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Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria
采用单细胞流式细胞术测定细菌的肽吸收   

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Abstract

Antimicrobial peptides (AMPs) can target the bacterial envelope or alternatively have intracellular targets. The latter requires uptake of the peptide by the bacterial cells. The bacterial internalization of an AMP can be evaluated by a fluorescence-based method that couples the use of the fluorescently labelled AMP to the fluorescence quencher trypan blue. Trypan blue is excluded from the interior of intact cells and the fluorescence of the extracellular peptide or of the peptide bound on the bacterial surface can be quenched by it, while the fluorescence of the internalized peptide is not affected. The uptake of the peptide by the bacteria is determined by measuring the fluorescence in individual cells by flow cytometry.

Keywords: Antimicrobial peptide(抗菌肽), Flow cytometry(流式细胞术), Peptide uptake(肽摄取), Peptide transporter(肽转运蛋白), Trypan blue(台盼蓝), Propidium iodide uptake(碘化丙啶摄取)

Background

AMPs consist of a broad and diverse class of potent antimicrobials that have potential as novel therapeutic agents (Wang et al., 2015). AMPs are part of innate immunity and are produced by organisms of all kingdoms. They are mobilized by these organisms to fight infecting microbes, that can be either bacteria, fungi or viruses. They do so by directly killing the microbes, but they can also act as sentinels that alert other immune pathways. Interestingly, it has also become clear that AMPs are not only agents against bad microbes, but that they also have key roles in the control of symbiotic bacterial populations in animal and plant hosts (Maróti et al., 2011; Kondorosi et al., 2013).

The diversity of AMPs in sequence and structure is so large that it is difficult to classify them. Moreover, AMPs of different origin have also highly diverse modes of action. They can be broadly divided in peptides that target the bacterial envelope, destroying its cell barrier function by permeabilizing cell membranes, and peptides that are internalized and target a vital intracellular function (Scocchi et al., 2016). Therefore, in the initial characterization of a novel antibacterial peptide, it is important to determine its major site of action. The protocol we described here is based on a flow cytometry method and enables a rapid determination if an AMP of interest is internalized by bacteria at sublethal concentrations (Benincasa et al., 2009). This characterization can be done prior to the biochemical identification of the cellular targets.

We have applied the method to Escherichia coli, Salmonella typhimurium, Sinorhizbobium meliloti and Bradyrhizobium spp. using different antibacterial peptides, including the mammalian Bac7 peptide which inhibits the ribosomes (Mardirossian et al., 2014), and the plant peptide NCR247 which permeabilizes bacterial membranes but can also be internalized and bind diverse intracellular targets (Farkas et al., 2014; Guefrachi et al., 2015). This simple method can be easily adapted for use in other bacteria and other AMPs or other types of bioactive peptides. The method is also suitable for testing the activity of peptide uptake transporters in bacteria as illustrated in an example (Mattiuzzo et al., 2007; Guefrachi et al., 2015).

Materials and reagents

  1. Eppendorf tubes
  2. Sterile membrane filters 0.2 µm (SARSTEDT, catalog number: 83.1826.001 )
  3. Microscopy slides and cover-glasses (Chance Propper LTD)
  4. 96-well microplates, black with transparent bottom, 400 µl (Greiner Bio One, catalog number: 655096 )
  5. Bacteria of interest: e.g., Escherichia coli HB101, BW25113 (Mattiuzzo et al. 2007; Benincasa et al., 2009; Runti et al., 2013; Guida et al., 2015), Salmonella typhimurium ATCC 14028 (Benincasa et al., 2015), Sinorhizobium meliloti Sm1021 (Arnold et al., 2013; Guefrachi et al., 2015), Bradyrhizobium sp. ORS285 (Guefrachi et al., 2015)
  6. Bacterial growth media:
    1. Mueller-Hinton broth, MHB (see Recipes) (BD, DifcoTM, catalog number: 275710 ), for E. coli or S. typhimurium
    2. Yeast extract broth, YEB (see Recipes), for S. meliloti
    3. Yeast extract mannitol broth, YMB (see Recipes), for Bradyrhizobium
  7. Chemicals and components for bacterial growth media preparation and buffer solutions:
    a. Technical agar (BD, DifcoTM, catalog number: 281230 )
    b. Yeast extract (BD, BactoTM, catalog number: 212750 )
    c. Peptone (BD, BactoTM, catalog number: 211677 )
    d. Beef extract (Conda, catalog number: 1700 )
    e. Saccharose (VWR, catalog number: 27483.363 )
    f. Mannitol (VWR, catalog number: 25311.297 )
    g. Sodium glutamate (VWR, catalog number: 27872.298 )
    h. Magnesium sulfate heptahydrate (MgSO4·7H2O) (EMD Millipore, catalog number: 105886 )
    i. Dibasic potassium phosphate (K2HPO4) (VWR, catalog number: 26930.362 )
    j. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: S9390 )
    k. Sodium phosphate monobasic dehydrate (NaH2PO4·2H2O) (Sigma-Aldrich, catalog number: 71505 )
    l. Iron(III) chloride (FeCl3) (Sigma-Aldrich, catalog number: 701122 )
    m. Calcium chloride dihydrate (CaCl2·2H2O) (EMD Millipore, catalog number: 102382 )
    n. Sodium chloride (NaCl) (EMD Millipore, catalog number: 106404 )
    o. Sodium hydroxide (NaOH) (VWR, catalog number: 567530-250 )
    p. HCl (CARLO ERBA Reagents, catalog number: 403871 )
    q. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
    r. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  8. Stock solution of fluorescently labelled peptides
    Note: Fluorophores successfully used to label peptides are BODIPY FL (Guida et al., 2015), fluorescein (FITC) (Guefrachi et al., 2015), Alexa dye (Benincasa et al., 2010). Before internalization studies, check that labelling does not affect the biological activity of the peptide using a Minimal Inhibitory Concentration assay. Labelled peptides can be synthesized in house if a peptide synthesizer is available or obtained from a commercial supplier offering a custom peptide synthesis service (http://www.proteogenix.science/custom-peptide-synthesis/).
  9. Buffered-saline (BS) (see Recipes)
  10. Buffered high salt solution (BHSS) (see Recipes)
  11. Phosphate buffer (PB) (see Recipes)
  12. PB supplemented with Tween 20 (PBT) (see Recipes)
  13. Trypan blue (Sigma-Aldrich, catalog number: T6146) stock solution (see Recipes)
  14. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4170) stock solution (see Recipes)

Equipment

  1. Incubator for bacterial growth (FIRLABO, Bioconcept)
  2. Thermostatic bath (Thermo Fisher Scientific, Thermo ScientificTM, model: TSGP02 )
  3. Flow cytometer (Beckman Coulter, model: Cytomics FC 500 equipped with an argon laser [488 nm, 5 mW]) or Moflo Astrios (Beckman-Coulter, model: Moflo Astrios) equipped with an argon laser (488 nm, 100 mW) and photomultiplier tube fluorescence detectors for filtered light set at 525 nm for BODIPY (BY) detection (filter 526/52 nm)
    Note: The product 'Cytomics FC 500' has been discontinued.
  4. HeraeusTM PicoTM and FrescoTM centrifuge for Eppendorf tubes (Thermo Fisher Scientific, Thermo ScientificTM, model: Heraeus Pico 17 )
  5. Spectrophotometer (Amersham Biosciences, Ultrospec 10 cell density meter)
  6. Confocal microscope with an oil immersion objective lens (Nikon Eclipse C1si or Leica TCS SP X)
  7. Fluorescence plate reader (Tecan Trading, Infinite®, model: M200 )

Software

  1. FCS Express 3 or later version (De Novo Software, Los Angeles, CA)
  2. Summit 6.2.2(Beckman-Coulter, Inc.)
  3. Leica Application Suite X
  4. EZ-C1 Free Viewer (Nikon Corporation)
  5. ImageJ (Wayne Resband, National Institutes of Health, USA)
  6. MagellanTM - Data Analysis Software (Tecan Trading AG)

Procedure

  1. Flow cytometry analysis
    This procedure uses flow cytometry to determine whether a fluorescently labelled peptide can be internalized by a bacterium of interest. Flow cytometry measures a large number of individual cells and therefore provides a strong support for the significance of peptide uptake.
    1. Pick up a single colony of the bacterial strains grown onto agar plates with the appropriate medium.
    2. Suspend the bacteria in 5 ml of sterile liquid medium in polypropylene tubes with ventilation cap and incubate with shaking (140 rpm) at 37 °C for E. coli and S. typhimurium, or 30 °C for S. meliloti and Bradyrhizobium. Growth is overnight (approximately 18 h) for E. coli, S. typhimurium and S. meliloti, and 3 days for the slow growing Bradyrhizobium.
    3. Dilute the pre-cultures 1:30 in fresh medium and incubated them by shaking at the appropriate temperature to obtain mid-log phase bacteria, approx. 2 h for E. coli and S. typhimurium, 4 h for S. meliloti and overnight for Bradyrhizobium.
      Note:
      1. The use of a mid-log phase culture (OD600 = 0.3-0.5) is important to obtain reproducible results since some bacterial species may become insensitive or less sensitive to certain peptides at later growth phases.
      2. Bradyrhizobium samples are washed 3 times with 1 volume sterile PBT before proceeding to the next step. This washing step allows the peptides to interact more efficiently with the bacterial membrane.
    4. Adjust the bacteria to 1 x 106 to 1 x 107 Colony Forming Units (CFU)/ml (OD600 ~0.01 to 0.05) in MHB for E. coli and S. typhimurium or PB for S. meliloti and Bradyrhizobium.
    5. Prepare 1 ml aliquots of bacterial suspension in Eppendorf tubes.
    6. Add the fluorescently-labelled peptide to the bacteria and incubate samples for chosen times (usually a few minutes to one hour, depending on the bacterial strain and the peptide) at 37 °C or 30 °C according to the bacterial strain used. Prepare one tube for each peptide concentration and time point and arrange a negative control without peptide.
      Note: To evaluate correctly peptide internalization, select a concentration that is below the membrane-permeabilizing concentration of the peptide. This information can be obtained by a PI-uptake assay as described in the supplementary Procedure C or Procedure D.
    7. Wash samples three times in BHSS (for E. coli and S. typhimurium) or PB (for S. meliloti and Bradyrhizobium) in order to remove the fraction of peptide that is bound weakly to the bacterial surface. Resuspend cells in 1 ml of BHSS or PB, respectively.
    8. Analyze samples by the flow cytometer. Gate on the bacterial population using the forward scatter (FSC) and side scatter (SSC) parameters. Use filter settings that are adapted for the fluorophore used. The detector is set to logarithmic amplification. A total number of 10,000 to 50,000 events are usually acquired for each sample.
    9. Plot the number of counted events as a function of the fluorescence intensity (Figure 1). Cytometric data analysis may be performed using the FCS Express software, the Summit 6.2.2 software or equivalent software.
    10. Add trypan blue to each bacterial sample at 1 mg/ml final concentration.
    11. Incubate 10 min at room temperature and then analyze all samples again as in steps A8 and A9.

  2. Confocal scanning laser microscopy
    This procedure is complementary to Procedure A. It can confirm for a smaller number of cells peptide uptake or membrane association.
    1. Prepare samples as described until step A7 in Procedure A.
    2. Place 1 to 10 µl of each treated bacterial suspension between slide and cover-glass to obtain an immobile monolayer of cells.
    3. Observe samples with a confocal laser scanning microscope using a 63x or 100x objective.
    4. Analyse the image stacks collected by the confocal microscope using appropriate software, e.g., for image acquisition Leica Application Suite X or EZ-C1 Free Viewer and for image processing ImageJ.
    5. Observe the distribution of fluorescence on the cell surface and inside the bacterial cells (Figure 2).

Supplementary procedures: if the membrane permeabilization activity of the peptide of interest is unknown, Procedures C and D can be used to determine a concentration at which the fluorescent peptide does not affect the bacterial membrane permeability, providing a working concentration to be used in Procedures A and B.

  1. Propidium iodide uptake assay by microplate reader
    1. Prepare samples as described until step A3 in Procedure A.
    2. Adjust the bacteria to 1 x 107 to 1 x 108 CFU/ml (OD600 ~0.1) in MHB for E. coli and S. typhimurium or PB for S. meliloti and Bradyrhizobium.
    3. Add PI to each bacterial sample at 10 µg/ml final concentration.
    4. Add samples to a microplate, 190 µl per well.
    5. Add 10 µl peptide to each sample at the required final concentration.
      Note: A range of concentrations should be tested from one tenth of the Minimal Inhibitory Concentration or lower till the minimal inhibitory concentration. Typical Minimal Inhibitory Concentrations for antimicrobial peptides are in the range of 1 to 10 µM.
    6. Immediately measure fluorescence in the fluorescence plate reader. Using Magellan software, acquire data every 2 min for 120 cycles. Filters for PI fluorescence are: excitation 536-539 nm filter; emission 617-620 nm filter.
    7. Analyse data in an Excel data sheet (Figure 3).

  2. Propidium iodide uptake assay by flow cytometry (alternative to Procedure C)
    1. Prepare samples as described until step A4 in Procedure A.
    2. Aliquot 1 ml of bacterial suspension into the tubes. Prepare one tube for each concentration of peptide.
    3. Add PI to each sample (final concentration 10 μg/ml).
    4. Add the peptide to the desired concentration and incubate in a thermostatic bath at 37 °C.
    5. Acquire each sample with the flow cytometer every 15 min (for maximum 2 h).
    6. Plot the number of cells as a function of the PI fluorescence signal (at 620 nm).
    7. For each incubation time, evaluate the percentage of permeabilised cells (Figure 4).

Data analysis

The flow cytometry analysis will reveal fluorescence positive and negative bacterial populations after treatment with a labelled peptide (Figure 1). Fluorescence negative bacteria can be recognized by comparison with an untreated sample which will help to determine the background signal. Uptake of peptide can be concluded by comparing the Mean Fluorescence Intensity (MFI) values obtained in the absence and in the presence of trypan blue of each sample (Figure 1). Trypan blue is a fluorescence quencher which absorbs light in the range of FITC or BODIPY-FL. Because trypan blue does not penetrate bacterial cells and quenching requires contact between the fluorochrome and the quencher, it will only quench the light emitted by peptides located outside the bacterial cell while light emitted by internalized fluorochrome will be detected. Thus, little or no difference between the MFI values of cells treated with or without trypan blue indicates that the peptide is mainly internalized into bacterial cells; conversely, a large difference between the MFI values indicates that the peptide is mainly located on the bacterial surface or in both the cytoplasm and membranes. In this case, further studies are required to precisely establish the peptide distribution in the cells. For example, bacterial samples treated with fluorescent peptides can be visualized by confocal laser scanning microscopy, without any fixation, and by following the same protocol used for the flow cytometric assay (Figure 2; see Procedure B).
Uptake of an AMP of interest is meaningful, suggesting an intracellular mode of action of the peptide, if it is taking place at a concentration at which the membrane permeability of the target bacteria is not affected by the peptide. Membrane integrity can be determined with PI-uptake assays (Procedure C or Procedure D). Dilution series of the peptide of interest will determine the maximum concentration of the peptide at which no PI uptake is observed (same fluorescence level in the AMP-treated sample and the untreated control; Figures 3 and 4). This AMP concentration is a usable concentration for the flow cytometry uptake experiments.

Representative data

  1. A representative example of a flow cytometry experiment demonstrating the uptake of the Bac7 peptide, labelled with BODIPY FL, by S. meliloti wild type and the inability of a mutant in the bacA transporter gene to take up the peptide.
     

    Figure 1. Bac71-16-BODYPI FL uptake by S. meliloti and its bacA mutant. A. The example shows that in the wild type strain, a bacterial population has high fluorescence (red arrow) which is almost 100 times higher than the negative population (bacteria that did not take up the peptide; black arrow). B. The MFI of this positive population (red arrow) is conserved in the presence of trypan blue demonstrating the uptake of the peptide by the bacteria. C. The S. meliloti ∆bacA mutant on the other hand shows only a high fluorescence signal in the absence of extracellular fluorescence quencher (red bracket). D. This fluorescence is completely quenched by the presence of trypan blue, demonstrating that this mutant cannot take up the peptide. This observation is in agreement with the bacA gene of S. meliloti encoding a transporter required for the uptake of the Bac7 peptide (Marlow et al., 2009). Note that the fluorescence levels in the x-axes are in logarithmic scale.

  2. Microscopy analysis shows the intracellular localization of the Bac71-16-BODYPI FL peptide.


    Figure 2. Localization of Bac71-16-BODYPI FL and Polymyxin B-BODYPI FL on E. coli cells observed by CSLM. A. Intracellular accumulation of the Bac71-16-BODYPI FL peptide in the cytosol of the bacterial cells. B. Accumulation on the bacterial surface of the Polymyxin B-BODYPI FL, a peptide that target the bacterial envelope.

  3. Typical example of a PI-uptake assay demonstrating membrane permeabilization provoked by the AMP NCR247 and measured using a fluorescence plate reader.


    Figure 3. PI-uptake assay with S. meliloti wild type cells treated with the NCR247 AMP. The bacteria were treated with the indicated concentrations of the peptide. At 4 µM and particularly at 10 µM PI uptake is observed but not at 1 µM. Thus the latter concentration is a usable working concentration for peptide uptake assays with Procedure A or B.

  4. Determination of the non-lytic condition for a peptide by cytometric PI-uptake assay.


    Figure 4. PI-uptake in E. coli cells after treatment with Bac71-16 and polymyxin B. A marker window is set on the basis of the untreated cell population (grey histograms) to indicate the interval of fluorescence intensity at which the cells are considered PI-positive. The example shows that fluorescence of E. coli cells treated with 0.25 µM polymyxin B for 10 min shifts to higher values (red histogram), indicating that membranes have been permeabilized. Conversely, fluorescence signal of bacteria incubated with 1 µM Bac71-16 for 30 min (green histogram) is superimposed to that of the negative control indicating a non-lytic activity for this peptide at these conditions.

Notes

The fraction of a bacterial population that is responsive and has taken up a fluorescently labelled AMP (relative fraction of positive and negative cells as in Figure 1) may vary between experiments and is depending on the studied bacterial strain and on the identity of the peptide.

Recipes

  1. Mueller-Hinton broth (MHB)
    21 g of dehydrated medium (DIFCO) dissolved in 1 L of MilliQ water
    Autoclave and store at room temperature
    For agar plates, add 1.5% agar before autoclaving
  2. Yeast extract broth (YEB, for 1 L medium)
    5 g Bacto beef extract
    1 g Bacto yeast extract
    5 g Bacto peptone
    5 g saccharose
    500 mg MgSO4·7H2O
    Dissolve in 900 ml MilliQ water
    Adjust the pH to 7.2 with 1 N NaOH, adjust the volume to 1 L, autoclave and store at room temperature
    For agar plates, add 1.5% agar before autoclaving
  3. Yeast extract mannitol broth (YMB, for 1 L medium)
    10 g mannitol
    500 mg K2HPO4
    500 mg sodium glutamate
    1 g yeast extract
    50 mg NaCl
    40 mg CaCl2·2H2O
    4 mg FeCl3
    100 mg MgSO4·7H2O
    Dissolve in 900 ml MilliQ water
    Adjust the pH to 6.8 with 1 N HCl, adjust the volume to 1 L, autoclave and store at room temperature
    For agar plates, add 1.5% agar before autoclaving
  4. Buffered-saline (BS)
    10 mM Na-phosphate buffer containing 150 mM NaCl, pH 7.4
    Filtered using a 0.2 µm membrane filter
  5. Buffered high salt solution (BHSS)
    10 mM Na-phosphate
    400 mM NaCl
    10 mM MgCl2
    Adjust the pH to 7.4
    Filtered using a 0.2 µm membrane filter
  6. Phosphate buffer (PB)
    50 mM Na-phosphate
    Adjust the pH to 7.0
    Filtered using a 0.2 µm membrane filter
  7. PB supplemented with Tween 20 (PBT)
    50 mM Na-phosphate
    0.05% (vol/vol) Tween 20
    Adjust the pH to 7.0
    Filtered using a 0.2 µm membrane filter
  8. Trypan blue stock solution
    10 mg/ml trypan blue in BS solution
    Filtered using a 0.2 µm membrane filter
  9. Propidium iodide (PI) stock solution
    1 mg/ml in MilliQ water or BS solution
    Filtered using a 0.2 µm membrane filter

Acknowledgments

The present work has benefited from the core facilities of Imagerie-Gif, (http://www.i2bc.paris-saclay.fr), member of IBiSA (http://www.ibisa.net), supported by ‘France-BioImaging’ (ANR-10-INBS-04-01), and the Labex ‘Saclay Plant Science’ (ANR-11-IDEX-0003-02). This work was funded by grant ANR-13-BSV7-0013 and by the University of Trieste grant FRA2014.

References

  1. Arnold, M. F. F., Haag, A. F., Capewell, S., Boshoff, H. I., James, E. K., McDonald, R., Mair, I., Mitchell, A. M., Kerscher, B., Mitchell, T. J., Mergaert, P., Barry 3rd, C. E. Scocchi, M., Zanda, M., Campopiano D. J. and Ferguson, G.P. (2013). Partial complementation of Sinorhizobium meliloti bacA mutant phenotypes by the Mycobacterium tuberculosis BacA protein. J Bacteriol 195(2): 389-398.
  2. Benincasa, M., Pacor, S., Gennaro, R. and Scocchi, M. (2009). Rapid and reliable detection of antimicrobial peptide penetration into Gram-negative bacteria based on fluorescence quenching. Antimicrob Agents Chemother 53(8): 3501-3504.
  3. Benincasa, M., Pelillo, C., Zorzet, S., Garrovo, C., Biffi, S., Gennaro, R. and Scocchi, M. (2010). The proline-rich peptide Bac7(1-35) reduces mortality from Salmonella typhimurium in a mouse model of infection. BMC Microbiol 10: 178.
  4. Benincasa, M., Zahariev, S., Pelillo, C., Milan, A., Gennaro, R. and Scocchi, M. (2015). PEGylation of the peptide Bac7(1-35) reduces renal clearance while retaining antibacterial activity and bacterial cell penetration capacity. Eur J Med Chem 95: 210-219.
  5. Farkas, A., Maroti, G., Durgo, H., Gyorgypal, Z., Lima, R. M., Medzihradszky, K. F., Kereszt, A., Mergaert, P. and Kondorosi, E. (2014). Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms. Proc Natl Acad Sci U S A 111(14): 5183-5188.
  6. Guefrachi, I., Pierre, O., Bourge, M., Timchenko, T., Alunni, B., Czernic, P., Villaécija-Aguilar, J.-A., Verly, C., Fardoux, J., Mars, M., Kondorosi, E., Giraud, E. and Mergaert, P. (2015). Bradyrhizobium BclA is a NCR peptide transporter required for bacterial differentiation in symbiosis with Aeschynomene. Mol Plant-Microbe Interact 28(11): 1155-1166.
  7. Guida, F., Benincasa, M., Zahariev, S., Scocchi, M., Berti, F., Gennaro, R. and Tossi, A. (2015). Effect of size and N-terminal residue characteristics on bacterial cell penetration and antibacterial activity of the proline-rich peptide Bac7. J Med Chem 58(3): 1195-1204.
  8. Kondorosi, E., Mergaert, P. and Kereszt, A. (2013). A paradigm for endosymbiotic life: Cell differentiation of Rhizobium bacteria provoked by host plant factors. Annu Rev Microbiol 67: 611-628.
  9. Mardirossian, M., Grzela, R., Giglione, C., Meinnel, T., Gennaro, R., Mergaert, P. and Scocchi, M. (2014). The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis. Chem Biol 21(12): 1639-1647.
  10. Marlow, V. L., Haag, A. F., Kobayashi, H., Fletcher, V., Scocchi, M., Walker, G. C. and Ferguson, G. P. (2009). Essential role for the BacA protein in the uptake of a truncated eukaryotic peptide in Sinorhizobium meliloti. J Bacteriol 191(5): 1519-1527.
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简介

抗微生物肽(AMP)可靶向细菌包膜或者具有细胞内靶标。后者需要细菌细胞摄取肽。 AMP的细菌内化可以通过基于荧光的方法来评估,所述方法将荧光标记的AMP的使用耦合到荧光猝灭剂台盼蓝。台盼蓝从完整细胞的内部排除,并且细胞外肽或结合在细菌表面上的肽的荧光可被其淬灭,而内化肽的荧光不受影响。通过用流式细胞术测量单个细胞中的荧光来测定细菌对肽的吸收。
关键词:抗微生物肽,流式细胞术,肽摄取,肽转运蛋白,台盼蓝,碘化物吸收量

[背景] AMP由广泛且多样的有效抗微生物剂组成,具有作为新型治疗剂的潜力(Wang等人)。 ,2015)。 AMP是天然免疫的一部​​分并且由所有王国的生物产生。它们被这些生物体动员以抵抗感染的微生物,其可以是细菌,真菌或病毒。他们这样做通过直接杀死微生物,但他们也可以作为哨兵,促进其他免疫途径。有趣的是,还已经清楚的是,AMP不仅是针对不良微生物的药剂,而且它们还在动物和植物宿主中共生细菌群体的控制中具有关键作用(Maróti等人,2011 ; Kondorosi et al 。,2013)。
    AMP在序列和结构上的多样性是如此之大,以至于难以对它们进行分类。此外,不同来源的AMP也具有高度不同的作用方式。它们可以大致分为靶向细菌包膜的肽,通过渗透细胞膜破坏其细胞屏障功能,以及内化并靶向重要的细胞内功能的肽(Scocchi等人,2016)。因此,在新型抗菌肽的初始表征中,重要的是确定其主要作用位点。我们在这里描述的协议是基于流式细胞术方法,并且使得能够快速确定感兴趣的AMP是否被亚致死浓度的细菌内化(Benincasa等人,2009)。这种表征可以在细胞靶标的生物化学鉴定之前进行。
   我们已将此方法应用于大肠杆菌,鼠伤寒沙门氏菌 ,中华根瘤菌和 Bradyrhizobium 使用不同的抗细菌肽,包括抑制核糖体的哺乳动物Bac7肽(Mardirossian等人,2014)和植物肽NCR247,其使细菌膜透化,但也可以内化并结合不同的细胞内靶标Farkas等人。,2014; Guefrachi 。,2015)。这种简单的方法可以容易地适用于其他细菌和其他AMP或其他类型的生物活性肽。该方法还适用于测试细菌中肽吸收转运蛋白的活性,如实施例中所示(Mattiuzzo等人,2007; Guefrachi等人,2015)。

关键字:抗菌肽, 流式细胞术, 肽摄取, 肽转运蛋白, 台盼蓝, 碘化丙啶摄取

材料和试剂

  1. Eppendorf管
  2. 无菌膜过滤器0.2μm(SARSTEDT,目录号:83.1826.001)
  3. 显微镜载玻片和盖玻片(Chance Propper LTD)
  4. 96孔微孔板,透明底部的黑色,400μl(Greiner Bio One,目录号:655096)
  5. 感兴趣的细菌:例如大肠杆菌HB101,BW25113(Mattiuzzo等人2007; Benincasa等人)。 。,2009; Runti等人,2013; Guida等人,2015),鼠伤寒沙门氏菌 ATCC 14028(Benincasa et al。 ,2015),中华根瘤菌Sml21(Arnold等人,2013; Guefrachi等人,2015),中华根瘤菌 Bradyrhizobium ORS285(Guefrachi等。,2015)
  6. 细菌生长培养基:
    1. Mueller-Hinton肉汤,MHB(参见Recipes)(BD,Difco ,目录号:275710)。大肠杆菌或 S。 typhimurium
    2. 酵母提取物肉汤,YEB(参见Recipes),用于S. meliloti
    3. 酵母提取物甘露醇肉汤,YMB(参见Recipes),Bradyrhizobium
  7. 细菌生长培养基制备和缓冲溶液的化学品和组分:
    一个。技术琼脂(BD,Difco TM ,目录号:281230)
    b。酵母提取物(BD,Bacto TM ,目录号:212750)
    C。蛋白胨(BD,Bacto TM ,目录号:211677)
    d。牛肉提取物(Conda,目录号:1700)
    e。蔗糖(VWR,目录号:27483.363)
    F。甘露醇(VWR,目录号:25311.297)
    G。谷氨酸钠(VWR,目录号:27872.298)
    H。硫酸镁七水合物(MgSO 4·7H 2 O)(EMD Millipore,目录号:105886)
    一世。磷酸氢二钾(K 2 HPO 4)(VWR,目录号:26930.362)
    j。磷酸氢二钠七水合物(Na 2 HPO 4·7H 2 O)(Sigma-Aldrich,目录号:S9390)
    k。磷酸二氢钠一水合物(NaH 2 PO 4·2H 2 O)(Sigma-Aldrich,目录号:71505)
    l。氯化铁(III)(FeCl 3)(Sigma-Aldrich,目录号:701122)
    m。氯化钙二水合物(CaCl 2·2H 2 O)(EMD Millipore,目录号:102382)
    n。氯化钠(NaCl)(EMD Millipore,目录号:106404)
    o。氢氧化钠(NaOH)(VWR,目录号:567530-250)
    p。 HCl(CARLO ERBA Reagents,目录号:403871)
    q。氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
    r。吐温20(Sigma-Aldrich,目录号:P1379)
  8. 荧光标记肽的储备溶液
    注意:成功用于标记肽的荧光团是BODIPY FL(Guida等人,2015),荧光素(FITC)(Guefrachi等人,2015),Alexa染料(Benincasa等人,2010)。在内化研究之前,使用最小抑制浓度测定法检查标记不影响肽的生物活性。如果肽合成仪可用,或者从提供定制肽合成服务的商业供应商获得( http://www.proteogenix.science/custom-peptide-synthesis/)。
  9. 缓冲盐水(BS)(参见配方)
  10. 缓冲高盐溶液(BHSS)(参见配方)
  11. 磷酸盐缓冲液(PB)(参见配方)
  12. PB补充Tween 20(PBT)(参见配方)
  13. 台盼蓝(Sigma-Aldrich,目录号:T6146)储备溶液(参见Recipes)
  14. 碘化丙啶(PI)(Sigma-Aldrich,目录号:P4170)储备溶液(参见Recipes)

设备

  1. 细菌生长孵育器(FIRLABO,Bioconcept)
  2. 恒温浴(Thermo Fisher Scientific,Thermo Scientific ,型号:TSGP02)
  3. 装备有氩激光器(488nm,100mW)的流式细胞仪(Beckman Coulter,型号:Cytomics FC 500,配备有氩激光器[488nm,5mW])或Moflo Astrios(Beckman-Coulter,型号:Moflo Astrios)用于BODIPY(BY)检测(过滤器526/52 nm)的设置在525nm的过滤光的光电倍增管荧光检测器
    注意:产品"Cytomics FC 500"已停产。
  4. 用于Eppendorf管(Thermo Fisher Scientific,Thermo Scientific TM ,model:型号)的Heraeus Pico TM和Fresco Heraeus Pico 17)
  5. 分光光度计(Amersham Biosciences,Ultrospec 10细胞密度计)
  6. 具有油浸物镜(Nikon Eclipse C1si或Leica TCS SP X)的共聚焦显微镜
  7. 荧光板读数器(Tecan Trading,Infinite ,型号:M200)

软件

  1. FCS Express   3或更高版本(De Novo Software,Los Angeles,CA )
  2. 峰会6.2.2 (Beckman-Coulter,Inc.)
  3. Leica Application Suite X
  4. EZ-C1免费查看者(尼康公司)
  5. ImageJ (Wayne Resband,美国国立卫生研究院)
  6. MagellanTM - 数据分析软件(Tecan Trading AG)

程序

  1. 流式细胞术分析
    该程序使用流式细胞术来确定荧光标记的肽是否可被感兴趣的细菌内化。流式细胞术测量大量的单个细胞,因此为肽摄取的重要性提供了强有力的支持
    1. 用适当的培养基取出在琼脂平板上生长的单菌落菌落
    2. 将细菌悬浮在5ml无菌液体培养基的聚丙烯管中,带有通风帽,并在37℃下振荡(140rpm)孵育。大肠杆菌和 S。鼠伤寒沙门氏菌 ,或30℃。 meliloti 和 Bradyrhizobium 。对于E,生长过夜(约18小时)。大肠杆菌,鼠伤寒沙门菌和 S。 meliloti ,而缓慢生长的Bradyrhizobium 则为3天。
    3. 在新鲜培养基中稀释预培养物1:30,并通过在适当温度下振荡孵育它们以获得中对数期细菌, E为2小时。 col 和 typhimurium ,4 h for meliloti ,并在 Bradyrhizobium 之间过夜。
      注意:
      1. 使用中对数期培养物(OD 600 = 0.3-0.5)对于获得可重复的结果是重要的,因为一些细菌物种可能变得对以后生长期的某些肽不敏感或较不敏感。
      2. 在进行下一步骤之前,将Bradyrhizobium样品用1体积无菌PBT洗涤3次。这种洗涤步骤使肽能更有效地与细菌膜相互作用。
    4. 将细菌调节至1×10 6至1×10 7菌落形成单位(CFU)/ml(OD 600〜0.01至0.05)在MHB中。大肠杆菌和 S。 typhimurium 或PB for meliloti 和 Bradyrhizobium 。
    5. 在Eppendorf管中制备1ml等分的细菌悬浮液
    6. 将荧光标记的肽添加到细菌中并根据使用的细菌菌株在37℃或30℃下孵育样品所选择的时间(通常几分钟至一小时,取决于细菌菌株和肽)。为每个肽浓度和时间点准备一个管,并安排没有肽的阴性对照。
      注意:为了正确评估肽内化,选择低于肽的膜透化浓度的浓度。该信息可以通过补充程序C或程序D中所述的PI摄取试验获得。
    7. 在BHSS(大肠杆菌和鼠伤寒沙门氏菌)或PB(用于苜蓿中华根瘤菌)和Bradyrhizobium(大肠杆菌)中洗涤样品三次,/em>),以除去与细菌表面弱结合的肽部分。将细胞分别重悬在1ml BHSS或PB中
    8. 通过流式细胞仪分析样品。使用前向散射(FSC)和侧向散射(SSC)参数对细菌群体进行门控。使用适用于所用荧光团的滤光片设置。检测器设置为对数放大。通常每个样品获得10,000到50,000个事件总数。
    9. 作为荧光强度的函数绘制计数事件的数量(图1)。可以使用FCS Express软件,Summit 6.2.2软件或等效软件进行细胞计数数据分析
    10. 向每个细菌样品中加入台盼蓝,最终浓度为1mg/ml
    11. 在室温下孵育10分钟,然后重新分析所有样品,如步骤A8和A9
  2. 共焦扫描激光显微镜
    该程序与方法A互补。它可以证实较少数量的细胞肽吸收或膜结合
    1. 按照所述制备样品,直到方法A中的步骤A7
    2. 在载玻片和盖玻片之间放置1至10μl的每种处理的细菌悬浮液以获得固定的单层细胞。
    3. 使用共聚焦激光扫描显微镜使用63x或100x物镜观察样品。
    4. 使用适当的软件,例如,用于图像采集Leica Application Suite X或EZ-C1 Free Viewer和用于图像处理ImageJ的分析由共焦显微镜收集的图像堆栈。
    5. 观察细胞表面和细菌细胞内荧光的分布(图2)

补充程序:如果目的肽的膜渗透活性未知,则程序C和D可用于确定荧光肽不影响细菌膜渗透性的浓度,从而提供待使用的工作浓度在程序A和B中。

  1. 酶标仪测定碘化丙锭吸收
    1. 按照所述制备样品,直到方法A中的步骤A3
    2. 将MHB中的细菌调节至1×10 7至1×10 8 CFU/ml(OD 600〜0.1),用于E 。大肠杆菌和 S。 typhimurium 或PB for meliloti 和 Bradyrhizobium 。
    3. 向每个细菌样品中加入10μg/ml终浓度的PI
    4. 将样品加入微量培养板,每孔190μl
    5. 每个样品加入10μl肽至所需的最终浓度。
      注意:浓度范围应该从最小抑制浓度的十分之一进行测试,或者更低,直到最小抑制浓度。抗微生物肽的典型最小抑制浓度在1至10μM的范围内。
    6. 立即测量荧光读板仪中的荧光。使用Magellan软件,每2分钟采集数据120个周期。 PI荧光的滤光片是:激发536-539nm滤光片;发射617-620nm滤光片
    7. 分析Excel数据表中的数据(图3)。

  2. 通过流式细胞术(方法C的替代)的碘化丙啶吸收测定
    1. 按照所述方法制备样品,直到方法A中的步骤A4
    2. 等分1毫升细菌悬浮液的管中。为每种浓度的肽制备一个管
    3. 向每个样品中加入PI(终浓度为10μg/ml)
    4. 将肽加入所需浓度,并在37℃的恒温浴中孵育
    5. 用流式细胞仪每15分钟获取一份样品(最多2小时)
    6. 绘制细胞数作为PI荧光信号(在620nm)的函数。
    7. 对于每个孵育时间,评估透化细胞的百分比(图4)

数据分析

流式细胞术分析将揭示用标记肽处理后的荧光阳性和阴性细菌群体(图1)。可以通过与未处理的样品比较来识别荧光阴性细菌,其将有助于确定背景信号。通过比较在每种样品的台盼蓝不存在和存在下获得的平均荧光强度(MFI)值,可以终止肽的摄取(图1)。台盼蓝是吸收FITC或BODIPY-FL范围内的光的荧光猝灭剂。因为台盼蓝不穿透细菌细胞并且淬灭需要荧光染料和淬灭剂之间的接触,所以它将仅淬灭位于细菌细胞外部的肽发射的光,而检测由内化荧光染料发射的光。因此,用或不用台盼蓝处理的细胞的MFI值之间很少或没有差异表明肽主要内化到细菌细胞中;相反,MFI值之间的大差异表明肽主要位于细菌表面上或细胞质和膜中。在这种情况下,需要进一步的研究以精确地建立细胞中的肽分布。例如,用荧光肽处理的细菌样品可以通过共聚焦激光扫描显微镜,没有任何固定,并通过遵循用于流式细胞术测定的相同方案(图2;参见程序B)可视化。
感兴趣的AMP的摄取是有意义的,这表明肽的细胞内作用模式,如果其以靶细菌的膜通透性不受肽影响的浓度发生。膜完整性可以用PI-摄取测定法(方法C或方法D)测定。感兴趣的肽的稀释系列将确定没有观察到PI摄取的肽的最大浓度(在AMP处理的样品和未处理的对照中相同的荧光水平;图3和4)。该AMP浓度是流式细胞摄取实验的可用浓度。

代表数据

  1. 流式细胞术实验的代表性实例,证明用BODIPY FL标记的Bac7肽的摄取。 meliloti 野生型,并且在bacA 转运蛋白基因中突变体不能携带肽。
     

    图1. Bac7 1-16 - BODYPI FL 摄取。 突变体。 A.该实施例显示在野生型菌株中,细菌群体具有高荧光(红色箭头),其几乎比阴性群体(未吸收肽的细菌;黑色箭头)。 B.该阳性群体的MFI(红色箭头)在存在台盼蓝的情况下是保守的,表明细菌对肽的吸收。 C. The。另一方面,meliloti突变体在不存在细胞外荧光淬灭剂(红色支架)的情况下仅显示高荧光信号。这种荧光被台盼蓝的存在完全淬灭,证明该突变体不能摄取肽。该观察结果与S的bacA 基因一致。 meliloti 编码摄取Bac7肽所需的转运蛋白(Marlow等人,2009)。注意x轴上的荧光水平是对数刻度。

  2. 显微镜分析显示Bac7 BODYPI FL 肽的细胞内定位。


    图2.在 E上的Bac7 1-16 - BODYPI FL 和多粘菌素B- BODYPI FL 通过CSLM观察到的大肠杆菌细胞。A.细菌细胞的细胞溶质中Bac7 1-16 - BODYPI FL 肽的细胞内积累。 B.在多粘菌素B - BODYPI FL的细菌表面上的累积,其是靶向细菌包膜的肽。

  3. PI吸收测定的典型实例,证明由AMP NCR247引起的并且使用荧光读板器测量的膜透化。


    图3.用S的PI吸收测定。用NCR247 AMP处理的苜蓿中华猕猴桃野生型细胞。用指定浓度的肽处理细菌。在4μM,特别是在10μM,观察到PI摄取,但不是在1μM。因此,后一浓度是用于程序A或B的肽摄取试验的可用工作浓度
  4. 通过细胞计数PI吸收测定确定肽的非溶解条件

    图4.E中的PI摄取。在用Bac7 1-16和多粘菌素B处理后的大肠杆菌细胞。基于未处理的细胞群(灰色直方图)设置标记窗口,将细胞视为PI阳性的荧光强度。该实施例显示了E的荧光。用0.25μM多粘菌素B处理10分钟的大肠杆菌细胞转移至更高的值(红色直方图),表明膜已被透化。相反,与1μMBac7 1-16孵育30分钟的细菌的荧光信号(绿色直方图)叠加到阴性对照的荧光信号,表明在这些条件下对该肽的非裂解活性。

笔记

响应并且摄取荧光标记的AMP(图1中的阳性和阴性细胞的相对分数)的细菌群体的分数可以在实验之间变化,并且取决于研究的细菌菌株和肽的特性。

食谱

  1. Mueller-Hinton肉汤(MHB)
    将21g溶解在1L MilliQ水中的脱水培养基(DIFCO)
    高压灭菌并在室温下贮存
    对于琼脂平板,在高压灭菌之前加入1.5%琼脂
  2. 酵母提取液(YEB,1L培养基)
    5克细菌用牛肉提取物
    1克细菌酵母提取物
    5克细菌蛋白胨 5克蔗糖
    500mg MgSO 4·7H 2 O·h/v 溶于900ml MilliQ水中
    用1N NaOH调节pH至7.2,将体积调节至1L,高压灭菌并在室温下贮存
    对于琼脂平板,在高压灭菌之前加入1.5%琼脂
  3. 酵母提取甘露醇肉汤(YMB,1L培养基)
    10g甘露醇 500mg K 2 HPO 4
    500mg谷氨酸钠 1g酵母提取物
    50mg NaCl
    40mg CaCl 2·2H 2 O 2·h/v 4mg FeCl 3
    100mg MgSO 4·7H 2 O·dm 2 溶于900ml MilliQ水中
    用1N HCl调节pH至6.8,将体积调节至1L,高压灭菌并在室温下保存
    对于琼脂平板,在高压灭菌之前加入1.5%琼脂
  4. 缓冲盐水(BS)
    含有150mM NaCl,pH7.4的10mM磷酸钠缓冲液 使用0.2μm膜过滤器过滤
  5. 缓冲高盐溶液(BHSS)
    10 mM磷酸钠 400 mM NaCl
    10mM MgCl 2/
    将pH调节至7.4
    使用0.2μm膜过滤器过滤
  6. 磷酸盐缓冲液(PB)
    50 mM磷酸钠 将pH调节至7.0
    使用0.2μm膜过滤器过滤
  7. PB补充有Tween 20(PBT)
    50 mM磷酸钠 0.05%(vol/vol)吐温20 将pH调节至7.0
    使用0.2μm膜过滤器过滤
  8. 台盼蓝储液
    10mg/ml台盼蓝在BS溶液中
    使用0.2μm膜过滤器过滤
  9. 碘化丙啶(PI)储液
    1mg/ml在MilliQ水或BS溶液中
    使用0.2μm膜过滤器过滤

致谢

目前的工作受益于Imagerie-Gif的核心设施( http://www.i2bc.paris-saclay.fr ),IBiSA的成员( http: //www.ibisa.net ),由"France-BioImaging"(ANR-10-INBS-04-01)和Labex'S Saclay Plant Science'(ANR-11-IDEX-0003-02 )。这项工作由资助ANR-13-BSV7-0013和由里雅斯特大学授予FRA2014。

参考文献

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引用:Benincasa, M., Barrière, Q., Runti, G., Pierre, O., Bourge, M., Scocchi, M. and Mergaert, P. (2016). Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria. Bio-protocol 6(23): e2038. DOI: 10.21769/BioProtoc.2038.
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