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Adhesion of Enteroaggregative E. coli Strains to HEK293 Cells
肠凝聚型大肠埃希杆菌对HEK293细胞的粘附测定   

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mBio
May 2017

Abstract

Enteroaggregative Escherichia coli (EAEC) is a recognized cause of acute diarrhea among both children and adults worldwide. EAEC strains are characterized by the presence of aggregative adherence fimbriae (AAF), which play a key role in pathogenesis by mediating attachment to the intestinal mucosa and by triggering host inflammatory responses. The aggregative adherence fimbria II (AAF/II) is the most important adherence factor of EAEC prototype strain 042 (EAEC042) to intestinal cells. Multiple receptors for AAF/II on epithelial cells have been identified including the transmembrane signaling mucin Muc1. This protocol describes a method to measure adherence of EAEC strains to HEK293 cells expressing the Muc1 glycoprotein.

Keywords: Muc1 (Muc1), EAEC (EAEC), Adherence (粘附), Mucins (粘蛋白), Glycoprotein (糖蛋白), Fimbria (菌毛), Hek293 (Hek293)

Background

EAEC is an important cause of endemic and epidemic diarrheal disease worldwide. Although most commonly associated with pediatric diarrhea in developing countries, EAEC is also linked to diarrhea in immunocompromised adults, travelers and food-borne outbreaks in the industrialized world, such as the large lethal outbreak caused by a Shiga toxin (Stx) type 2a-producing EAEC strain of serotype O104:H4 in Northern Europe in 2011 (Harrington et al., 2006; Rasko et al., 2011). EAEC pathogenesis is determined by the organism’s ability to adhere to intestinal cells, produce enterotoxins and cytotoxins, and ultimately to induce inflammation (Harrington et al., 2006). EAEC adherence to intestinal cells is mediated by AAF fimbrial adhesins (Czeczulin et al., 1997). To date, at least five variants of the AAF fimbriae have been described, all encoded in virulence plasmids ranging from 55 to 65 MDa (Jonsson et al., 2015). The AAF structure is comprised by a positively charged major subunit and a putative minor subunit at the tip of fimbrial structures (Berry et al., 2014).

The prototype EAEC strain 042 that exhibits the AAF/II variant has been shown to produce diarrhea in adult volunteers (Nataro et al., 1995). Clinical and laboratory data suggest that EAEC induces inflammatory enteritis, while studies using polarized T84 monolayers indicates that release of IL-8 is associated to the presence of AAF/II adhesin (Harrington et al., 2005). Although the importance of the adherence of EAEC to intestinal cells has been established, the cell receptors involved in the inflammatory response mediated by AAF fimbriae have not been fully characterized. Several receptors on epithelial cells have been identified for AAF/II including extracellular matrix (ECM) proteins such as fibronectin and laminin, and cytokeratin 8 (Farfan et al., 2008; Izquierdo et al., 2014). However, these receptors are localized on the basolateral side of intestinal cells. Thus, it is unlikely that these proteins play an important role during the initial infection with EAEC. Furthermore, it has been shown that fibronectin does not participate in the inflammatory response mediated by AAF/II (Yanez et al., 2016). We have recently found that EAEC also binds to the signaling Muc1 glycoprotein, and such binding is dependent on the sialylation of the protein (Boll et al., 2017). Mucins (MUC) are large (> 200 kDa) secreted and transmembrane glycoproteins with a high carbohydrate content (50-90% by weight) expressed by a variety of normal and malignant secretory epithelial cells (Corfield et al., 2001). Muc1 is a polymorphic transmembrane mucin-like protein that contains a large extracellular domain consisting of a glycosylated polypeptide made up of 30-100 tandem repeats of a 20-amino acid sequence, a transmembrane domain, and a cytoplasmic tail of 72 amino acids (Nath and Mukherjee, 2014). Muc1 is associated to numerous signaling pathways in malignant and inflammatory processes (Nath and Mukherjee, 2014). Our recent study shows that Muc1 is associated with the inflammatory response mediated by AAF/II. Moreover, EAEC 042 up-regulates epithelial Muc1 expression dependent on the presence of AAF (Boll et al., 2017).

The existence of multiple receptors for EAEC in intestinal cells complicates the identification and characterization of specific receptors in this cell lineage. The use of HEK293 cells, which differ from enterocytes, allows the evaluation of new potential receptors through transfection of these cells with plasmids encoding the candidate receptor. Likewise, binding assays performed with cells in suspension minimizes the binding of EAEC strains to non-abiotic surfaces. This protocol could be used to find out potential receptors for other enteropathogens. Here, we described in detail the method we previously used to visualize the adherence of EAEC to HEK293 cells transfected with a Muc1-encoding plasmid.

Materials and Reagents

  1. Cell culture flasks (Corning, catalog number: 3275 )
  2. 20, 200 and 1,000 μl Pipette tips (Barrier Low-Retention tips, Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 2149P-05-RT , 2069-HR and 2279 )
  3. 24-well plate
  4. Circle cover glasses (Fisher Scientific, Fisherbrand, catalog number: 12-545-80 )
  5. Tissue paper
  6. 15 ml conical culture tubes (Corning, catalog number: 430052 )
  7. Serological pipettes (1, 5, 10, 25, 50 ml) (Corning, catalog numbers: 4012 , 4051 , 4492 , 4251 , 4501 )
  8. Glass slides (Fisher Scientific, Fisherbrand, catalog number: 12-550-123 )
  9. Coverslip (Fisher Scientific, Fisherbrand, catalog number: 12-543C )
  10. Cell lines: HEK293-pcDNA3.1 and HEK293-pcDNA3.1-Muc1
    HEK293 cells (ATCC, catalog number: CRL-1573 )
    pcDNA3.1 (Thermo Fisher Scientific, InvitrogenTM, catalog number: V79020 )
    Note: pcDNA3.1-Muc1 clone was provided by Dr. Erik P. Lillehoj (Lillehoj et al., 2002).
  11. EAEC042 strain is available from our lab strain collection (Nataro et al., 1995)
  12. pGFP vector (Takara Bio, Clontech, catalog number: 632370 )
  13. LB medium (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10855001 )
  14. Glycerol (Fisher Scientific, Fisherbrand, catalog number: BP229-4 )
  15. Carbenicillin
  16. DMEM-High glucose (no phenol red), used to culture EAEC (Thermo Fisher Scientific, GibcoTM, catalog number: 31053028 )
  17. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 70011044 )
  18. Dulbecco’s modified Eagle’s medium (DMEM), used to culture HEK293 cells (Thermo Fisher Scientific, GibcoTM, catalog number: 11965118 )
  19. Fetal bovine serum, certified, heat inactivated (FBS) (Sigma-Aldrich, catalog number: F4135 )
  20. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  21. G418 (Sigma-Aldrich, catalog number: G418-RO)
    Manufacturer: Roche Diagnostics, catalog number: 4727878001 .
  22. 0.5 M EDTA (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9260G )
  23. Methanol (Sigma-Aldrich, catalog number: 322415-1L )
  24. Glacial acetic acid (Fisher Scientific, Fisher Chemicals, catalog number: A38-500 )
  25. FITC Mouse anti-Human Muc1 (CD227) (BD, BD Biosciences, catalog number: 559774 )
  26. Coverslip sealant (Biotium, catalog number: 23005 )
  27. FM 4-64FX (Thermo Fisher Scientific, InvitrogenTM, catalog number: F34653 )
  28. ProLongTM Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36935 )
  29. 4’,6-Diamidino-2-Phenylindole (DAPI, Dilactate) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D3571 )
  30. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14175103 )
  31. Formalin (Sigma-Aldrich, catalog number: HT501128-4L )

Equipment

  1. 10, 20, 200, 1,000 μl pipettes (Pipette Pack Set, Eppendorf, catalog number: 2231300006 )
  2. CO2 incubator (NuAire, model: NU-4750 )
  3. -80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: Forma Model 900 )
  4. Rotary shaker (ATR, model: AJ118 )
  5. Laminar flow hood (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series A2 Model 1375 )
  6. UV-Vis Spectrophotometer (GE Healthcare, Amersham Biosciences, model: Ultrospec 2100 Pro )
  7. Liquid nitrogen storage container (Worthington, model: LD35 )
  8. Centrifuge (Eppendorf, model: 5430 )
  9. Microcentrifuge (Fisher Scientific, model: accuSpinTM Micro 17R )
  10. Epifluorescence microscope (Olympus, model: BX51 )

Procedure

  1. Bacterial cell culture
    1. Streak out a loopful of EAEC strains (previously transformed with the pGFP plasmid and preserved at -80 °C in LB medium containing 20% glycerol) on LB agar plates containing 100 μg/ml of carbenicillin and incubate at 37 °C for 16-18 h.
    2. Inoculate 3-5 EAEC colonies into 5 ml LB medium and incubate at 37 °C for 16-18 h with shaking (200 rpm) using a rotary shaker. Made 1:100 dilution from this culture using fresh DMEM-High glucose (no red phenol) to induce the expression of AAF fimbria (e.g., 100 µl of bacterial culture in 9,900 µl DMEM-HG), and incubate at 37 °C with shaking (200 rpm) until reaching the mid logarithmic phase (OD600 = 0.6).
      Note: DMEM-HG is known to upregulate the expression of AAF fimbriae. DMEM-HG without red phenol is used to facilitate monitoring of EAEC growth in this medium by the naked eye and spectrophotometrically. EAEC takes approximately 4 h to reach an OD600 = 0.6 when grown in DMEM-HG.
    3. Centrifuge EAEC cultures at 5,000 x g for 10 min, wash with 10 ml 1x PBS, and resuspend in fresh DMEM (DMEM used to grow HEK293 cells) at a concentration of 1 x 108 colony forming units (CFU). We have previously determined that OD600 = 0.35 corresponds to approximately 1 x 108 EAEC CFU per ml.

  2. HEK293 cell culture
    1. Grow the human embryonic kidney cell line HEK293 (ATCC) transfected with pcDNA3.1 and pcDNA3.1-Muc1 plasmids in 75 or 150 cm2 flasks with DMEM (Gibco) supplemented with 10% FBS (Sigma-Aldrich), penicillin-streptomycin, and 200 µg/ml of G418 (Sigma-Aldrich) (to ensure maintenance of pCDNA3.1 plasmid derivatives).
    2. For passage and seeding purposes, wash HEK293 cells with 10 ml of sterile PBS when near to 80-90% confluency and detach using 0.35 mM EDTA in PBS as follows: add 3 ml EDTA per 75 cm2 culture flask, incubate for 15 min at 37 °C and 5% CO2 until cells have rounded and detached from the bottom, collect with 7 ml fresh medium, count cells, spin down cells for 5 min at 400 x g, and resuspend cells in fresh medium to the desired cell concentration (2 x 106/ml for binding assays).

  3. Detection of Muc1 on HEK293-Muc1 cells by immunofluorescence
    1. Split cells onto a 24-well plate containing circle cover glasses. Pipette up and down to make sure cells are not concentrating in the center of the dish well. Grow cells to 70-90% confluency.
    2. Fix cells in Clark’s solution (90% methanol; 10% glacial acetic acid) for 3 h at RT.
    3. Remove fixative and block with 2% BSA in PBS for 30 min.
    4. Incubate with FITC conjugated anti Muc1 antibody (1:200 dilution) in PBS 2% BSA for 2 h at room temperature in the dark.
    5. Wash with PBS three times for 5 min each.
    6. Take out the coverslip and place it on top of a tissue paper and let it dry out. The coverslip must be facing up.
    7. Once the coverslip has dried out, add a drop of anti-fade with DAPI onto the coverslip.
    8. Mount the coverslip on the slide inverting it so the sample will be in direct contact with the slide.
    9. Add coverslip sealant around the coverslip to seal it on the slide. Keep the samples at -20 °C in the dark until analysis by using a fluorescent microscope.

  4. EAEC binding assay in HEK293 cell suspension

    EAEC strains bind avidly to biotic and abiotic surfaces (such as plastic surfaces). To minimize unspecific binding to abiotic surfaces, cell-EAEC binding assays are performed in cell suspensions.
    Binding assays are performed with cell suspensions in 15 ml conical tubes as follows:

    1. Stain approximately a total of 2 x 106 cells in suspension by adding 300 µl of 5 µg/ml of FM 4-64FX (Thermo Scientific) and 4’,6’-diamidino-2-phenylindole (DAPI) for nucleus visualization for 5 min, followed by incubation with EAEC expressing GFP.
      Note: DAPI can also be added at the end of the experiment as part of the anti-fade solution. HEK293 cell membranes are stained with FM 4-64FX before EAEC infection to avoid staining of bacterial membranes. If wish, FM 4-64FX staining can be skipped and proceed with the infection with fluorescent bacteria.
    2. Remove the excess of FM 4-64FX dye on cells by adding 10 ml of HBSS to the tubes and spinning cells down at 250 x g (~1,500 rpm) for 5 min.
    3. Mix 1 x 106 FM 4-64FX-stained HEK293 cell derivatives expressing or not Muc1 with approximately 2 x 107 CFUs of GFP-EAEC strains to a multiplicity of infection (MOI) of 1:20 (cell:bacteria) in 1 ml of fresh DMEM medium. Incubate the cells for 1 h at 37 °C and 5% CO2.
    4. After 1 h incubation, wash the cells three times by adding 10 ml of HBSS buffer and by gently inverting the tube 2-3 times. Centrifuge the cells at 100 x g (~1,000 rpm) for 2 min to allow cell precipitation, but minimizing unbound bacteria sedimentation. Repeat HBSS-washes two more times.
      Note: Perform washes of HEK293-Muc1 cells by gentle inversion of the tube back and forth to avoid shedding of the highly glycosylated extracellular domain of Muc1 (and disengagement of bacteria), which is not covalently attached to its transmembrane C-terminal domain.
    5. Remove the supernatant by aspiration using a disposable transfer pipette or a vacuum system (care should be taken not to disturb or aspirate the cells at the bottom of the tube; most unbound bacteria settle at later times and lower speeds).
    6. Fix cells by resuspending in 100 µl of PBS-5% formalin (e.g., 50 µl PBS + 50 formalin at 5% [v:v]). Examine the cells immediately or save at 4 °C to analyze it later, preferably during the first 48 h.
    7. Use 10 µl of cell suspension to prepare a smear on glass slides by extending the cell suspension with a coverslip. Let smear to dry out for about 10 min, then coat smear with a drop of anti-fade-DAPI (Thermo Fisher Scientific).
    8. Analyze the slides by using a fluorescence microscope equipped with wavelength filters for blue (365/10 excitation/420LP emission), green (480/20 excitation/510LP emission), and red (535/30 excitation/580LP emission). You will see DNA stained in blue, cells membranes stained in red and bacteria in fluorescent green. A representative example of a binding assay with the prototype EAEC 042 strain and its isogenic fimbria mutant (042aafA) is illustrated in Figure 1.


      Figure 1. Binding of EAEC to HEK293-Muc1 cells. A. Production of Muc1 glycoprotein in HEK293 cells transfected with pcDNA3 (HEK293) or pcDNA3-MUC1 (HEK293-Muc1) was assessed with a FITC-conjugated monoclonal antibody to Muc1 by fluorescence microscopy (100x). B. HEK293 and HEK293-Muc1 cells were infected in suspension with EAEC strain 042 or with its isogenic fimbria mutant (042aafA), each expressing a GFP reporter and analyzed by fluorescence microscopy (40x). EAEC042 is in green, cell membranes are stained in red with FM 4-64FX, and DNA is stained in blue with DAPI. C. Bacteria adhered to cells were enumerated microscopically and data were analyzed by comparison of means in a paired t-test with significant differences P < 0.05 (*). Bars represent the means of at least three independent experiments, with the error bars indicating standard deviations.

    Data analysis

    EAEC adhered to HEK293 derivatives are enumerated microscopically (40x) by counting HEK293 cells and bacteria bound to cells in 20 randomized fields per coverslip from at least three independent experiments. Results are expressed as the number of bacteria per 100 cells. Data are analyzed by comparison of means in a paired t-test with significant differences when P < 0.05.

    Acknowledgments

    This protocol was partially reported in our previously published study (Boll et al., 2017). This work was supported by Departmental funds to FR-P (Pediatrics Department, University of Virginia). The authors declare no conflicts of interest or competing interests.

    References

    1. Berry, A. A., Yang, Y., Pakharukova, N., Garnett, J. A., Lee, W. C., Cota, E., Marchant, J., Roy, S., Tuittila, M., Liu, B., Inman, K. G., Ruiz-Perez, F., Mandomando, I., Nataro, J. P., Zavialov, A. V. and Matthews, S. (2014). Structural insight into host recognition by aggregative adherence fimbriae of enteroaggregative Escherichia coli. PLoS Pathog 10(9): e1004404.
    2. Boll, E. J., Ayala-Lujan, J., Szabady, R. L., Louissaint, C., Smith, R. Z., Krogfelt, K. A., Nataro, J. P., Ruiz-Perez, F. and McCormick, B. A. (2017). Enteroaggregative Escherichia coli adherence fimbriae drive inflammatory cell recruitment via interactions with epithelial MUC1. MBio 8(3).
    3. Corfield, A. P., Carroll, D., Myerscough, N. and Probert, C. S. (2001). Mucins in the gastrointestinal tract in health and disease. Front Biosci 6: D1321-1357.
    4. Czeczulin, J. R., Balepur, S., Hicks, S., Phillips, A., Hall, R., Kothary, M. H., Navarro-Garcia, F. and Nataro, J. P. (1997). Aggregative adherence fimbria II, a second fimbrial antigen mediating aggregative adherence in enteroaggregative Escherichia coli. Infect Immun 65(10): 4135-4145.
    5. Farfan, M. J., Inman, K. G. and Nataro, J. P. (2008). The major pilin subunit of the AAF/II fimbriae from enteroaggregative Escherichia coli mediates binding to extracellular matrix proteins. Infect Immun 76(10): 4378-4384.
    6. Harrington, S. M., Dudley, E. G. and Nataro, J. P. (2006). Pathogenesis of enteroaggregative Escherichia coli infection. FEMS Microbiol Lett 254(1): 12-18.
    7. Harrington, S. M., Strauman, M. C., Abe, C. M. and Nataro, J. P. (2005). Aggregative adherence fimbriae contribute to the inflammatory response of epithelial cells infected with enteroaggregative Escherichia coli. Cell Microbiol 7(11): 1565-1578.
    8. Izquierdo, M., Navarro-Garcia, F., Nava-Acosta, R., Nataro, J. P., Ruiz-Perez, F. and Farfan, M. J. (2014). Identification of cell surface-exposed proteins involved in the fimbria-mediated adherence of enteroaggregative Escherichia coli to intestinal cells. Infect Immun 82(4): 1719-1724.
    9. Jonsson, R., Struve, C., Boisen, N., Mateiu, R. V., Santiago, A. E., Jenssen, H., Nataro, J. P. and Krogfelt, K. A. (2015). Novel aggregative adherence fimbria variant of enteroaggregative Escherichia coli. Infect Immun 83(4): 1396-1405.
    10. Lillehoj, E. P., Kim, B. T. and Kim, K. C. (2002). Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin. Am J Physiol Lung Cell Mol Physiol 282(4): L751-756.
    11. Nataro, J. P., Deng, Y., Cookson, S., Cravioto, A., Savarino, S. J., Guers, L. D., Levine, M. M. and Tacket, C. O. (1995). Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers. J Infect Dis 171(2): 465-468.
    12. Nath, S. and Mukherjee, P. (2014). MUC1: a multifaceted oncoprotein with a key role in cancer progression. Trends Mol Med 20(6): 332-342.
    13. Rasko, D. A., Webster, D. R., Sahl, J. W., Bashir, A., Boisen, N., Scheutz, F., Paxinos, E. E., Sebra, R., Chin, C. S., Iliopoulos, D., Klammer, A., Peluso, P., Lee, L., Kislyuk, A. O., Bullard, J., Kasarskis, A., Wang, S., Eid, J., Rank, D., Redman, J. C., Steyert, S. R., Frimodt-Moller, J., Struve, C., Petersen, A. M., Krogfelt, K. A., Nataro, J. P., Schadt, E. E. and Waldor, M. K. (2011). Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany. N Engl J Med 365(8): 709-717.
    14. Yanez, D., Izquierdo, M., Ruiz-Perez, F., Nataro, J. P., Giron, J. A., Vidal, R. M. and Farfan, M. J. (2016). The role of fibronectin in the adherence and inflammatory response induced by enteroaggregative Escherichia coli on epithelial cells. Front Cell Infect Microbiol 6: 166.

简介

肠道集聚性大肠杆菌(EAEC)是全球儿童和成人急性腹泻的公认原因。 EAEC菌株的特征在于存在聚集粘附菌毛(AAF),其通过介导与肠粘膜的附着和通过引发宿主炎症反应而在发病机制中起关键作用。 聚合粘附菌毛II(AAF / II)是EAEC原型菌株042(EAEC042)对肠细胞最重要的粘附因子。 已经鉴定了上皮细胞上AAF / II的多种受体,包括跨膜信号传导粘蛋白Muc1。 该协议描述了测量EAEC菌株对表达Muc1糖蛋白的HEK293细胞的依从性的方法。

【背景】EAEC是世界范围内地方性和流行性腹泻病的重要原因。尽管发展中国家儿童腹泻最常见,但EAEC还与免疫受损成人腹泻,旅行者和工业化国家的食源性疾病有关,例如由志贺毒素(Stx)2a型产生的大致致命爆发2011年在北欧的血清型O104:H4的EAEC菌株(Harrington等人,2006; Rasko等人,2011)。 EAEC发病机制由生物体粘附肠细胞,产生肠毒素和细胞毒素并最终诱导炎症的能力决定(Harrington等,2006)。 EAEC对肠细胞的依从性由AAF菌毛粘附素介导(Czeczulin等人,1997)。迄今为止,已经描述了至少5种AAF菌毛的变体,全部编码在范围为55至65MDa的毒力质粒中(Jonsson等人,2015)。 AAF结构由带正电荷的主要亚基和在假伞结构顶端的推定的次要亚基组成(Berry等人,2014年)。

已显示展示AAF / II变体的原型EAEC菌株042在成年志愿者中产生腹泻(Nataro等,1995)。临床和实验室数据表明EAEC诱导炎性肠炎,而使用极化T84单层的研究表明IL-8的释放与AAF / II粘附素的存在相关(Harrington等,2005)。尽管EAEC对肠细胞的依从性的重要性已经确立,但参与由AAF菌毛介导的炎症反应的细胞受体尚未得到充分表征。针对AAF / II已经鉴定了上皮细胞上的几种受体,包括细胞外基质(ECM)蛋白质,例如纤连蛋白和层粘连蛋白,以及细胞角蛋白8(Farfan et al。,2008; Izquierdo et al。 ,2014)。然而,这些受体位于肠细胞的基底外侧。因此,这些蛋白质在初始感染EAEC过程中不可能发挥重要作用。此外,已经显示纤连蛋白不参与由AAF / II介导的炎症反应(Yanez等人,2016)。最近我们发现EAEC也与信号传导的Muc1糖蛋白结合,这种结合依赖于蛋白质的唾液酸化(Boll等人,2017)。粘蛋白(MUC)是由多种正常和恶性分泌上皮细胞表达的具有高碳水化合物含量(50-90%重量)的大分泌和跨膜糖蛋白(> 200kDa)(Corfield等人,2001)。 Muc1是一种多态跨膜粘蛋白样蛋白,其含有由糖基化多肽组成的大的胞外结构域,所述糖基化多肽由20个氨基酸序列的30-100个串联重复,跨膜结构域和72个氨基酸的细胞质尾(Nath和Mukherjee,2014)。 Muc1与许多恶性和炎症过程中的信号通路相关(Nath和Mukherjee,2014)。我们最近的研究显示Muc1与AAF / II介导的炎症反应有关。此外,EAEC 042上调依赖于AAF存在的上皮Muc1表达(Boll等人,2017)。

肠细胞中EAEC的多种受体的存在使该细胞谱系中特定受体的鉴定和表征复杂化。不同于肠细胞的HEK293细胞的使用允许通过用编码候选受体的质粒转染这些细胞来评估新的潜在受体。同样,用悬浮细胞进行的结合分析使EAEC菌株与非 - 非生物表面的结合最小化。该协议可用于找出其他肠道致病菌的潜在受体。在这里,我们详细描述了我们以前用来可视化EAEC与用Muc1编码质粒转染的HEK293细胞的依从性的方法。

关键字:Muc1, EAEC, 粘附, 粘蛋白, 糖蛋白, 菌毛, Hek293

材料和试剂

  1. 细胞培养瓶(康宁,目录号:3275)
  2. 20,200和1,000μl移液器吸头(Barrier Low-Retention tips,Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:2149P-05-RT,2069-HR和2279)
  3. 24孔板
  4. 圆盖玻璃(Fisher Scientific,Fisherbrand,目录号:12-545-80)
  5. 纸巾

  6. 15 ml锥形培养管(Corning,目录号:430052)
  7. 血清移液管(1,5,10,25,50 ml)(Corning,产品目录号:4012,4051,4482,4251,4501)
  8. 玻璃载玻片(Fisher Scientific,Fisherbrand,目录号:12-550-123)
  9. Coverslip(Fisher Scientific,Fisherbrand,目录号:12-543C)
  10. 细胞系:HEK293-pcDNA3.1和HEK293-pcDNA3.1-Muc1
    HEK293细胞(ATCC,目录号:CRL-1573)
    pcDNA3.1(Thermo Fisher Scientific,Invitrogen TM,目录号:V79020)
    注:pcDNA3.1-Muc1克隆由Dr. Erik P. Lillehoj < (Lillehoj et al。,2002)。
  11. EAEC042菌株可从我们的实验室菌种集合中获得(Nataro等,1995)
  12. pGFP载体(Takara Bio,Clontech,目录号:632370)
  13. LB培养基(Thermo Fisher Scientific,Invitrogen TM,目录号:10855001)
  14. 甘油(Fisher Scientific,Fisherbrand,目录号:BP229-4)
  15. Carbenicillin
  16. DMEM-高葡萄糖(无酚红),用于培养EAEC(Thermo Fisher Scientific,Gibco TM,产品目录号:31053028)
  17. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:70011044)
  18. 用于培养HEK293细胞(Thermo Fisher Scientific,Gibco TM,目录号:11965118)的Dulbecco's改良伊格尔培养基(DMEM)
  19. 经过认证的热灭活胎牛血清(FBS)(Sigma-Aldrich,目录号:F4135)
  20. 青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  21. G418(Sigma-Aldrich,目录号:G418-RO)
    制造商:Roche Diagnostics,目录号:4727878001。
  22. 0.5M EDTA(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9260G)
  23. 甲醇(Sigma-Aldrich,目录号:322415-1L)
  24. 冰醋酸(Fisher Scientific,Fisher Chemicals,目录号:A38-500)
  25. FITC小鼠抗人Muc1(CD227)(BD,BD Biosciences,目录号:559774)
  26. Coverslip密封胶(Biotium,目录号:23005)
  27. FM 4-64FX(Thermo Fisher Scientific,Invitrogen TM,目录号:F34653)
  28. 具有DAPI的ProLong TM Gold Antifade Mountant(Thermo Fisher Scientific,Invitrogen TM,产品目录号:P36935)
  29. 4',6-二脒基-2-苯基吲哚(DAPI,Dilactate)(Thermo Fisher Scientific,Invitrogen TM,目录号:D3571)
  30. Hank平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14175103)
  31. 福尔马林(Sigma-Aldrich,目录号:HT501128-4L)

设备

  1. 10,20,200,1000μl移液器(Pipette Pack Set,Eppendorf,目录号:2231300006)
  2. CO 2培养箱(NuAire,型号:NU-4750)
  3. -80°C冷冻箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma Model 900)
  4. 旋转摇床(ATR,型号:AJ118)
  5. 层流罩(Thermo Fisher Scientific,Thermo Scientific TM,型号:1300系列A2型号1375)
  6. 紫外可见分光光度计(GE Healthcare,Amersham Biosciences,型号:Ultrospec 2100 Pro)
  7. 液氮储存容器(Worthington,型号:LD35)
  8. 离心机(Eppendorf,型号:5430)
  9. 微量离心机(Fisher Scientific,型号:accuSpin TM Micro 17R)
  10. 落射荧光显微镜(奥林巴斯,型号:BX51)

程序

  1. 细菌细胞培养
    1. 在含有100μg/ ml羧苄青霉素的LB琼脂平板上划出一连串EAEC菌株(预先用pGFP质粒转化并在-80℃保存在含20%甘油的LB培养基中),并在37℃下温育16-18 h。
    2. 将3-5个EAEC菌落接种到5ml LB培养基中,并使用旋转摇动器在37℃下振荡(200rpm)孵育16-18小时。用新鲜的DMEM-高葡萄糖(无红色苯酚)从该培养物中进行1:100稀释以诱导AAF菌毛(例如100μl细菌培养物在9,900μlDMEM-HG中)的表达,并在37℃振荡(200rpm)孵育直至达到对数中期(OD 600 = 0.6)。
      注意:已知DMEM-HG上调AAF菌毛的表达。使用不含红色苯酚的DMEM-HG来促进肉眼和分光光度法监测该培养基中的EAEC生长。在DMEM-HG中生长时,EAEC需要约4小时才能达到OD 600 = 0.6。
    3. 以5,000xg离心EAEC培养物10分钟,用10ml 1x PBS洗涤,并以1×10 8个细胞/ ml的浓度重新悬浮于新鲜的DMEM(用于生长HEK293细胞的DMEM)中。菌落形成单位(CFU)。我们先前已经确定OD 600 = 0.35对应于每毫升约1×10 8 EAEC CFU。

  2. HEK293细胞培养
    1. 用补充有10%FBS(Sigma-Aldrich)的DMEM(Gibco)在75或150cm 2培养瓶中培养用pcDNA3.1和pcDNA3.1-Muc1质粒转染的人胚胎肾细胞系HEK293(ATCC) Aldrich),青霉素 - 链霉素和200μg/ ml G418(Sigma-Aldrich)(以确保维持pCDNA3.1质粒衍生物)。
    2. 为了传代和接种目的,当接近80-90%铺满时用10ml无菌PBS洗涤HEK293细胞,并使用PBS中的0.35mM EDTA分离,如下:每75cm2培养物加入3ml EDTA在37℃和5%CO 2下温育15分钟直到细胞已经圆化并从底部分离,用7ml新鲜培养基收集,计数细胞,在400下旋转细胞5分钟并将细胞重悬于新鲜培养基中达到所需的细胞浓度(2×10 6 / ml用于结合测定)。

  3. 免疫荧光检测HEK293-Muc1细胞上Muc1的表达
    1. 将细胞分装到含有圆圈盖玻片的24孔板上。上下移动以确保细胞不会集中在培养皿的中心。将细胞培养至70-90%汇合。

    2. 在Clark的溶液(90%甲醇; 10%冰醋酸)中固定细胞3小时

    3. 用PBS中的2%BSA移除固定剂并封闭30分钟
    4. 在室温下在黑暗中用FITC缀合的抗Muc1抗体(1:200稀释)在PBS 2%BSA中孵育2小时。
    5. 用PBS清洗三次,每次5分钟。
    6. 取出盖玻片,放在薄纸上,让它变干。盖玻片必须朝上。
    7. 一旦盖玻片干透了,将DAPI防褪色液滴加到盖玻片上。

    8. 将盖玻片安装在玻片上,使其与玻片直接接触。
    9. 在盖玻片周围添加盖玻片密封胶,将其密封在玻片上。将样品保存在-20℃的黑暗中,直到使用荧光显微镜进行分析。

  4. 在HEK293细胞悬液中的EAEC结合测定

    EAEC菌株可以与生物和非生物表面(如塑料表面)紧密结合。为了最小化对非生物表面的非特异性结合,细胞-EAEC结合测定在细胞悬浮液中进行。
    如下用15ml锥形管中的细胞悬液进行结合测定:

    1. 通过加入300μl5μg/ ml的FM 4-64FX(赛默飞世尔科技)和4',6'-二脒基-2-苯基吲哚(2μM),将大约2×10 6个细胞悬浮于悬浮液中DAPI)进行核显影5分钟,然后与表达EAEC的GFP一起温育。
      注意:DAPI也可以在实验结束时添加,作为防褪色解决方案的一部分。在EAEC感染之前用FM 4-64FX染色HEK293细胞膜以避免细菌膜染色。如果愿意,可以跳过FM 4-64FX染色并继续用荧光细菌感染。
    2. 通过向管中加入10ml HBSS并将细胞以250μgxg(〜1,500rpm)向下旋转5分钟来除去细胞上过量的FM 4-64FX染料。
    3. 将表达或不表达Muc1的1×10 6 FM 4-64FX染色的HEK293细胞衍生物与约2×10 7个CFU的GFP-EAEC菌株混合至多重感染( MOI)为1:20(细胞:细菌)的1ml新鲜DMEM培养基中。
      在37°C和5%CO 2下孵育细胞1小时。
    4. 孵育1小时后,通过加入10ml HBSS缓冲液洗涤细胞三次并轻轻翻转管2-3次。将细胞以100μg/ g(〜1,000rpm)离心2分钟以使细胞沉淀,但使未结合的细菌沉淀最小化。重复HBSS洗涤两次。
      注:通过来回轻柔倒转管来进行HEK293-Muc1细胞的洗涤以避免高度糖基化的Muc1细胞外结构域脱落(并且脱离细菌),其不共价连接到其跨膜C端域名。
    5. 使用一次性移液管或真空系统吸取上清液(注意不要打扰或抽吸管底部的细胞;大部分未结合的细菌会在稍后时间和较低的速度下沉降)。
    6. 通过重悬于100μlPBS-5%福尔马林(例如5%[v:v]的50μlPBS + 50福尔马林)中来固定细胞。立即检查细胞或保存在4°C以后分析,最好在前48小时。
    7. 用10μl细胞悬液通过用盖玻片延长细胞悬液来在载玻片上制备涂片。涂抹干燥约10分钟,然后涂抹一滴防褪色DAPI(赛默飞世尔科技)。
    8. 使用配备有蓝色(365/10激发/ 420LP发射),绿色(480/20激发/ 510LP发射)和红色(535/30激发/ 580LP发射)的波长滤光片的荧光显微镜分析载玻片。你会看到DNA染成蓝色,细胞膜染成红色,细菌呈荧光绿色。图1说明了使用原型EAEC 042菌株及其同基因菌毛变种(042aafA)进行结合分析的代表性实例。


      图1.EAEC与HEK293-Muc1细胞的结合A.在用pcDNA3(HEK293)或pcDNA3-MUC1(HEK293-Muc1)转染的HEK293细胞中用FITC缀合物评估Muc1糖蛋白的产生通过荧光显微镜检查Muc1的单克隆抗体(100x)。 B.将HEK293和HEK293-Muc1细胞悬浮于EAEC菌株042或其等基因菌毛突变体(042.eafA)的悬浮液中,各自表达GFP报道分子并通过荧光显微镜术(40x)分析。 EAEC042呈绿色,细胞膜用FM 4-64FX染成红色,DNA用DAPI染成蓝色。 C.通过显微镜计数粘附于细胞的细菌,并通过比较配对试验中的平均值和显着性差异来分析数据。 0.05(*)。条代表至少三次独立实验的方法,误差线表示标准偏差。

    数据分析

    至少从三次独立实验中,通过计数HEK293细胞并将细菌结合到20个随机化场中的细胞(每个盖玻片),在显微镜下(40x)枚举粘附于HEK293衍生物的EAEC衍生物。结果表示为每100个细胞的细菌数量。数据通过比较配对 t - 检验中的平均值,当 p


    致谢

    该协议部分在我们以前发表的研究中报道(Boll et al。,2017)。这项工作得到了部门资金支持FR-P(弗吉尼亚大学儿科)的支持。作者声明不存在利益冲突或利益冲突。

    参考

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    2. Boll,E.J.,Ayala-Lujan,J.,Szabady,R.L.,Louissaint,C.,Smith,R.Z.,Krogfelt,K.A.,Nataro,J.P.,Ruiz-Perez,F.and McCormick,B.A。(2017)。 Enteroaggregative 大肠杆菌粘附菌毛通过与上皮MUC1相互作用驱动炎症细胞募集。 MBio 8(3)。
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    13. Rasko,DA,Webster,DR,Sahl,JW,Bashir,A.,Boisen,N.,Scheutz,F.,Paxinos,EE,Sebra,R.,Chin,CS,Iliopoulos,D.,Klammer, Peluso,P.,Lee,L.,Kislyuk,AO,Bullard,J.,Kasarskis,A.,Wang,S.,Eid,J.,Rank,D.,Redman,JC,Steyert,SR,Frimodt-Moller ,J.,Struve,C.,Petersen,AM,Krogfelt,KA,Nataro,JP,Schadt,EE和Waldor,MK(2011)。 E的起源。大肠杆菌菌株导致德国溶血性尿毒症综合征的暴发。 N Engl J Med 365(8):709-717。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ayala-Lujan, J. L. and Ruiz-Perez, F. (2018). Adhesion of Enteroaggregative E. coli Strains to HEK293 Cells. Bio-protocol 8(8): e2802. DOI: 10.21769/BioProtoc.2802.
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