解物中确定存活细菌(保护庆大霉素是细胞内的)的数量来评估侵袭性。相对于野生型,存活细菌数量较少表明存在侵袭或细胞内存活的缺陷。如果在庆大霉素处理后(通常1小时)使用短的感染时间,则庆大霉素保护测定允许比较仅进入宿主细胞的细菌能力。然而,如果在庆大霉素处理(2小时或更长时间)后使用更长的感染时间,则该测定还将反映细菌在靶细胞内存活和繁殖的能力,除了侵入的能力之外。因此,如果使用庆大霉素保护试验来研究通过允许长的感染时间来存活和繁殖的能力,那么这些实验应该总是在短的感染时间进行测试以确保正确解释结果。然而,庆大霉素保护测定不适于评估细菌在整个单层中的细胞间传播程度,这可通过噬斑形成测定方便地确定。虽然细菌病原体的侵袭性也可以通过荧光显微镜技术研究,如免疫荧光和FACS;它需要使用昂贵的试剂,如标记探针,特殊报告菌株和复杂的设备。庆大霉素保护试验不仅相对容易操作且价格便宜,而且相当快速且劳动强度较低,使其适应更高的通量。

这里介绍的方案重现了经典的庆大霉素保护试验,该试验已经优化用于福氏志贺菌和TC7肠上皮细胞,但可以被修饰以与其他细胞内病原体和其他宿主细胞类型一起使用。

关键字:志贺氏菌, 胞内致病菌, 侵染, iii型分泌系统, 肠上皮细胞

材料和试剂

  1. 锥形瓶(Duran,目录号:2121628)
  2. 微量离心管(Eppendorf,Safe-Lock管,目录号:0030120086,0030120094)
  3. 离心管(Sarstedt,目录号:62.554.502,62.547.254)
  4. 培养管(TPP,目录号:91016)
  5. 移液器吸头(VWR,目录号:89041-404,89041-412,89041-400)
  6. 血清移液器(VWR,目录号:612-3702,612-3700,612-3698)
  7. 6孔组织培养试板(TPP,目录号:92406)
  8. TC7细胞(人细胞系,Caco-2结肠腺癌细胞的衍生物)(Chantret et al。,1994) 
  9. 福氏志贺菌 M90T(Sansonetti et al。,1982)
  10. Dulbecco改良Eagle培养基(DMEM)(Gibco,目录号:21885-025)
  11. 青霉素 - 链霉素溶液(10,000 U / ml)(Gibco,目录号:15140122)
  12. 最低必需培养基非必需氨基酸溶液(100x)(Gibco,目录号:11140-035)
  13. Dulbecco的磷酸盐缓冲盐水(PBS)解决方案(Gibco,目录号:14190-144)
  14. 胎牛血清(Gibco,目录号:10500056)
  15. HEPES 1 M解决方案(Gibco,目录号:15630-080)
  16. 胰蛋白酶-EDTA(0.05%),酚红(Gibco,目录号:25300-054)
  17. 台盼蓝染色(0.4%)(Thermo Scientific,目录号:T10282)
  18. 琼脂糖(VWR生命科学,目录号:35-1020)
  19. 胰蛋白酶大豆汤(TSB)即用型粉末(默克,目录号:105459)
  20. 胰蛋白酶大豆琼脂(TSA)即用型粉末(默克,目录号:105458)
  21. 乙醇(VWR chemicals,目录号:20821.558)
  22. 刚果红(西格玛,目录号:C6277) 
  23. 脱氧胆酸钠(西格玛,目录号:30970)
  24. 硫酸庆大霉素(西格玛,目录号:G1264)
  25. 无菌一次性petriplates(西格玛,目录号:P5606-400EA)
  26. 刚果红解决方案(见食谱)
  27. 生长培养基(见食谱)
  28. 感染缓冲液(见食谱)
  29. 脱氧胆酸钠溶液(见食谱)
  30. 庆大霉素溶液(见食谱)

设备

  1. 生物安全柜(Thermo Scientific,HERAsafe KS 18)
  2. 分光光度计(Amersham,Utrospec 2100 pro)
  3. 移液器控制器(VWR,Accurpette)
  4. CO 2 培养箱(Thermo Scientific,Heracell VIOS 160i)
  5. 台式离心机(VWR,Micro Star 17R)
  6. 离心机(Eppendorf,5810R带转子A-4-81用于板)
  7. 水浴(格兰特,JBA 12)
  8. 移液器(Eppendorf,Research plus)
  9. 倒置显微镜(Motic,AE2000双目)
  10. 细胞计数器(Thermo Scientific,Countess II FL)
  11. 细胞计数器载玻片(Thermo Scientific,Countess Cell Counting Chamber Slides,C10228)
  12. 轨道振动筛(EdmundBühler,Swip SM25)
  13. 真空泵(VWR,迷你隔膜真空泵VP 86)

软件

  1. Prism 7(GraphPad, https://www.graphpad.com )

程序

注意:实验应在生物安全2级实验室进行。

  1. 细菌的制备
    参考协议 - 噬斑测定的程序A,以确定TC7人肠上皮细胞中弗氏志贺氏菌的侵袭和细胞间传播(Sharma和Puhar,2019)。

  2. TC7细胞的制备
    参考方案 - 斑块试验的程序B,以确定TC7人肠上皮细胞中弗氏志贺氏菌的侵袭和细胞间传播(Sharma和Puhar,2019)。

  3. 感染和细胞裂解
    第3天
    1. 在第3天,当细菌达到OD 600nm = 0.3-0.4时,将1-1.5ml培养物移液到微量离心管中。
    2. 将管在3,000 x g 下旋转5分钟。吸出上清液。在室温下加入等体积的PBS,并通过轻敲微量离心管或通过轻轻涡旋重悬沉淀。
    3. 重复步骤C2至少两次,最后在室温下将沉淀重悬于感染缓冲液(参见食谱)中。记录细菌悬浮液的OD 600nm 。
      推荐的该实验感染复数(MOI)为5.由于每个孔中有1×10 6 细胞,每孔所需的细菌数量为5×10 6 。
      OD 600nm = 1时志贺氏杆菌细胞的数量为0.5×10 9 / ml;因此,在OD = z时,细菌细胞的数量= z×0.5×10 9 。使用以下公式计算感染每个孔所需的细菌悬浮液的体积:

      所需的细菌悬浮液体积(ml)= 5 x 10 6 / zx 0.5 x 10 9
    4. 从细胞中吸出培养基,并在室温下向每个孔中加入2ml无菌PBS。
    5. 轻轻旋转盘子,小心地吸出所有液体。在至少2次洗涤后,在室温下向每个孔中加入2ml感染缓冲液(参见食谱)。
    6. 将所需体积的细菌悬浮液(通常约10μl,在步骤C3中计算)添加到6孔板的每个孔中。在室温下将板在180 x g 下离心10分钟。
    7. 将板在37℃,10%CO 2 下孵育所需的感染时间,通常为1小时。当志贺氏菌在37°C(35°C及以上)的温育温度下与细胞接触时,通过T3SS分泌效应蛋白,从而介导细菌入侵。
    8. 孵育后,如步骤C4和C5,用无菌PBS洗涤孔三次。
    9. 向每个孔中加入2ml庆大霉素溶液(参见配方),并将板在37℃和10%CO 2 下再孵育1小时。如果评估细胞内细菌存活和繁殖,则可以延长该孵育时间。
    10. 与庆大霉素一起温育后,用PBS洗涤孔三次(如步骤C4和C5)。
    11. 从孔中吸出PBS并加入1ml脱氧胆酸钠溶液(参见配方)以裂解TC7细胞(细菌抵抗脱氧胆酸盐)。
    12. 使用1 ml移液器,将细胞从表面刮下并用吸管上下移动以有效裂解细胞并使裂解液均质化。

  4. 电镀
    1. 如下在无菌PBS中制备该裂解物的对数连续稀释液。
    2. 在1.5 ml微量离心管中分配450μl无菌PBS。
    3. 向一个试管中加入50μl细胞裂解液,并将其标记为10 -1 稀释液(如图1所示)。将管涡旋几秒钟,并将50μl悬浮液从该管转移至含有450μl无菌PBS的下一个管中。将该新管标记为10 -2 稀释液。重复此步骤以获得裂解物的进一步稀释液(图1)。
    4. 对于较短的感染时间点,较低的稀释度将提供显着的结果。例如,如果感染时间为1小时,10 -1 稀释就足够了;但如果感染时间增加到2小时,由于细菌繁殖,10 -2 稀释将更合适。同样,考虑更高的稀释度,甚至更长的感染时间。


      图1.细胞裂解物的对数连续稀释的示意图

    5. 标记琼脂平板上的部分(在板的底部),如图2所示。
    6. 放入10μl稀释的悬浮液(10 -1 / 10 -2 / 10 -3 / 10 -4
    7. 每种稀释液至少滴三滴(图2中的R1,R2和R3;代表技术重复)。 
    8. 在另一个平板上,以相似的方式标记扇区(在平板的底部)并放入由复制孔的裂解物制备的悬浮液滴。该板用作另一技术复制品。
    9. 将板在37℃孵育过夜。
    10. 计算掉落中的殖民地。只考虑其中菌落数在可计数范围内(3-30个菌落)的稀释液。
    11. 同时,对起始细菌悬浮液进行对数连续稀释,并以与上述类似的方式在TSA板上点10μl滴。 
    12. 将板在37℃下孵育过夜,与其他板一起孵育,并计数滴中的菌落,类似于步骤D10。接种物的菌落计数用作对步骤C3中进行的计算的验证。


      图2. TSA板上的区域,用于将多达四种不同稀释液滴入一式三份

数据分析

  1. 从原始菌落计数中,使用以下公式确定CFU / ml:

    CFU / ml =菌落数×稀释倍数×100
    例如,如果在10 -2 稀释液中计数15个菌落,则使用上述公式计算CFU / ml如下:

    CFU / ml = 15(菌落数)×100(稀释因子,10 -2 )×100 = 150,000或1.5×10 5

  2. 使用Prism软件绘制每个待分析菌株的以CFU / ml表示的细菌数量与时间的关系,并使用相同的软件进行统计分析(图3)。要在固定时间点仅比较两个样本值,请使用学生的 t - test。然而,为了研究多个样本在多个时间点的统计显着性,使用双向ANOVA和Tukey的事后检验。


    图3.两种不同菌株在不同感染时间内CFU / ml的细胞内细菌数量的示例图。每个点代表从复制品获得的独立值。

笔记

  1. 为了制备弗氏志贺氏菌的甘油储液,在新鲜的TSB中将过夜培养物稀释200倍,并在37℃和200rpm摇动下孵育。定期监测培养物的OD 600 。在OD 600 = 0.6时,在2ml冷冻管中取出1ml培养物并加入无菌甘油至浓度为15-50%(v / v)。轻轻混合并将管储存在-80°C。冷冻原料在-80°C下稳定多年,然而,多次冻融循环可能会缩短保质期。
  2. 仔细选择刚果红琼脂上的红色菌落进行实验。灰白色菌落失去了毒力质粒并且是入侵缺陷的。如果在过夜培养后不立即使用平板,但保持在4°C(可能长达3周),需要标记白色菌落,因为刚果红的非特异性结合它们会变红。侵袭性菌落的红色染色仅在35°C以上的生长期间发生。
  3. 刚果红能够通过志贺氏菌中的III型分泌系统(T3SS)诱导分泌。染料与具有活性T3SS的细菌菌落结合,使其呈现红色。这是一种简单快速的筛选方法,用于区分毒性志贺氏菌菌落与无毒菌株(由于毒力质粒的固化)。不幸的是,刚果红的作用机制尚不清楚。
  4. 培养TC7细胞并在青霉素和链霉素(100U / ml)存在下维持。然而,感染缓冲液中不含任何抗生素(可抑制细菌生长)。这也强调了在开始感染之前洗涤细胞单层的重要性。
  5. 加入细菌后,将6孔板离心以帮助结合,因为志贺氏菌缺乏粘附因子。
  6. 保留入侵后所需的所有缓冲液和试剂,然后在使用前加热至37°C。介导入侵的T3SS效应子的分泌是温度依赖性的。它在35°C以上有效,但在室温下无效。为了使入侵同步,重要的是在入侵前将细菌保持在室温下的缓冲液中。
  7. 含有庆大霉素的培养基应在使用当天制备。
  8. 即使在较长时间的孵育期间,庆大霉素也不能穿透细胞并杀死细胞内的细菌。细胞内细菌一直保持“保护”状态。
  9. 脱氧胆酸盐是一种去污剂,因此TC7细胞裂解液应该用很少的移液来制造,以避免气泡。
  10. 使用专用的孵化器进行感染。
  11. 使用移液器控制器时,轻轻地用重力(Accurpipette中的“G”设置)或以最低流速将溶液分配到6孔板中以避免分离细胞单层。
  12. 在旋转平板,清洗细胞和离心过程中要小心,因为融合的细胞单层很容易脱落。
  13. TC7细胞可以在没有血清的培养基中存活约6小时。因此,感染时间长达约6小时不需要额外的血清。然而,对于更长的孵育时间,可以在庆大霉素处理后加入血清。
  14. 如需进一步阅读和详细介绍本手稿中未详述的基本方法(如细菌接种,倒板,等),建议读者阅读以下书籍:实验室手册(卡布奇诺和威尔士,2017)和动物细胞培养:基础技术和专业应用手册(Freshney,2010)。

食谱

  1. 刚果红解决方案
    1%(w / v)刚果红在水中
    称取1g刚果红染料,并将其溶于70ml水中。将溶液的体积调至100 ml,并使用0.2μm膜过滤器对溶液进行过滤灭菌。刚果红溶液在室温下长时间稳定
  2. 生长媒介
    DMEM
    1x氨基酸溶液
    100 U / ml Pen-Strep溶液
    10%热灭活FBS
    向500ml DMEM中加入5ml氨基酸溶液,5ml Pen-Strep溶液和50ml FBS(通过在55℃温育30分钟灭活)
    注意:生长培养基必须在4°C下储存,可以使用3-4周。
  3. 感染缓冲区
    DMEM(无血清)
    20 mM HEPES,pH 7.4
    向50ml无血清DMEM中加入1ml 1M无菌HEPES pH 7.4
    注意:感染缓冲液在4°C下稳定数月。
  4. 脱氧胆酸钠溶液
    PBS中0.5%脱氧胆酸钠
    向40毫升PBS中加入200毫克脱氧胆酸钠并过滤灭菌 注意:该溶液在室温下稳定数月。
  5. 庆大霉素溶液
    感染缓冲液中50μg/ ml庆大霉素
    向15 ml感染缓冲液中加入25 mg / ml(水中制备)的25 mg庆大霉素
    注意:庆大霉素的储备溶液必须在-20°C下以小等分试样储存。虽然冷冻储备溶液可稳定数月,但反复冻融循环可能使抗生素效率低下。工作的庆大霉素溶液不应存放,并且总是做好新鲜的准备。

致谢

我们感谢Puhar实验室的成员进行批判性阅读。如。在Emmanuelle Charpentier的赞助下,获得MIMS卓越选择博士后计划的津贴。该计划由Kempe Foundations和Knut以及Alice Wallenberg基金会资助。 A.P.承认来自MIMS,UCMR,Umeå大学和Wallenberg Academy Fellow计划的慷慨资助。该方案用于Puhar 等人(2013)。

利益争夺

作者宣称没有利益冲突。

参考

  1. Ashida,H.,Mimuro,H。和Sasakawa,C。(2015)。 Shigella 通过提供具有特定作用的效应蛋白来操纵宿主免疫反应。< / a> 前免疫 6:219。
  2. Cappuccino,J.G。和Welsh,C.T(2017)。 微生物学:实验室手册,第11版。 ISBN:978-0134298672。
  3. Chantret,I.,Rodolosse,A.,Barbat,A.,Dussaulx,E.,Brot-Laroche,E.,Zweibaum,A。和Rousset,M。(1994)。 从细胞系Caco-2的早期和晚期分离的克隆中蔗糖酶 - 异麦芽糖酶的差异表达:葡萄糖依赖性负调节的证据。 J Cell Sci 107(Pt 1):213-225。
  4. Freshney,R。I.(2010)。 动物细胞培养:基础技术手册和专业应用。第六版。 ISBN:9780470528129。
  5. Puhar,A。和Sansonetti,P。J.(2014)。 III型分泌系统。 Curr Biol 24(17 ):R784-791。
  6. Puhar,A.,Tronchere,H.,Payrastre,B.,Nhieu,G。T.和Sansonetti,P。J.(2013)。 志贺氏菌效应器通过调节危险信号ATP的上皮释放来抑制炎症产生脂质介质PtdIns5P。 Immunity 39(6):1121-1131。
  7. Sansonetti,P.J。,Kopecko,D.J。和Formal,S.B。(1982)。 参与质粒对弗氏志贺氏菌的侵袭能力。 感染免疫 35(3):852-860。
  8. Shigellosis-第3-2018章黄皮书|旅行者的健康| CDC。可在以下网址获得: https://wwwnc.cdc。 gov / travel / yellowbook / 2018 / infectious-diseases-related-to-travel / shigellosis 。
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引用:Sharma, A. and Puhar, A. (2019). Gentamicin Protection Assay to Determine the Number of Intracellular Bacteria during Infection of Human TC7 Intestinal Epithelial Cells by Shigella flexneri. Bio-protocol 9(13): e3292. DOI: 10.21769/BioProtoc.3292.
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