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May 2020

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Assay for Assessing Mucin Binding to Bacteria and Bacterial Proteins
评估粘蛋白与细菌和细菌蛋白结合的实验   

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Abstract

Legionella pneumophila, a Gram-negative bacterium and the causative agent of Legionnaires’ disease, exports over 300 effector proteins/virulence factors, through its type II (T2SS) and type IV secretion systems (T4SS). One such T2SS virulence factor, ChiA, not only functions as a chitinase, but also as a novel mucinase, which we believe aids ChiA-dependent virulence during lung infection. Previously published protocols manipulated wild-type L. pneumophila strain 130b and its chiA mutant to express plasmid-encoded GFP. Similarly, earlier studies demonstrated that wheat germ agglutinin (WGA) can be fluorescently labeled and can bind to mucins. In the current protocol, GFP-labeled bacteria were incubated with type II and type III porcine stomach mucins, which were then labeled with TexasRed-tagged WGA and analyzed by flow-cytometry to measure the binding of bacteria to mucins in the presence or absence of endogenous ChiA. In addition, we analysed binding of purified ChiA to type II and type III porcine stomach mucins. This protocol couples both bacterial and direct protein binding to mucins and is the first to measure Gram-negative bacterial binding to mucins using WGA and flow-cytometric analysis.


Graphic abstract:



Strategy for assessing bacterial and protein binding to mucins


Keywords: Mucin-bacteria binding (粘蛋白-细菌结合), Mucin (粘蛋白), ELISA (酶联免疫吸附试验), Mucin-binding proteins (粘蛋白结合蛋白), Bacterial flow-cytometry (细菌流式细胞术), Legionella pneumophila (嗜肺军团菌)

Background

Legionella pneumophila (Lpn), a Gram-negative bacterium, is the causative agent of Legionnaires’ disease, a severe form of pneumonia. Lpn is an intracellular pathogen that produces over 300 protein effectors that it secretes through either a type II secretion system (T2SS), or through a type IV secretion system (T4SS) (Hubber and Roy, 2010; White and Cianciotto, 2019). ChiA is one such T2SS protein effector. ChiA is an 81-kDa endochitinase that has a role in Lpn virulence during lung infection (Rehman et al., 2020). Lpn carrying a deletion of the ChiA gene (ΔchiA) shows decreased survival in the lungs of mice, compared to WT Lpn (DebRoy et al., 2006). While humans do not produce chitin, they do produce analogous glycoproteins, mucins, that have known properties in interacting with and blocking infection of other pathogens. In our study, we showed, for the first time, that ChiA is able to both bind to and degrade mucins (Rehman et al., 2020). To determine if live bacteria were able to bind to mucins, we utilized our previously published protocol to manipulate wild-type Lpn strain 130b and its chiA mutant to express a GFP-producing plasmid (DebRoy et al., 2006; Rondelet and Condemine, 2013). Furthermore, earlier studies showed that wheat germ agglutinin (WGA) binds to mucins (Bhavanandan and Katlic, 1979; Valdizan et al., 1992). Therefore, we utilized porcine stomach mucins which we labeled with TexasRed-conjugated WGA (Model et al., 2009). To determine whether ChiA directly binds to these mucins, we also used an ELISA based-assay with purified recombinant (N-terminally His-tagged) ChiA and detected binding using anti-His6 antibodies.


Although mucin binding to bacteria has been studied previously (Naughton et al., 2014), and fluorescently labeled WGA has been studied in the context of Gram-positive bacteria (Fife et al., 2000) this is the first protocol to directly label mucins and bacteria and then utilize flow cytometry to measure mucin binding to a Gram-negative bacterium. Furthermore, by analyzing both bacterial binding to mucins and the binding of purified proteins to mucins, this protocol provides insight into synergistic binding of different surface exposed bacterial proteins to mucins. This protocol can be further applied to study mucin binding to other Gram-negative bacteria.


Part I: Bacterial Mucin Binding with Flow Cytometry


Materials and Reagents

  1. Legionella pneumophila Brenner et al. (1979) (ATCC® BAA-74TM)

    1. WT 130b purchased from ATCC (see above)

    2. ChiA mutant as previously reported (DebRoy et al., 2006)

    3. GFP plasmid (pMGFP), as previously reported, was derived from pMMB-GRN/pMMB-Gent (addgene 45475). GFP is expressed from a Ptac promotor and therefore IPTG is required (White and Cianciotto, 2016) (Sturgill-Koszycki and Swanson, 2000). While copy number in Legionella is unknown, the PMMB-Gent derives from PMMB67EH (ATCC 37622)

  2. 1× Phosphate Buffered Saline (PBS) (Corning, catalog number: 21-040-CM)

  3. IPTG (Sigma-Aldrich, catalog number: I6758)

  4. Type II porcine stomach mucin (Sigma-Aldrich, catalog number: M1778)

  5. Type III porcine stomach mucin (Sigma-Aldrich, catalog number: M2378)

  6. TexasRed-tagged WGA (ThermoFisher Scientific, catalog number: W21405)

  7. Sodium carbonate, Na2CO3 (Sigma-Aldrich, catalog number: S7795)

  8. Sodium bicarbonate, NaHCO3 (Sigma-Aldrich, catalog number: S5761)

  9. Buffered Charcoal Yeast Extract (BCYE) agar plates (see Recipes)

    1. ACES (Sigma-Aldrich, catalog number: A9758)

    2. KOH (Sigma-Aldrich, catalog number: 221473)

    3. Yeast Extract (Sigma-Aldrich, catalog number: Y1625)

    4. α-Ketoglutaric acid sodium salt (Sigma-Aldrich, catalog number: K1875)

    5. Activated Charcoal (Sigma-Aldrich, catalog number: C9157)

    6. Bacteriological Agar (Sigma-Aldrich, catalog number: A5306)

    7. L-cysteine HCl (Sigma-Aldrich, catalog number: C1276)

    8. Ferric pyrophosphate (Sigma-Aldrich, catalog number: P6526)

  10. 50 mM Carb/Bicarb Buffer (see Recipes)

  11. Mucin solution (see Recipes)

Equipment

  1. Flow Cytometer (https://www.bdbiosciences.com/en-us/go-campaign/lsr-ii-comp-cont) using Blue Laser (488 nm), Long pass Filter 600 and 505, Band Pass Filter 600-620 and 500-550 (BD Biosciences, model: LSR II)

  2. General Purpose UV/Vis Spectrophotometer (Beckman Coulter, model: DU720)

  3. Forced Air Microbiological Incubators (VWR, catalog number: 89511-430)

Software

  1. GraphPad Prism version 8.0.0 for Mac (GraphPad Software, San Diego, California USA, www.graphpad.com)

  2. FlowJoTM Software for Mac, Version 8.8.6. (Ashland, OR: Becton, Dickinson and Company, www.flowjo.com)

Procedure

  1. Lpn Preparation

    1. Streak out Lpn strain 130b and ChiA mutant NU319, both harboring a GFP-expressing plasmid, onto BCYE agar plates containing IPTG at 1 mM.

    2. Incubate the plates for 3 days in a 37 °C incubator to grow out a lawn of bacteria.

    3. Resuspend ~3 swabs of scraped bacteria into 5 ml of PBS and read the optical density (OD) of each suspension at 660 nm using a DU720 spectrophotometer. Use PBS to dilute the bacterial suspension to an OD of 0.3, which corresponds to approx. 1 × 109 CFU/ml.


  2. Mucin Preparation

    Prepare both type II and type III porcine stomach mucin solutions in carb/bicarb buffer according to the recipe below.


  3. Mucin/Lpn Incubation

    1. Statically incubate 1 ml of the Lpn suspension with 100 μl of either type II porcine stomach mucin, type III porcine stomach mucins, or PBS for 1 h in a 25 °C or 37 °C incubator. Repeat every condition in triplicate.

      Note: Keep an aliquot of the type II and type III porcine stomach mucin solutions (no bacteria) as controls for the flow-cytometry analysis.

    2. Centrifuge, at room temperature, the mixtures for 5 min at 4,000 × g (for the 37 °C samples) or 8,000 × g (for the 25 °C samples).

    3. Wash each pellet in 1 ml of PBS

    4. Repeat centrifugation and wash step (Steps C2 and C3) three times.

    5. Resuspend pellet in 1 ml of PBS containing 7.5 μg of TexasRed-tagged WGA and incubate statically for 15 min at 25 °C.

    6. Repeat the centrifugation and wash steps (Steps C2 and C3) three times.

    7. Resuspend the pellet in 500 µl of PBS.


  4. Flow Cytometry

    Use BD LSRII flow cytometer to analyze suspensions via a TexasRed filter (Blue Laser – 488 nm: Long Pass 600, Band Pass 600-620) and GFP Filter (Blue Laser – 488 nm: Long Pass 505, Band Pass 500-550).

    1. Set gating TexasRed and GFP parameters using only mucin and only bacteria respectively.

    2. Collect a minimum of 500,000 events.

Data analysis

The experiment was replicated a minimum of three times, and each experiment had three technical replicates. FlowJo software was used to analyze percent of mucin binding to Lpn. Bacteria capable of expressing GFP and isolated mucin were used to set thresholds for both the TexasRed- and GFP-negative populations (unlabeled mucins and no IPTG-dependent GFP expression) and the TexasRed- and GFP-positive populations (TexasRed labeled mucins and IPTG-induced GFP expression). After setting these thresholds (see below for the four histograms on the left), the percent population (see below for quadrant analysis in the right-most flow-plot) of single GFP-positive (Quadrant [Q]3), single TexasRed-positive (Q1), and double GFP and TexasRed positive (Q2) were calculated using FloJo quadrant tool. Thus, Q2 was used as a calculation of percent of mucin binding to bacteria. Due to variability in mean fluorescence between experimental days, data were normalized to GFP-Lpn incubated with TexasRed fluorophore alone (background), see sample data table below and Figure 4D (Rehman et al., 2020). GraphPad Prism8 statistical software was used to graph and analyze the percent binding of bacteria to mucins. Prism8 analysis quantified standard deviation and used the two-tailed Student’s t-test function to test for significant differences between groups tested. Only P-values less then 0.05 were determined to be significant.


Flow Cytometry Protocol (Figure 1)



Figure 1. Flow Cytometry. Thresholds were set using single-positive samples 1. IPTG induced GFP production in Lpn (top left histogram) and 2. non-GFP bacteria with TexasRed labeled mucins (top middle histogram). Single negative populations were set using 1. Bacteria cultured without IPTG (bottom left histogram) 2. Non-GFP bacteria with un-labeled mucins (bottom middle histogram). For sample analysis, IPTG induced, GFP-expressing bacteria were co-incubated with TexasRed-labeled mucins. Gating parameters were used to setup quadrant percentiles for analysis. All events collected were partitioned into four quadrants using FloJo tool. Percentage of total population that were only TexasRed positive were labeled Q1, double GFP and TexasRed bacterial populations were labeled Q2, single GFP-positive populations were designated Q3 and non-fluorescent, all negative, populations were designated Q4. Percent of population that was GFP and TexasRed double positive (Q2) was used as a metric for mucin-bacteria binding and was analyzed in the sample table (Table 1) below.


Sample Data (Table 1)


Table 1. Percent of population that was double positive for TexasRed-mucin and GFP-Lpn (Q2) was used as a value for mucin binding to bacteria. In the single example of WT behavior in the flow plot shown above, this value was 85% (right-most plot in Figure 1). This value plus the values obtained from two additional independent replicates testing WT bacteria were added to the third row in the first three columns on the left, i.e., under headers Rep1, Rep2, Rep3. The raw values for three technical replicates obtained from analysis of another bacterial sample; i.e., a ChiA mutant, appear in the three left-most lanes in row-4. The value for each of the replicates was then normalized to GFP-bacteria (WT or mutant) incubated with TexasRed alone (as noted in rows 1 and 2), to quantify background TexasRed fluorophore binding to bacteria. For the two bacterial samples incubated with mucin, these values appear in columns 5, 6, and 7 for rows 3 and 4. Finally, the normalized values were averaged, and standard deviations calculated, as indicated in the last two columns. In this sample dataset, 22.5 + 5.4% of WT bacteria significantly bound the mucin.

Recipes

  1. BCYE agar plates

    1. Add 10 g of ACES to ~900 ml of double distilled water. Stir into solution

    2. Add ~2.2 g KOH. Stir into solution

    3. Add 10 g of Yeast Extract and 1 g α-ketoglutaric acid. Stir into solution

    4. Adjust the pH of the broth to 6.85-6.95. Use concentrated HCl or 10 N KOH as required. Add double distilled water to 1 L

    5. Dispense the broth into a 2-L flask

    6. Add 1.5 g activated charcoal and 15 g of agar

    7. Autoclave 20 min at 121 °C

    8. Prepare cysteine and iron solutions respectively by adding 0.4 g of L-cysteine HCl to 10 ml double distilled water and 0.25 g ferric pyrophosphate to 10 ml double distilled water. Once each solution is made, sterilize the solution by filter (0.22 µm).

    9. Cool the medium to 50 °C in a water bath after autoclaving

    10. Add separately 10 ml filter-sterilized (0.22 µm) cysteine and 10 ml filter-sterilized (0.22 µm) ferric pyrophosphate to the medium

    11. If making plates with IPTG, add 1 ml of 1 M Filter Sterilized (0.22 µm) IPTG

    12. Cool to 40 °CPour plates and store at 4 °C

    13. once solidified

  2. 50 mM Carb/Bicarb Buffer
    1. Dissolve 1.59 g Na2CO3 and 2.93 g NaHCO3 in 1 L of deionized water

    2. Adjust the pH to 9.6 using HCl or NaOH

  3. Mucin solution
    Stir for 30 min to dissolve 10 mg of either type II or type III porcine stomach mucin in 100 ml of 50 mM Carb/Bicarb buffer above. Autoclave for 15 min at 135 °C to sterilize and to further dissolve the mucin*.
    *Note: Mucin is difficult to dissolve and will not fully go into solution by mixing alone; thus, autoclaving is required to get a homogeneous solution. Fresh preparation of mucin is recommended for each experiment.

Part II: Recombinant Protein Mucin Binding with ELISA

Materials and Reagents

  1. Immulon 2-HB 96-well plates (VWR, catalog number: 735-0464)

  2. Purified N-terminally His-tagged protein samples

  3. Anti-His HRP-conjugated antibody (Sigma-Aldrich, catalog number: SAB4301134)

  4. Mucins from porcine stomachs type II (Sigma-Aldrich, catalog number: M2378)

  5. Mucins from porcine stomachs type III (Sigma-Aldrich, catalog number: M1778)

  6. Sodium carbonate, Na2CO3(Sigma-Aldrich, catalog number: S7795)

  7. Sodium bicarbonate, NaHCO3(Sigma-Aldrich, catalog number: S5761)

  8. Bovine serum albumin (BSA) (Melford, catalog number: A30075) Phosphate buffered saline (PBS) (Fisher Scientific, catalog number: BP399-1)

  9. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884) Zinc chloride, ZnCl2(Sigma-Aldrich, catalog number: 229997)

  10. Tween 20 (Sigma-Aldrich, catalog number: P1379)

  11. o-Phenylenediamine dihydrochloride tablets (OPD) (Sigma-Aldrich, catalog number: P9187)

  12. SnakeSkin® dialysis tubing (Thermo Scientific, catalog number: 68100)

  13. Incubation buffer (see Recipes)

  14. 50 mM Carb/Bicarb Buffer (see Recipes)

  15. Mucin solution (see Recipes)Blocking buffer (see Recipes)

Equipment

  1. Plate reader (LabSystems iEMS Reader MF, catalog number: 5921200) using 450 nm filter

  2. Incubator (New Brunswick Innova 4230)

  3. Centrifuge (Eppendorf, model: 5810R)

Software

  1. Ascent Microplate Reader Software version 2.7.0 for Windows

  2. Microsoft Excel version 16.43 for Mac; although any database analysis software is appropriate

Procedure

  1. Protein preparation
    Dialyze sample proteins at 100 μM into incubation buffer, prepared according to the recipe below. Adjust the final protein concentration after dialysis to 10 μM.


  2. Mucin preparation
    Prepare both type II and type III porcine stomach mucin solutions in carb/bicarb buffer according to the recipe below.


  3. Incubation

    1. Statically incubate an Immulon 2-HB 96-well plate with 50 μl of either type II porcine stomach mucin, type III porcine stomach mucins, or PBS overnight in a 4 °C incubator. Repeat every condition in triplicate.

    2. Remove the mucin solution and block the wells with 200 μl of blocking buffer, according to the recipe below, statically for 1 h in a 24 °C incubator.

    3. Remove the blocking buffer and wash once by adding 200 μl of incubation buffer and incubating statically on the bench for 5 min. Next incubate the wells statically for 3 h in a 24 °C incubator with 50 μl of purified protein samples or PBS.

    4. Wash with 200 μl of incubation buffer as described above and repeat for a total number of 4 washing steps.


  4. Detection
    1. Remove the buffer and incubate the wells with 50 μl of anti-His-HRP antibody, diluted 1:2,000 in incubation buffer, statically for 1 h in a 24 °C incubator.

    2. Wash four times as described above with 200 μl of incubation buffer.

    3. Add 150 μl of o-Phenylenediamine dihydrochloride to each well and leave the plate in a 24 °C incubator without shaking for 30 min in the dark.

    4. Record at 450 nm using a plate reader.

Data analysis

The experiment was replicated a minimum of three times, and each experiment had three technical replicates. Data were imported into Microsoft Excel software and baseline corrected with the incubation with PBS alone samples. Sample data for ChiA, NttE (negative control) and SslE (positive control) is given below for binding to type II porcine stomach mucin (also see Figure 4C, Rehman et al., 2020). Excel analysis provided standard deviation values and a two-tailed Student’s t-test function was used to test for significant differences between groups tested. Only P-values less then 0.05 were determined to be significant.

Sample Data (Table 2)

Table 2. Example data for proten binding to type II stomach mucin extracts

Rep1 Rep2 Rep3 Mean (PBS) PBS subtracted Rep 1 PBS subtracted Rep 2 PBS subtracted Rep 3 Mean
(mucin)
Stdev
(mucin)
ChiA_PBS 0.239 0.190 0.181 0.203 - - - - -
NttE_PBS (-) 0.084 0.074 0.062 0.073 - - - - -
SslE_PBS (+) 0.130 0.164 0.156 0.150 - - - - -
ChiA_mucin 0.933 0.846 0.900 - 0.730 0.643 0.697 0.690 0.044
NttE_mucin (-) 0.138 0.119 0.151 - 0.065 0.046 0.078 0.063 0.016
SslE_mucin (+) 0.718 0.912 0.910 - 0.568 0.762 0.760 0.697 0.111


Recipes

  1. Incubation buffer (prepare fresh and store at 4 °C)

    1. Mix/dissolve 0.5 g BSA and 0.5 ml Tween-20 for every 1 L of PBS buffer

    2. Cool to 4 °C

  2. 50 mM Carb/Bicarb Buffer (can be prepared in advance and stored at room temperature)
    1. Dissolve 1.59 g Na2CO3 and 2.93 g NaHCO3 in 1 L of deionized water

    2. Adjust the pH to 9.6 using HCl or NaOH

  3. Mucin solution (can be prepared the day before and stored at 4 °C)
    1. Stir for 30 min to dissolve 10 mg of either type II or type III porcine stomach mucin in 100 ml of 50 mM Carb/Bicarb buffer above.

    2. Autoclave for 15 min at 135 °C to sterilize and to further dissolve the mucin*.

    *Note: Mucin is difficult to dissolve and will not fully go into solution by mixing alone; thus, autoclaving is required to get a homogeneous solution.

  1. Blocking buffer (can be prepared the day before and stored at 4 °C)

    1. Mix/dissolve 0.1 g BSA and 0.5 ml Tween-20 for every 1 L of PBS buffer

    2. Cool to 4 °C

Acknowledgments

Work in the Cianciotto lab was supported by a National Institutes of Health grant R01AI 043987. LSG and RCW were also partly supported by National Institutes of Health training grants T32 GM08061 and T32 AI0007476, respectively. We thank the Northwestern ImmunoBiology Flow Cytometry Core for the maintenance and use of their equipment. Work in the Garnett lab was supported by MRC grants MR/M009920/1 and MR/R017662/1.

Competing interests

NPC and JAG have a pending patent application (63/005,592) that describes the use of ChiA for therapeutic applications.

Ethics

No human or animal subjects were used in this protocol.

References

  1. Bhavanandan, V. P. and Katlic, A. W. (1979). The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid. J Biol Chem 254(10): 4000-4008.

  2. Brenner, D. J., Steigerwalt, A. G. and McDade, J. E. (1979). Classification of the Legionnaires' disease bacterium: Legionella pneumophila, genus novum, species nova, of the family Legionellaceae, familia nova. Ann Intern Med 90(4): 656-658.
  3. DebRoy, S., Dao, J., Soderberg, M., Rossier, O. and Cianciotto, N. P. (2006). Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung. Proc Natl Acad Sci U S A 103(50): 19146-19151.
  4. Fife, D. J., Bruhn, D. F., Miller, K. S. and Stoner, D. L. (2000). Evaluation of a fluorescent lectin-based staining technique for some acidophilic mining bacteria.Appl Environ Microbiol 66(5): 2208-2210.
  5. Hubber, A. and Roy, C. R. (2010). Modulation of host cell function by Legionella pneumophila type IV effectors. Annu Rev Cell Dev Biol 26: 261-283.
  6. Model, M. A., Reese, J. L. and Fraizer, G. C. (2009). Measurement of wheat germ agglutinin binding with a fluorescence microscope. Cytometry A 75(10): 874-881.
  7. Naughton, J., Duggan, G., Bourke, B. and Clyne, M. (2014). Interaction of microbes with mucus and mucins: recent developments. Gut Microbes 5(1): 48-52.
  8. Rehman, S., Grigoryeva, L. S., Richardson, K. H., Corsini, P., White, R. C., Shaw, R., Portlock, T. J., Dorgan, B., Zanjani, Z. S., Fornili, A., Cianciotto, N. P. and Garnett, J. A. (2020). Structure and functional analysis of the Legionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation. PLoS Pathog 16(5): e1008342.
  9. Rondelet, A. and Condemine, G. (2013). Type II secretion: the substrates that won't go away. Res Microbiol 164(6): 556-561.
  10. Sturgill-Koszycki, S. and Swanson, M. S. (2000). Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles. J Exp Med 192(9): 1261-1272.
  11. Valdizan, M. C., Julian, J. and Carson, D. D. (1992). WGA-binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells. J Cell Physiol 151(3): 451-465.
  12. White, R. C. and Cianciotto, N. P. (2019). Assessing the impact, genomics and evolution of type II secretion across a large, medically important genus: the Legionella type II secretion paradigm. Microb Genom 5(6): e000273.
  13. White, R. C., and Cianciotto, N. P. (2016). Type II secretion is necessary for optimal association of the Legionella-containing vacuole with macrophage Rab1B but enhances intracellular replication mainly by Rab1B-independent mechanisms.Infect Immun 84 (12): 3313-3327.
  • 简介

    [摘要]嗜肺军团杆菌是革兰氏阴性细菌,是军团菌病的病原体,通过其II型(T2SS)和IV型分泌系统(T4SS)出口了300多种效应蛋白/毒力因子。一种这样的T2SS毒力因子ChiA不仅起几丁质酶的作用,而且还起新型粘蛋白酶的作用,我们认为它可以在肺部感染期间帮助ChiA依赖性毒力。以前发表的协议操纵野生型肺炎嗜血杆菌130b菌株及其chiA突变体,以表达质粒编码的GFP。同样,较早的研究表明,小麦胚芽凝集素(WGA)可以进行荧光标记,并可以与粘蛋白结合。 在当前方案中,将GFP标记的细菌与II型和III型猪胃粘蛋白孵育,然后用TexasRed标记的WGA进行标记,并通过流式细胞术进行分析,以测量在有或没有HLA的情况下细菌与粘蛋白的结合。内源性ChiA。另外,我们分析了纯化的ChiA与II型和III型猪胃粘蛋白的结合。该方案将细菌和直接蛋白结合到粘蛋白上,并且是第一个使用WGA和流式细胞术分析革兰氏阴性细菌与粘蛋白结合的方法。



    图形摘要:

    自动生成手机说明的屏幕截图

    评估细菌和蛋白质与粘蛋白结合的策略


    [背景技术]嗜肺军团菌(LPN ),革兰氏阴性细菌,是军团病,肺炎的严重形式的病原体。L pn是一种细胞内病原体,会产生300多种蛋白质效应子,可通过II型分泌系统(T2SS)或IV型分泌系统(T4SS)分泌(Hubber和Roy ,2010; White和Cianciotto ,2019 )。ChiA是一种这样的T2SS蛋白效应子。ChiA是一种81 kDa的内切几丁质内切酶,在肺部感染期间对Lpn毒力起作用(Rehman等人,2020年)。LPN携带缺失嘉基因的(Δ嘉)的节目在小鼠的肺中存活率降低,与WT相比LPN (德布鲁瓦等人,2006年)。尽管人类不产生几丁质,但它们确实产生类似的糖蛋白,粘蛋白,它们具有与其他病原体相互作用和阻断其感染的已知特性。在我们的研究中,我们首次证明了ChiA能够结合并降解粘蛋白(Rehman等,2020 )。为了确定活细菌是否能够与粘蛋白结合,我们利用了先前发表的方案来操纵野生型e Lpn菌株130b及其chiA突变体来表达产生GFP的质粒(DebRoy等,2006; Rondelet和Condemine , 2013 )。此外,较早的研究表明,小麦胚芽凝集素(WGA)与粘蛋白结合(Bhavanandan和Katlic ,1979; Valdizan等,1992 )。因此,我们利用了猪胃粘蛋白,将其用TexasRed共轭的WGA进行了标记(Model等,2009 )。为了确定她的ChiA是否直接与这些粘蛋白结合,我们还使用了基于ELISA的测定与纯化的重组(N末端带有His标签的)ChiA,并使用抗His 6抗体检测了结合。

    尽管粘蛋白与细菌的结合已有研究(Naughton等,2014 ),荧光标记的WGA已在革兰氏阳性细菌的背景下进行了研究(Fife等,2000 ),这是直接标记粘蛋白的第一个方案和细菌,然后利用流式细胞仪测量粘蛋白与革兰氏阴性细菌的结合。此外,通过分析细菌与粘蛋白的结合以及纯化的蛋白与粘蛋白的结合,该方案提供了对不同表面暴露的细菌蛋白与粘蛋白的协同结合的见解。该方案可进一步用于研究粘蛋白与其他革兰氏阴性细菌的结合。

    关键字:粘蛋白-细菌结合, 粘蛋白, 酶联免疫吸附试验, 粘蛋白结合蛋白, 细菌流式细胞术, 嗜肺军团菌



    第一部分:B流式细胞术与细菌黏蛋白结合


    材料和试剂


    1.嗜肺军团菌Brenner等。(1979 )(ATCC ® BAA-74 TM )     

    WT 130b从ATC C购买(见上文)
    如先前报道的ChiA突变体(DebRoy et al。,2006)
    如先前报道,GFP质粒(pMGFP)衍生自pMMB-GRN / pMMB-Gent(addgene 45475)。GFP是由Ptac启动子表达的,因此需要IPTG(White和Cianciotto ,2016)(Sturgill-Koszycki和Swanson ,200 0)。虽然军团菌中的拷贝数未知,但PMMB-Gent源自PMMB67EH(ATCC 37622)
    2. 1       ×磷酸盐缓冲盐水(PBS)(Corning,目录号:21-040-CM)


    3. IPTG(Sigma-Aldrich,目录号:I6758)     

    4. II型猪胃粘蛋白(Sigma-Aldrich,目录号:M1778)     

    5. III型猪胃粘蛋白(Sigma-Aldrich,目录号:M2378)     

    6. Texas带红色标签的WGA(ThermoFisher Scientific,目录号:W21405)     

    7.碳酸钠Na 2 CO 3 (西格玛奥德里奇,目录号:S7795)     

    8.碳酸氢钠NaHCO 3 (西格玛奥德里奇,目录编号:S5761)     

    9.缓冲木炭酵母提取物(BCYE)琼脂板(请参见食谱)     

    ACES(Sigma- Aldrich,目录号:A9758)
    KOH(Sigma-Aldrich,目录号:221473)
    酵母提取物(西格玛奥德里奇,目录号:Y1625)
    α-酮戊二酸钠盐(Sigma-Aldrich,目录号:K1875 )
    活性炭(西格玛奥德里奇,目录号:C9157)
    细菌琼脂(Sigma-Aldrich,目录号:A5306)
    L-半胱氨酸HCl(Sigma-Aldrich,目录号:C1276)
    焦磷酸铁(Sigma-Aldrich,目录号:P6526)
    10. 50 mM Carb / Bicarb缓冲液(请参阅食谱)   

    11.粘蛋白溶液(请参阅食谱)   



    设备


    使用蓝色激光(488 nm),长通滤光片600和505,带通滤光片600-620的流式细胞仪(https://www.bdbiosciences.com/zh-cn/go-campaign/lsr-ii-comp-cont)和500-550 (BD Biosciences,型号:LSR II )
    通用紫外/可见分光光度计(贝克曼库尔特,型号:DU720)
    强制空气微生物培养箱(VWR,目录号:89511-430)


    软件


    适用于Mac的GraphPad Prism版本8.0.0 (GraphPad软件,美国加利福尼亚圣迭戈,www.graphpad.com)
    适用于Mac的FlowJo TM软件,版本8.8.6 。(俄勒冈州阿什兰:Becton,Dickinson and Company ,www.flowjo.com)


    程序


    Lpn制备
    将都带有GFP表达质粒的Lpn菌株130b和ChiA突变体NU319划线到BCYE琼脂平板上,该平板含有1 mM的IPTG。
    在37℃下孵育平板3天 °C孵化器可以长出细菌的草坪。
    重悬〜3个拭子刮下细菌分成5米升的PBS,并且使用分光光度计DU720读取在660nm处的每个悬浮液的光密度(OD)。使用PBS稀释细菌悬液至OD为0.3,对应于大约 1 × 10 9 CFU /毫升。


    粘蛋白制备
    根据以下配方,在carb / bicarb缓冲液中准备II型和III型猪胃粘蛋白溶液。


    粘蛋白/ Lpn孵育
    在25 °C或37 °C的培养箱中,将1 ml的Lpn悬液与100μl的II型猪胃粘蛋白,III型猪胃粘蛋白或PBS静态孵育1小时。一式三份重复每个条件。
    注意:保留等分的II型和III型猪胃粘蛋白溶液(无细菌)作为流式细胞仪分析的对照。


    离心机,在室温下,5分钟的混合物在4 ,000 ×克(对于37 ℃下的样品)或8 ,000 ×克(对于25 ℃下的样品)。
    用1毫升PBS洗涤每个沉淀
    重复离心和洗涤步骤(小号TEP小号Ç 2和C ^ 3)三次。
    将沉淀重悬于1 ml含7.5的PBS中 μg带有TexasRed标签的WGA,在25 °C静态孵育15分钟。
    重复三次离心和洗涤步骤(步骤C2和C3 )。
    将沉淀重悬于500 µl PBS中。


    流式细胞仪
    我们è BD LSRII流式细胞仪来分析悬液通过一个TexasRed过滤器(蓝色激光- 488纳米:长通600,带通滤波器600-620)和GFP过滤器(蓝色激光- 488纳米:长通505,带通滤波器500-550) 。


    仅使用粘蛋白和仅细菌设置门控TexasRed和GFP参数。
    至少收集500,000个事件。


    数据分析


    该实验至少重复3次,每个实验有3个技术重复。使用FlowJo软件分析粘蛋白与Lpn的结合百分比。使用能够表达GFP和分离的粘蛋白的细菌为TexasRed和GFP阴性群体(未标记的粘蛋白,且无IPTG依赖的GFP表达)以及TexasRed和GFP阳性群体(TexasRed标记的粘蛋白和IPTG-)设置阈值诱导的GFP表达)。设置了这些阈值(左侧的四个直方图,请参见下文)后,单个GFP阳性(象限[Q] 3),单个TexasRed-使用FloJo象限工具计算阳性(Q1),双GFP和TexasRed阳性(Q2)。因此,Q2被用作粘蛋白与细菌结合的百分比的计算。由于实验日之间平均荧光的差异,将数据标准化为单独用TexasRed荧光团孵育的GFP-Lpn(背景),请参见下面的样品数据表和图4D (Rehman等,2020 )。使用GraphPad Prism8统计软件对细菌与粘蛋白的结合百分比进行绘图和分析。Prism8分析量化了标准偏差,并使用了两尾学生t检验函数来检验测试组之间的显着差异。仅小于0.05的P值被确定为显着。


    流式细胞仪协议(图1)




    图1 。流式细胞术。使用单阳性样品设置阈值。1. IPTG在Lpn中诱导GFP产生(左上方直方图),以及2.具有TexasRed标记粘蛋白的非GFP细菌(顶部中部直方图)。使用1.设置无IPTG的细菌(左下直方图)。2.带有未标记粘蛋白的非GFP细菌(下中直方图)。为了进行样品分析,将IPTG诱导的,表达GFP的细菌与TexasRed标记的粘蛋白共孵育。门控参数用于设置用于分析的象限百分位。使用FloJo工具将收集的所有事件划分为四个象限。将仅德克萨斯红阳性的总人口的百分比标记为Q1,将双GFP和将德克萨斯红细菌的种群标记为Q2,将单个GFP阳性群体标记为Q3,将所有非荧光的阴性群体标记为Q4。GFP和TexasRed双阳性(Q2)的人口百分比用作黏蛋白与细菌结合的指标,并在下面的样本表中进行了分析(表1)。


    样品沓TA (表1)


    表1 。对德克萨斯红粘蛋白和GFP- Lpn (Q2)双阳性的人口百分比用作粘蛋白与细菌结合的值。在上述所示的流程情节WT行为单例如,该值是85%(最右边的曲线图图URE 1)。该值加上从另外两个测试WT细菌的独立重复获得的值添加到左侧前三列的第三行,即标题Rep1,Rep2,Rep3下。通过对另一种细菌样品的分析获得的三个技术重复样品的原始值;即ChiA突变体出现在第4行的最左侧的三个泳道中。然后将每个重复样本的值标准化为单独与TexasRed一起孵育的GFP细菌(WT或突变体)(如第1和2行所述),以量化背景TexasRed荧光团与细菌的结合。对于用粘蛋白孵育的两个细菌样品,这些值出现在第3行和第4行的第5、6和7列中。最后,对标准化值进行平均,并计算标准差,如最后两列所示。在此样本数据集中,22.5 + 5.4%的WT细菌显着结合了粘蛋白。




    菜谱


    BCYE琼脂板
    将10克ACES加入约900毫升双蒸馏水中。搅拌入溶液
    加入〜2.2 g KOH。搅拌入溶液
    加入10克酵母提取物和1克α-酮戊二酸。搅拌入溶液             
    调整的pH值的肉汤6.85-6.95。使用浓缩HC升或10 -KOH作为必需的。加双倍蒸馏水至1升
    将肉汤分配到2升烧瓶中
    加入1.5克活性炭和15克琼脂
    在121 °C下高压灭菌20分钟
    制备半胱氨酸和IR分别通过添加0.4克L-半胱氨酸HC对溶液升至10ml双蒸水和0.25g的焦磷酸铁至10ml双蒸水。一旦每个溶液制成,消毒用溶液滤波器(0 0.22微米)。
    高压灭菌后,在水浴中将培养基冷却至50 °C
    分别添加10米升过滤灭菌的(0 0.22微米)半胱氨酸和10米升过滤灭菌的(0 0.22微米)焦磷酸铁到介质
    如果用IPTG使板,添加1米升1个的灭菌微米的过滤器(0 0.22微米)IPTG
    冷却至40 °C
    凝固后将板倒入并储存在4 °C下
    50 mM Carb / Bicarb缓冲液
    将1.59 g Na 2 CO 3和2.93 g NaHCO 3溶解在1 L去离子水中
    使用HCl或NaOH将pH调节至9.6
    粘蛋白溶液
    搅拌30分钟,以将10 mg II型或III型猪胃粘蛋白溶解在100 ml的上述50 mM Carb / Bicarb缓冲液中。在135 °C下高压灭菌15分钟以灭菌并进一步溶解粘蛋白*。


    *注意:粘蛋白很难溶解,仅靠混合不能完全溶解。因此,需要高压灭菌以获得均匀的解决方案。建议为每个实验新鲜制备粘蛋白。


    第二部分:[R ecombinant蛋白粘蛋白与ELISA的结合


    材料和试剂


    Immulon 2-HB 96孔板(VWR,目录号:735-0464 )
    纯化的N端带有His标签的蛋白质样品
    抗His HRP偶联抗体(Sigma-Aldrich,目录号:SAB4301134 )
    来自II型猪胃的粘蛋白(Sigma-Aldrich,目录号:M2378 )
    来自III型猪胃的粘蛋白(Sigma-Aldrich,目录号:M1778)
    碳酸钠Na 2 CO 3 (Sigma-Aldrich,目录号:S7795)
    碳酸氢钠NaHCO 3 (Sigma-Aldrich,目录号:S5761)
    牛血清白蛋白(BSA)(Melford,目录号:A30075)
    磷酸盐缓冲盐水(PBS)(Fisher Scientific,目录号BP399-1)
    乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
    氯化锌ZnCl 2 (Sigma-Aldrich,目录号:229997)
    吐温20(Sigma-Aldrich,目录号:P1379)
    邻苯二胺二盐酸盐片(OPD)(Sigma-Aldrich,目录号:P9187)
    蛇皮®透析管(Thermo Scientific的,目录号:68100 )
    孵育缓冲液(请参阅食谱)
    50 mM Carb / Bicarb缓冲液(请参阅食谱)
    粘蛋白溶液(请参阅食谱)
    阻塞缓冲区(请参见食谱)


    设备


    P后期读取器(LABSYSTEMS读者的iEMS MF ,:目录号5921200)使用450nm的过滤器
    孵化器(New Brunswick Innova 4230)
    离心机(Eppendorf ,型号:5810R)


    软件


    适用于Windows的Ascent Microplate Reader软件版本2.7.0
    适用于Mac的Microsoft Excel版本16.43;尽管任何数据库分析软件都适用


    程序


    蛋白质制备
    将样品蛋白质以100μM的浓度注入根据以下配方制备的孵育缓冲液中。将透析后的最终蛋白质浓度调整为10μM。


    粘蛋白制剂
    根据以下配方,在carb / bicarb缓冲液中准备II型和III型猪胃粘蛋白溶液。


    孵化
    将Immulon 2-HB 96孔板与50μlII型猪胃粘蛋白,III型猪胃粘蛋白或PBS在4°C的培养箱中静态孵育过夜。一式三份重复每个条件。
    除去粘蛋白溶液和用200μl阻断缓冲液阻断的孔中,根据下面的配方,静态1 ħ在一个24 ℃的培养箱中。
    除去封闭缓冲液,通过添加200μl孵育缓冲液并在长凳上静态孵育5分钟来洗涤一次。接下来,将孔在24°C的培养箱中与50μl纯化的蛋白质样品或PBS静态孵育3 h 。
    如上所述,用200μl孵育缓冲液洗涤,并重复进行总共4个洗涤步骤。


    侦查
    2:删除缓冲器,并用50微升抗His-HRP抗体,稀释1的孵育的孔,在培养缓冲液000,静态1个小时在24℃下孵化。
    如上所述用200μl孵育缓冲液洗涤四次。
    向每个孔中加入150μl邻苯二甲胺二盐酸盐,然后将板置于24°C的培养箱中,在黑暗中不摇动30分钟。
    使用酶标仪在450 nm处记录。




    数据一nalysis


    该实验至少重复3次,每个实验有3个技术重复。数据将导入到Microsoft Excel软件中,并通过与PBS单独的样品一起孵育来校正基线。下面给出了ChiA,NttE(阴性对照)和SslE(阳性对照)与II型猪胃粘蛋白结合的样品数据(另请参见图4C ,Rehman等,2020)。Excel分析提供标准偏差值,并使用两尾学生t检验函数来检验测试组之间的显着差异。仅小于0.05的P值被确定为显着。



    样本数据(表1)


    表1.与II型胃粘蛋白提取物的蛋白结合的示例数据


    菜谱


    孵育缓冲液(准备新鲜并在4 ° C下储存)
    每1 L PBS缓冲液混合/溶解0.5 g BSA和0.5 ml Tween - 20
    冷却至4 °C
    50 mM Carb / Bicarb缓冲液(可预先制备并在室温下保存)
    将1.59 g Na 2 CO 3和2.93 g NaHCO 3溶解在1 L去离子水中
    使用HCl或NaOH将pH调节至9.6
    粘蛋白溶液(可以在前一天制备并在4 °C下保存)
    搅拌30分钟以将10 mg II型或III型猪胃粘蛋白溶解在100 ml的上述50 mM Carb / Bicarb缓冲液中。
    在135 °C下高压灭菌15分钟以灭菌并进一步溶解粘蛋白*。
    *注意:粘蛋白很难溶解,仅靠混合不能完全溶解。因此,需要高压灭菌以获得均匀的溶液。


    封闭缓冲液(可在前一天制备并保存在4 °C )
    每1 L PBS缓冲液混合/溶解0.1 g BSA和0.5 ml Tween - 20
    冷却至4 °C




    致谢


    Cianciotto实验室的工作得到了美国国立卫生研究院拨款R01AI 043987的支持。LSG和RCW也分别得到了美国国立卫生研究院培训补助金T32 GM08061和T32 AI0007476的部分支持。我们感谢西北免疫生物学流式细胞仪核心设备的维护和使用。Garnett实验室的工作得到了MRC赠款MR / M009920 / 1和MR / R017662 / 1的支持。


    利益争夺


    NPC和JAG拥有待决的专利申请(63 / 005,592),其中描述了ChiA在治疗应用中的用途。


    伦理


    此协议中未使用任何人类或动物受试者。


    参考


    Bhavanandan,副总裁和Katlic,AW(1979)。小麦胚芽凝集素与唾液糖蛋白的相互作用。唾液酸的作用。生物化学杂志254(10):4000-4008。
    Brenner,DJ,Steigerwalt,AG和McDade,JE(1979)。军团菌病细菌的分类:军团菌,肺炎军团菌,军团菌科新星种,家族新星。实习生医学90(4):656-658。
    DebRoy,S.,Dao,J.,Soderberg,M.,Rossier,O.和Cianciotto,NP(2006)。II型肺炎军团菌分泌基因组显示独特的外蛋白和几丁质酶,可促进细菌在肺部的持久性。美国国家科学院院刊103(50):19146-19151。
    法夫,DJ,布鲁恩,DF,米勒,堪萨斯州和斯托纳,DL(2000年)。对某些嗜酸性采矿细菌的基于荧光凝集素的染色技术的评估。应用环境微生物学66(5):2208-2210。
    Hubber,A. and Roy,CR(2010)。IV型嗜肺军团菌效应子对宿主细胞功能的调节。Annu Rev Cell Dev Biol 26:261-283。
    模型,硕士,里斯,JL和弗雷泽,GC(2009年)。用荧光显微镜测量小麦胚芽凝集素的结合。细胞计数法A 75(10):874-881。
    Naughton,J.,Duggan,G.,Bourke,B.和Clyne,M.(2014年)。微生物与粘液和粘蛋白的相互作用:最新进展。肠道微生物5(1):48-52。
    Rehman,S.,Grigoryeva,LS,Richardson,KH,Corsini,P.,White,RC,Shaw,R.,Portlock,TJ,Dorgan,B.,Zanjani,ZS,Fornili,A.,Cianciotto,NP和Garnett ,JA(2020)。嗜肺军团菌几丁质酶ChiA的结构和功能分析揭示了金属依赖性粘蛋白降解的新机制。PLoS Pathog 16(5):e1008342。
    Rondelet,A.和Condemine,G.(2013年)。II型分泌物:不会消失的底物。Res Microbiol 164(6):556-561。
    Sturgill-Koszycki,S.和Swanson,MS(2000)。嗜肺军团菌复制液泡成熟成酸性内吞细胞器。实验医学杂志192(9):1261-1272。
    Valdizan,MC,Julian,J.和Carson,DD(1992)。WGA结合的粘蛋白糖蛋白可保护小鼠子宫上皮细胞的顶端细胞表面。J细胞生理学151(3):451-465。
    White,RC和Cianciotto,NP(2019)。在医学上重要的一个大类中评估II型分泌的影响,基因组学和进化:军团菌II型分泌范例。微型B GENOM 5(6):e000273。
    White,RC和Cianciotto,NP(2016)。II型分泌对于含军团菌的液泡与巨噬细胞Rab1B的最佳结合是必需的,但主要通过Rab1B独立机制增强细胞内复制。感染免疫84(12):3313-3327。
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    引用:Grigoryeva, L. S., Rehman, S., White, R. C., Garnett, J. A. and Cianciotto, N. P. (2021). Assay for Assessing Mucin Binding to Bacteria and Bacterial Proteins. Bio-protocol 11(5): e3933. DOI: 10.21769/BioProtoc.3933.
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