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Preparation and Analysis of Crude Autolytic Enzyme Extracts from Staphylococcus aureus

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Apr 2014



The metabolism of the cell surface during bacterial cell division involves synthesis and degradation of peptidoglycan (PGN), the major component of the bacterial cell wall. Bacteria have to ensure that their surface remains capable of withstanding high turgor pressures and, simultaneously, that the PGN at their surface is concealed from receptors produced by the host innate immune system. For cell separation to occur, and for PGN to be kept concealed, “old” PGN is degraded by specific PGN hydrolases, also known as autolysins, that are found at the bacterial cell surface or that are secreted into the growth medium.

Bacterial PGN hydrolases are cell wall lytic enzymes that comprise a broad and diverse group of proteins. It is often difficult to assign a specific function to a PGN hydrolase mainly because an organism can have a large number of hydrolases with redundant activities and one hydrolase can have more than one enzymatic activity and participate in various cell processes (Vollmer et al. 2008). Bacillus subtilis has ca. 35 known or hypothetical PGN hydrolases, whereas Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) have, respectively, ca.16 and 19 PGN hydrolases (Vollmer, 2012; Heidrich et al., 2001; Singh et al., 2012).

PGN hydrolases can be classified in three main classes: glycosidases, amidases and peptidases. Glycosidases cleave the glycan backbone and are divided into N-acetylglucosaminidases and N-acetylmuramidases. Amidases cleave the linkage between the peptide chain and the N-acetylmuramic residue of the glycan chain. Peptidases, such as endopeptidases and carboxypeptidases, are able to cleave peptide bonds between different amino acids of the PGN stem peptide.

Here we describe a method to extract PGN hydrolases, which are non-covalently linked to the S. aureus cell wall (Vollmer, 2008). Analysis of extracts containing denatured PGN hydrolytic enzymes is performed by running a zymogram gel (a SDS-PAGE gel containing crude bacterial cell walls or substrate cells), which is then incubated in a non-denaturing buffer to allow renaturation of the PGN hydrolases. These renatured enzymes can then be identified through the production of clear bands that are observed where cell wall digestion has occurred. The protocol is divided into three steps: A) Preparation of the crude autolytic extracts from S. aureus cells; B) Preparation of substrate cells for gel zymograms; C) Analysis of crude autolytic extracts by gel zymography.

We also show that this method can be used to determine the absence or altered activity of PGN hydrolases produced by different S. aureus mutant strains.

Keywords: Murein (胞壁质), Peptidoglycan (肽聚糖), PGN hydrolases (PGN水解酶), Zymogram (酶谱), Staphylococcus aureus (金黄色葡萄球菌)

Materials and Reagents

  1. Petri dishes (Sarstedt AG, catalog number: 82.1473 )
  2. 1 μl loops (Sarstedt AG, catalog number: 86.1567.010 )
  3. 25 ml glass (or plastic disposable) pipettes (Normax, catalog number: 4.5434334 )
  4. 50 ml Falcon tubes (Sarstedt AG, catalog number: 62.548.004 )
  5. 2 ml micro tubes (Sarstedt AG, catalog number: 72.691 )
  6. JA-14 centrifuge tubes (Thermo Fisher Scientific, NalgeneTM, catalog number: 3120-0250 )
  7. JA-20 centrifuge tubes (Thermo Fisher Scientific, NalgeneTM, catalog number: 3114-0050 )
  8. Spacer plates with 0.75 mm integrated spacers (Bio-Rad Laboratories, catalog number: 165-3310 )
  9. Mini-PROTEAN® Comb, 10-well, 0.75 mm (Bio-Rad Laboratories, catalog number: 165-3354 )
  10. Staphylococcus aureus strains to analyze regarding the cell wall lytic activity of their PGN hydrolases.
    Note: In order to produce substrate cells, researchers may use the Staphylococcus aureus NCTC8325-4 strain, which has been cured from prophages and it can be obtained from BEI Resources under the reference NR-45937, and Micrococcus luteus DSM20030 strain, which can be obtained from DSMZ stock center.
  11. Tryptic Soy Agar plates (TSA) (BD, Difco, catalog number: 236950 )
  12. Tryptic Soy Broth (TSB) (BD, Bacto, catalog number: 211825 )
  13. Luria Agar (Miller's LB agar) (LA) (Conda, catalog number: 1552 )
  14. Luria Broth (Miller's LB broth) (LB) (Conda, catalog number: 1551 )
  15. Ethanol (Merck Millipore Corporation, catalog number: 1.02371.1000 )
  16. Ice (homemade)
  17. Liquid nitrogen (Air Liquide)
  18. Tris (Trizma® base) (Sigma-Aldrich, catalog number: T1503 )
  19. Sodium chloride (Merck Millipore Corporation, catalog number: 1.06444.1000 )
  20. Hydrochloric acid (Merck Millipore Corporation, catalog number: 1.01834.2500 )
  21. Sodium Dodecyl Sulfate (Sigma-Aldrich, catalog number: L5750 )
  22. Glycine (Sigma-Aldrich, catalog number: G8898 )
  23. 30% Acrylamide/Bis Solution (Bio-Rad Laboratories, catalog number: 161-0158 )
  24. Dithiothreitol (DTT) (VWR International, catalog number: V3155 )
  25. Ammonium persulfate (APS) [(NH4)2S2O8] (Sigma-Aldrich, catalog number: A3678 )
  26. Bromophenol Blue sodium salt (Sigma-Aldrich, catalog number: B8026 )
  27. Precision Plus ProteinTM Dual Color Standards (PPPS) (Bio-Rad Laboratories, catalog number: 1610374 )
  28. Methylene Blue hydrate (Sigma-Aldrich, catalog number: 66720 )
  29. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  30. Magnesium chloride hexahydrate (Sigma-Aldrich, catalog number: M9272 )
  31. Calcium chloride dihydrate (CaCl2.2H2O) (Sigma-Aldrich, catalog number: C3306 )
  32. Potassium hydroxide (KOH) (Sigma-Aldrich ,catalog number: P5958 )
  33. N, N, N′, N′-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
  34. 70% ethanol (see Recipes)
  35. 500 mM Tris-HCl (pH 7.5)
(see Recipes)
  36. Washing buffer (see Recipes)

  37. 4% SDS (w/v) (see Recipes)
  38. 10% SDS (w/v) (see Recipes)
  39. 1.5 M Tris-HCl (pH 8.8)
(see Recipes)
  40. 0.5 M Tris-HCl (pH 6.8)
(see Recipes)
  41. 10% APS (w/v)
(see Recipes)
  42. Zymogram SDS-PAGE gels (see Recipes)
  43. 5x Laemmli loading buffer (see Recipes)
  44. Tris-Glycine-SDS buffer (see Recipes)
  45. Renaturation buffer (see Recipes)
  46. Methylene Blue solution (see Recipes)


  1. Erlenmeyer flasks of 100 ml capacity (Normax, catalog number: 2121624N )
  2. Erlenmeyer flasks of 1 L capacity (Normax, catalog number: 2121654N )
  3. Cuvettes (Sarstedt AG, catalog number: 67.742 )
  4. 1.5 ml micro tubes (Sarstedt AG, catalog number: 72.690.001 )
  5. Micropipette (Gilson, model: Pipetman P1000 )
  6. Magnetic stir bars (VWR International, catalog number: 442-0361 )
  7. 30 °C/37 °C Incubator shaker (Eppendorf, New Brunswick Scientific, model: Innova® 40 )
  8. 30 °C /37 °C Incubator (BINDER GmbH, model: WTB )
  9. Burner
    Note: In this work we used a wall-attached burner, but a portable burner also works.
  10. Spectrophotometer (GE Healthcare, Amersham, model: Novaspec Plus Visible Spectrophotometer )
  11. Centrifuge (Beckman Coulter, model: Avanti J-26 XPI )
  12. JA-14 rotor (Beckman Coulter, model: 339247 )
  13. JA-20 rotor (Beckman Coulter, model: 334831 )
  14. Bench centrifuge (Eppendorf, model: 5430R )
  15. Rotor for 1.5 ml tubes (Thermo Fisher Scientific, Eppendorf, model: FA-45-24-11-HS )
  16. Shaker for 1.5 ml tubes (Eppendorf, model: Thermomixer® Comfort )
  17. Nanodrop (Thermo Fisher Scientific, model: ND-2000C )
  18. Autoclave
  19. Speed vac (Labconco, model: 78100 )
  20. Short plates (Bio-Rad Laboratories, catalog number: 165-3308 )
  21. Power Pac HV Power Supply (Bio-Rad Laboratories, catalog number: 164-5056 )
  22. Mini-PROTEAN Casting Stand Gaskets (Bio-Rad Laboratories, catalog number: 165-3305 )
  23. Mini-PROTEAN® Casting Frame (Bio-Rad Laboratories, catalog number: 165-3304 )
  24. Mini-PROTEAN® Casting Stand (Bio-Rad Laboratories, catalog number: 165-3303 )
  25. Mini-PROTEAN® Tetra Cell (Bio-Rad Laboratories, catalog number: 165-8000 )


  1. Preparation of the crude autolytic extracts from S. aureus cells
    1. The working area was decontaminated with 70% ethanol (v/v) using paper towels and the burner was turned on. This procedure was done throughout the protocol every time sterile conditions were needed (Note 1).
    2. The bacterial S. aureus strains of interest were inoculated on TSA plates by streak plating using 1 μl loops and incubated overnight in a 30 °C incubator (Note 2).
    3. Under sterile conditions, a single isolated colony was picked with a 1 μl loop and inoculated in 10 ml of TSB in a 100 ml Erlenmeyer flask. Cultures were grown overnight (16 h-18 h) in a 30 °C incubator shaker at 200 rpm.
    4. The optical densities at λ = 600 nm of the overnight cultures were measured after making a 1/10 dilution (100 μl of culture in 900 μl of TSB) in a cuvette, under sterile conditions.
    5. The volume required for a starting OD600 of ~ 0.05 was taken from the overnight cultures and inoculated in 250 ml of TSB in a 1 L Erlenmeyer flask. The cultures were grown in a 30 °C incubator shaker at 200 rpm.
    6. The centrifuge JA-26 XPI was prepared by placing the JA-14 rotor and cooling it down to 4 °C.
    7. An ice/ethanol bath was prepared and kept at 4 °C: The ice was poured into a container and absolute ethanol was homogenously spread onto it. The flask of Washing buffer solution, the JA-14 and JA-20 centrifuge tubes were placed in it to cool down.
    8. When the cultures reached an OD600 ~ 0.3 (which should take about 3 h), they were put in the ice/ethanol bath (Note 3).
    9. The cultures were transferred into cold JA-14 centrifuge tubes. The tubes were balanced and the cells were pelleted at 4 ºC, 15,050 x g for 15 min.
    10. After centrifugation, the JA-14 rotor was replaced by the JA-20, which was cooled down to 4 °C.
    11. The supernatants were discarded by inversion of the tubes.
    12. The cells were resuspended in 20 ml of ice-cold Washing buffer and transferred to cold JA-20 centrifuge tubes. Washing buffer was added to the maximum capacity of the tubes (~46 ml).
    13. Cells were centrifuged as before (step A9).
    14. The supernatants were carefully removed using a 25 ml glass (or plastic) pipette until a semidry pellet was left at the bottom of the tubes.
    15. The cells were carefully resuspended in 250 μl of 4% SDS (w/v), transferred to a 1.5 ml micro tube and incubated in a thermomixer at 25 °C, 700 rpm for 30 min (Note 4).
    16. After incubation, the cells were harvested using a bench centrifuge at 22,380 x g, for 15 min, room temperature (RT).
    17. The supernatants, containing the crude enzyme autolytic extracts, were transferred to clean micro tubes.
    18. The extracts were quantified in the Nanodrop (Proteins-280 nm) using a 2 μl aliquot. Mili-Q water was used as a blank.
    19. Each extract was divided into 50 μl aliquots, frozen in liquid nitrogen and stored at -80 °C until further use (Note 5).

  2. Preparation of substrate cells for gel zymograms
    1. For preparation of substrate cells, the S. aureus NCTC8325-4 and the M. luteus DSM20030 strains were plated on TSA and LA plates, respectively, by streak plating using 1 μl loops, and incubated in a 37 °C incubator. S. aureus was incubated overnight and M. luteus for at least 24 h (Note 2).

    2. Under sterile conditions, a single isolated colony of each strain was picked with a 1 μl loop and inoculated into 10 ml of TSB or LB in 100 ml Erlenmeyer flasks. Cultures were grown overnight (16 h-18 h) in a 37 °C incubator shaker at 200 rpm.
    3. The optical densities at λ = 600 nm of the overnight cultures were measured after 1/10 dilution, using as blanks 1 ml of the sterile TSB or LB.

    4. The volume required for a starting OD600 of ~ 0.05 was taken from the overnight cultures and inoculated in 250 ml of TSB or LB in a 1 L Erlenmeyer flask. The cultures were grown using a 37 °C incubator shaker at 200 rpm.
    5. When the cultures reached an OD600 ~ 1.0 the cells were harvested by centrifugation in a JA-14 tube for 15 min at 15,050 x g at room temperature. M. luteus culture should take one full day while S. aureus should take about 3 h to reach the desired OD (Note 6).

    6. The supernatants were discarded and cells were washed with 250 ml of Milli-Q water.
    7. The cells were centrifuged as above and the supernatants were discarded.
    8. The cells were resuspended in 30 ml of Milli-Q water and transferred to 50 ml Falcon tubes.
    9. The cell suspension was autoclaved for 15 min, 121 °C (Note 7).
    10. The autoclaved cells were transferred to JA-20 tubes and centrifuged for 15 min at 15,050 x g, RT.
    11. The supernatants were discarded and pellets were stored overnight at -20 °C (Note 8).
    12. The pellets were defrosted at room temperature, resuspended in 3 ml of Milli-Q water and divided in half into two pre-weighted 2 ml tubes.
    13. The suspension was lyophilized overnight in the speed vac.
    14. The weight of the tubes containing the cells was determined and the dry weight was calculated.
    15. Bacterial substrate cells were resuspended thoroughly in Milli-Q water at 50 mg/ml final concentration and stored at -20 °C (Note 9).

  3. Analysis of the crude autolytic extracts by gel zymography
    1. Substrate cells were thawed and, to ensure a complete resuspension, kept overnight at room temperature with agitation using a magnetic stirrer at maximum speed (Note 10).

    2. Two resolving Acrylamide/0.2% Bisacrylamide SDS-PAGE gels (Laemmli, 1970) were prepared containing a final concentration of 2 mg/ml of substrate cells from S. aureus (10 % gel) and M. luteus (8% gel) (Note 11 and Figure 1).
    3. A standard stacking gel, lacking substrate cells, was placed on top of the resolving gels (Laemmli, 1970).

    4. 10 μg (for the M. luteus gel) and 15 μg (for the S. aureus gel) of crude autolytic enzyme extracts (Note 12) were mixed with Laemmli loading buffer (Laemmli, 1970) at a final concentration of 2x (samples were not heated, see Recipes).
    5. The electrophoresis conditions used were as follows:
      1. Temperature: Room Temperature.
      2. Voltage constant: 70 V (per gel).
      3. Running buffer: Tris-Glycine-SDS buffer.
    6. After electrophoresis, the gels were rinsed once with Milli-Q water.

    7. The gels were washed 3 times with Milli-Q for 15 min at RT with gentle agitation.

    8. The gels were incubated overnight at 37 °C with gentle agitation in Renaturation buffer (Note 13).
    9. Zymograms were stained in Methylene Blue Solution for an hour and destained in water until the bands were clear (Note 14 and Figure 2).

      Figure 1. A 10% zymogram gel and SDS-PAGE gel. Left panel shows a zymogram gel, which is slightly opaque. Right panel shows a regular SDS-PAGE gel that is completely transparent due to the lack of substrate cells.

      Figure 2. Zymography analysis of S. aureus autolytic enzymes. A. Gel zymography analysis of crude autolytic enzyme extracts of S. aureus. M. luteus and S. aureus zymogram gels were used to analyze the autolytic enzymatic extracts harvested from the parental S. aureus NCTC8325-4 strain and from mutants that lack different autolysins (NCTCΔatl, which lacks the major Atl autolysin; NCTCΔlytM, which lacks the glycyl-glycine endopeptidase LytM; NCTCΔlytN, which lacks the putative cell wall hydrolase LytN and NCTCΔsle1, which lacks the N-acetylmuramoyl-L-alanine amidase Sle1). Five different forms of the major autolysin Atl were seen with either substrate cells. However, only the S. aureus substrate cells allowed detection of Sle1 activity, hence showing Sle1 substrate specificity for S. aureus peptidoglycan. B. Representation of the post-translational cleavage (black arrows) of the atl encoded protein (left). The processed amidase (AM) releases stem peptides from PGN by cutting the bond between the stem peptides and the N-acetylmuramic acid residue. The glucosaminidase (GL) releases muropeptides by cutting the glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine of glycan strands. Representation of Sle1 (right), which includes three LysM domains (responsible for the interaction with PGN) and a CHAP domain (associated with amidase activity of different PGN hydrolases).


  1. The working area should be a safe place to work near a flame, i.e., no air currents and away from explosives, flammable materials and solvents. For this protocol, work next to the flame can be substituted by working in a biological safety cabinet. Diligently follow all recommended safety guidelines and waste disposal regulations recommended by your host institution.
  2. Plating of the strains should be done from a glycerol stock onto a fresh media plate to ensure the viability of bacterial culture and to prevent selection of suppressor mutants. Growth of the S. aureus strains used in this work is not affected by light/dark conditions.
  3. Incubating the bacterial culture to an OD600 ~ 0.3 ensures that bacteria are harvested during the exponential growth phase. The fast transfer of bacteria to the ice/ethanol bath will reduce the activity of autolysins, which would modify the PGN present at the surface of the substrate cells, and prevent lysis of bacteria, which would result in the loss of specific PGN hydrolases to the growth medium.
  4. This step is required to extract proteins that are associated with the bacterial cell surface without inducing the lysis of bacteria.

  5. Dividing the extract in different aliquots will prevent the consecutive thawing and freezing steps that would result in the loss of activity of certain PGN hydrolases (3 cycles of thawing and freezing did not result in loss of cell wall lytic activity of Atl and Sle1).
  6. To harvest S. aureus and M. luteus at the same time please take in account their different growth rates.
  7. This step ensures that enzymes in this cell suspension lose their activity, and therefore do not cause lysis of the substrate cells during gel zymography analysis.

  8. We have obtained better results when autoclaved cells were stored at -20 °C prior the step of lyophilization. We assume that freezing step may help the resuspension of the substrate.
  9. The use of different bacteria as substrate cells in the gel zymography analysis may result in differences in the intensity and number of bands observed in the zymogram.
  10. This is a critical step as if substrate cells are not well resuspended, the resulting zymogram will be grainy, lowering the sensitivity of the gel zymography analysis.

  11. The M. luteus cell wall is considerably more sensitive to PGN lytic enzymes than S. aureus cells. Therefore when M. luteus cells are used, there is an increase in the intensity and in the number of bands that are detected in a Zymogram. It is preferable to use an 8% gel when using in M. luteus as substrate cells so that there is a better separation of the high molecular weight. In S. aureus gels, it is preferable to run a more concentrated gel to detect the activity of Sle1 that runs close to the 37 kDa protein standard.
  12. Also due to the high susceptibility of M. luteus cells to PGN hydrolases, the optimum quantity of extracts protein is 5-10 μg whereas for S. aureus gel the minimum extracts protein amount to be used should be 15 μg.
  13. This step is required to regain the activity of the PGN hydrolases, which was lost during treatment with the SDS solution.

  14. In this step, clear bands are observed due to the presence of autolysins. Intact substrate cells will retain the dye, while no color will be observed where substrate bacterial cells have lysed.
    In the gel containing M. luteus cells, the clear bands start to be seen after a 15 min wash and a picture of the gel should be taken before changing the water. The washing step should be repeated twice, taking pictures of the gel with every change of the water.
    The gel with S. aureus cells will take at least three 15 min washes before the bands start to be seen. We usually take a picture after three washes, then after a fourth or fifth wash of about 3 h-5 h. A final overnight wash is recommended to destain the gel as much as possible so that less active lytic proteins may be better seen (e.g. GL activity).


  1. 70% ethanol
    Into a spray bottle, mix 70 ml of ethanol with 30 ml of water
  2. 500 mM Tris-HCl (pH 7.5)

    Add 30.3 g of Trizma® base to 300 ml of Milli-Q water
    Let it stir until solubilisation occurs
    Adjust the pH to 7.5 with 1 M HCl

    Fill up to 500 ml with Mili-Q water
    Confirm pH and autoclave
    Use it as a sterile stock solution
    Stored at RT
  3. Washing buffer

    50 mM Tris-HCl (pH 7.5)/150 mM NaCl
    To 4.4 g of NaCl add 50 ml of 500 mM Tris-HCl (pH 7.5) and 400 ml of Milli-Q water
    After solubilisation, confirm pH and add Milli-Q water to 500 ml
    Filter sterilize (0.2 μm) or autoclave
    Stored at RT until use
  4. 4% SDS (w/v)

    Dissolve 4 g of SDS in 80 ml of Milli-Q water by stirring
    SDS is an irritant agent
    Wear a mask while weighting the powder
    Fill up to 100 ml with Mili-Q water

    Filter sterilize (0.2 μm)
    Stored at RT
  5. 10% SDS (w/v)

    Weight 10 g and dissolve in 80 ml of Milli-Q water by stirring
    SDS is an irritant agent
    Wear a mask while weighting the powder
    Fill up to 100 ml with Milli-Q water
    Filter sterilize (0.2 μm)
    Stored at RT
  6. 1.5 M Tris-HCl (pH 8.8)

    Add 18.2 g of Trizma® base to 60 ml of Milli-Q water
    Let it stir until solubilisation occurs
    Adjust the pH to 8.8 with 1 M HCl
    Fill up to 100 ml with Milli-Q water

    Confirm pH and filter sterilize (0.2 um)
    Keep it as a sterile stock solution-use an aliquot (~20 ml) as a working solution
    Stored at RT
  7. 0.5 M Tris-HCl (pH 6.8)

    Add 6.06 g of Trizma® base to 60 ml of Milli-Q water
    Let it stir until solubilisation occurs
    Adjust the pH to 6.8 with 1 M HCl
    Fill up to 100 ml with Mili-Q water
    Confirm pH and filter sterilize (0.2 um)
    Keep it as a sterile stock solution-use an aliquot (~20 ml) as a working solution.
    Stored at RT
  8. 10% APS (w/v)

    Weight 1 g and dissolve in 10 ml of Milli-Q water
    Stored in 500 μl aliquots at -20 °C
  9. Zymogram SDS-PAGE gels

  10. 5x Laemmli loading buffer
    Weight 0.77 g DTT, 4.3 g Glycerol, 2 mg Bromophenol Blue
    Add 5 ml 0.5 M Tris (pH 6.8) and fill up to 10 ml with Milli-Q water
    Stored at -20 °C in 50 μl aliquots
  11. Tris-Glycine-SDS buffer
    Dissolve 30.0 g of Tris base, 144.0 g of glycine, and 10.0 g of SDS in 1,000 ml of water
    Stored at RT
  12. Renaturation buffer
    50 mM Tris-HCl (pH 7.5), 0.1% (v/v) Triton X-100, 10 mM CaCl2, 10 mM MgCl2:
    Weight 0.74 g of calcium chloride dihydrate and 1.02 g of magnesium chloride hexahydrate
    Take 50 ml of 500 mM Tris-HCl (pH 7.5) and 500 μl of Triton X-100
    Stir until solubilisation
    Always use fresh
  13. Methylene blue solution

    0.1% (w/v) methylene blue in 0.01% potassium hydroxide:
    Dissolve 0.5 g of methylene blue and 0.05 g of potassium hydroxide in 500 ml Milli-Q water
    Stored at RT


This protocol, which was adapted or modified from Grilo et al. (2014) and Yokoi et al. (2008), has been used to describe that autolysins can prevent detection of the PGN at the bacterial cell surface (Atilano et al., 2014). This work was supported by fellowship SFRH/BD/78748/2011 to FV and project PTDC/BIA-PLA/3432/2012 to SRF from Fundação para a Ciência e Tecnologia. We thank Inês Grilo for suggestions and Teresa Baptista da Silva for technical support.


  1. Atilano, M. L., Pereira, P. M., Vaz, F., Catalao, M. J., Reed, P., Grilo, I. R., Sobral, R. G., Ligoxygakis, P., Pinho, M. G. and Filipe, S. R. (2014). Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system. Elife 3: e02277.
  2. Gotz, F., Heilmann, C. and Stehle, T. (2014). Functional and structural analysis of the major amidase (Atl) in Staphylococcus. Int J Med Microbiol 304(2): 156-163.
  3. Grilo, I. R., Ludovice, A. M., Tomasz, A., de Lencastre, H. and Sobral, R. G. (2014). The glucosaminidase domain of Atl - the major Staphylococcus aureus autolysin - has DNA-binding activity. Microbiologyopen 3(2): 247-256.
  4. Heidrich, C., Templin, M. F., Ursinus, A., Merdanovic, M., Berger, J., Schwarz, H., de Pedro, M. A. and Holtje, J. V. (2001). Involvement of N-acetylmuramyl-L-alanine amidases in cell separation and antibiotic-induced autolysis of Escherichia coli. Mol Microbiol 41(1): 167-178.
  5. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
  6. Oshida, T., Sugai, M., Komatsuzawa, H., Hong, Y. M., Suginaka, H. and Tomasz, A. (1995). A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization. Proc Natl Acad Sci U S A 92(1): 285-289.
  7. Singh, S. K., SaiSree, L., Amrutha, R. N. and Reddy, M. (2012). Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coli K12. Mol Microbiol 86(5): 1036-1051.
  8. Yokoi, K. J., Sugahara, K., Iguchi, A., Nishitani, G., Ikeda, M., Shimada, T., Inagaki, N., Yamakawa, A., Taketo, A. and Kodaira, K. (2008). Molecular properties of the putative autolysin Atl(WM) encoded by Staphylococcus warneri M: mutational and biochemical analyses of the amidase and glucosaminidase domains. Gene 416(1-2): 66-76.
  9. Vollmer, W., Joris, B., Charlier, P. and Foster, S. (2008). Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol Rev 32(2): 259-286.
  10. Vollmer, W. (2012). Bacterial growth does require peptidoglycan hydrolases. Mol Microbiol 86(5): 1031-1035.


细菌PGN水解酶是包含广泛和多样化蛋白质组的细胞壁溶解酶。主要是因为生物体可以具有大量具有冗余活性的水解酶,并且一种水解酶可以具有多种酶活性并参与各种细胞过程(Vollmer等,2008),通常难以将特定功能分配给PGN水解酶。 。枯草芽孢杆菌35种已知或假设的PGN水解酶,而大肠杆菌(E.coli)和金黄色葡萄球菌(金黄色葡萄球菌)分别具有约16和19个PGN水解酶(Vollmer,2012; Heidrich等,2001; Singh et al。 ,2012)。
在这里,我们描述了提取与金黄色葡萄球菌细胞壁非共价连接的PGN水解酶的方法(Vollmer,2008)。含有变性PGN水解酶的提取物的分析是通过运行Zymogram凝胶(含有粗细菌细胞壁或底物细胞的SDS-PAGE凝胶)进行的,然后将其在非变性缓冲液中温育以允许PGN水解酶复性。然后可以通过产生在细胞壁消化发生时观察到的清除条带来鉴定这些复性酶。方案分为三个步骤:A)从金黄色葡萄球菌细胞中制备粗自动提取物; B)制备用于凝胶酶谱的底物细胞; C)通过凝胶酶谱分析粗自动萃取物。

关键字:胞壁质, 肽聚糖, PGN水解酶, 酶谱, 金黄色葡萄球菌


  1. 培养皿(Sarstedt AG,目录号:82.1473)
  2. 1μl循环(Sarstedt AG,目录号:86.1567.010)
  3. 25 ml玻璃(或一次性塑料)移液管(Normax,目录号:4.5434334)
  4. 50ml Falcon管(Sarstedt AG,目录号:62.548.004)
  5. 2ml微量管(Sarstedt AG,目录号:72.691)
  6. JA-14离心管(Thermo Fisher Scientific,Nalgene TM ,目录号:3120-0250)
  7. JA-20离心管(Thermo Fisher Scientific,Nalgene TM ,目录号:3114-0050)
  8. 具有0.75mm集成间隔件的间隔板(Bio-Rad Laboratories,目录号:165-3310)
  9. Mini-PROTEAN Comb,10孔,0.75mm(Bio-Rad Laboratories,目录号:165-3354)
  10. 金黄色葡萄球菌菌株来分析其PGN水解酶的细胞壁溶解活性。
    注意: 为了产生底物细胞,研究人员可以使用 金黄色葡萄球菌 NCTC8325-4菌株,并且它可以从可以从DSMZ储存中心获得的BEI资源以参考号NR-45937和藤黄微球菌 DSM20030菌株获得。
  11. 胰蛋白酶大豆琼脂平板(TSA)(BD,Difco,目录号:236950)
  12. 胰蛋白酶大豆肉汤(TSB)(BD,Bacto,目录号:211825)
  13. Luria琼脂(Miller LB琼脂)(LA)(Conda,目录号:1552)
  14. Luria Broth(Miller's LB肉汤)(LB)(Conda,目录号:1551)
  15. 乙醇(Merck Millipore Corporation,目录号:1.02371.1000)
  16. 冰(自制)
  17. 液氮(液空)
  18. Tris(Trizma底物)(Sigma-Aldrich,目录号:T1503)
  19. 氯化钠(Merck Millipore Corporation,目录号:1.06444.1000)
  20. 盐酸(Merck Millipore Corporation,目录号:1.01834.2500)
  21. 十二烷基硫酸钠(Sigma-Aldrich,目录号:L5750)
  22. 甘氨酸(Sigma-Aldrich,目录号:G8898)
  23. 30%丙烯酰胺/Bis溶液(Bio-Rad Laboratories,目录号:161-0158)
  24. 二硫苏糖醇(DTT)(VWR International,目录号:V3155)
  25. 过硫酸铵(APS)[(NH 4)2 S 2 O 8](Sigma-Aldrich,目录号码:A3678)
  26. 溴酚蓝钠盐(Sigma-Aldrich,目录号:B8026)
  27. Precision Plus Protein TM 双色标准(PPPS)(Bio-Rad Laboratories,目录号:1610374)
  28. 亚甲基蓝水合物(Sigma-Aldrich,目录号:66720)
  29. Triton X-100(Sigma-Aldrich,目录号:T8787)
  30. 氯化镁六水合物(Sigma-Aldrich,目录号:M9272)
  31. 氯化钙二水合物(CaCl 2·2H 2 O·2H 2 O)(Sigma-Aldrich,目录号:C3306)
  32. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:P5958)
  33. N,N,N',N'-四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T9281)
  34. 70%乙醇(见配方)
  35. 500 mM Tris-HCl(pH 7.5)(参见配方)
  36. 洗涤缓冲液(见配方)
  37. 4%SDS(w/v)(参见配方)
  38. 10%SDS(w/v)(参见配方)
  39. 1.5 M Tris-HCl(pH 8.8)(参见配方)
  40. 0.5 M Tris-HCl(pH 6.8)(参见配方)
  41. 10%APS(w/v)(参见配方)
  42. Zymogram SDS-PAGE凝胶(参见配方)
  43. 5x Laemmli加载缓冲区(参见配方)
  44. Tris-甘氨酸-SDS缓冲液(参见配方)
  45. 复性缓冲液(参见配方)
  46. 亚甲蓝溶液(见配方)


  1. 100ml容量的Erlenmeyer烧瓶(Normax,目录号:2121624N)
  2. 1L容量的锥形烧瓶(Normax,目录号:2121654N)
  3. 比色杯(Sarstedt AG,目录号:67.742)
  4. 1.5ml微量管(Sarstedt AG,目录号:72.690.001)
  5. Micropipette(Gilson,型号:Pipetman P1000)
  6. 磁力搅拌棒(VWR International,目录号:442-0361)
  7. 30℃/37℃孵育振荡器(Eppendorf,New Brunswick Scientific,型号:Innova 40)
  8. 30℃/37℃孵育器(BINDER GmbH,型号:WTB)
  9. 燃烧器
  10. 分光光度计(GE Healthcare,Amersham,型号:Novaspec Plus可见分光光度计)
  11. 离心机(Beckman Coulter,型号:Avanti J-26 XPI)
  12. JA-14转子(Beckman Coulter,型号:339247)
  13. JA-20转子(Beckman Coulter,型号:334831)
  14. 台式离心机(Eppendorf,型号:5430R)
  15. 用于1.5ml管(Thermo Fisher Scientific,Eppendorf,型号:FA-45-24-11-HS)的转子
  16. 用于1.5ml管的振荡器(Eppendorf,型号:Thermomixer Comfort)
  17. Nanodrop(Thermo Fisher Scientific,型号:ND-2000C)
  18. 高压灭菌器
  19. Speed vac(Labconco,型号:78100)
  20. 短板(Bio-Rad Laboratories,目录号:165-3308)
  21. Power Pac HV电源(Bio-Rad Laboratories,目录号:164-5056)
  22. Mini-PROTEAN铸造支架垫片(Bio-Rad Laboratories,目录号:165-3305)
  23. Mini-PROTEAN ?铸造框架(Bio-Rad Laboratories,目录号:165-3304)
  24. Mini-PROTEAN ?铸造架(Bio-Rad Laboratories,目录号:165-3303)
  25. Mini-PROTEAN Tetra Cell(Bio-Rad Laboratories,目录号:165-8000)


  1. 制备来自S的粗自溶提取物。 aureus 细胞
    1. 使用纸用70%乙醇(v/v)对工作区域进行去污 毛巾和燃烧器打开。这个过程贯穿始终 该协议每次需要无菌条件(注1)。
    2. 细菌。金黄色葡萄球菌感兴趣的菌株接种在TSA上 通过使用1μl环的条纹铺板并在37℃下孵育过夜 30℃培养箱(注2)
    3. 在无菌条件下,单 用1μl环挑取分离的菌落,并接种于10ml TSB在100ml锥形烧瓶中。培养物生长过夜(16h-18 ?h)在30℃培养箱振荡器中以200rpm
    4. 光密度 ?在λ= 600nm处测量过夜培养物 1/10稀释(100μl培养物在900μlTSB中) 无菌条件
    5. 起始OD <600>所需的体积? ?从过夜培养物中取出0.05,并接种在250ml TSB在1L锥形瓶中。将培养物在30℃下生长 孵育摇床以200rpm
    6. 通过放置JA-14转子并将其冷却至4℃制备离心机JA-26 XPI
    7. 制备冰/乙醇浴并保持在4℃:冰 倒入容器中,并且将无水乙醇均匀铺展 到它。烧瓶洗涤缓冲液,JA-14和JA-20 将离心管置于其中以冷却
    8. 当培养物达到OD 600?0.3(其应该需要约3小时)时,将它们置于冰/乙醇浴中(注3)。
    9. 将培养物转移到冷JA-14离心管中。的 将管平衡,并将细胞在4℃,15,050xg下沉淀 15分钟。
    10. 离心后,用JA-20代替JA-14转子,将其冷却至4℃
    11. 通过倒置管子弃去上清液
    12. 将细胞重悬浮于20ml冰冷的洗涤缓冲液中 转移至冷JA-20离心管中。加入洗涤缓冲液 管的最大容量(?46ml)
    13. 如前所述离心细胞(步骤A9)
    14. 使用25ml玻璃小心地除去上清液 塑料)移液管,直到半成品球团留在底部
    15. 将细胞小心地重悬于250μl的4%SDS中 (w/v),转移到1.5ml微管中并在热混合器中温育 ?在25℃,700rpm,30分钟(注4)
    16. 孵育后,使用台式离心机在22,380×g下收获细胞15分钟,室温(RT)。
    17. 将含有粗酶自溶提取物的上清液转移到干净的微管中
    18. 使用2μl等分试样在Nanodrop(蛋白质-280nm)中定量提取物。使用Mili-Q水作为空白
    19. 将各提取物分成50μl等分试样,在液氮中冷冻并储存在-80℃直至进一步使用(注释5)。

  2. 制备用于凝胶酶谱的底物细胞
    1. 对于底物细胞的制备, aureus NCTC8325-4和 M。 藤黄细胞DSM20030菌株分别接种在TSA和LA板上, 通过使用1μl环的条纹铺板,并在37℃培养箱中孵育。

      将金黄色葡萄球菌温育过夜,并将M.luteus培养过夜至少24小时(注意 2)。
    2. 在无菌条件下,每个的单个分离的菌落 菌株用1μl环接种,并接种到10ml TSB或 LB在100ml锥形烧瓶中。培养物生长过夜(16h-18 h)在37℃培养箱振荡器中以200rpm
    3. 光密度 在1/10后测量过夜培养物在λ= 600nm处的光密度 稀释,使用1ml无菌TSB或LB作为空白。
    4. 的 从起始OD 600?0.05所需的体积 过夜培养并接种在1L的250ml TSB或LB中 锥形瓶。使用37℃培养摇床使培养物生长 ?以200rpm
    5. 当培养物达到OD 600?1.0时,细胞 通过在室温下在15,050×g下在JA-14管中离心15分钟收获。 M。 luteus 文化应该需要一整天,而 s。 aureus 应该需要约3小时才能达到所需的OD(注6)。
    6. 弃去上清液,用250ml Milli-Q水洗涤细胞
    7. 如上离心细胞,弃去上清液
    8. 将细胞重悬于30ml Milli-Q水中,并转移至50ml Falcon管中
    9. 将细胞悬浮液高压灭菌15分钟,121℃(注7)
    10. 将高压灭菌的细胞转移到JA-20管中,并在15,050×g,RT下离心15分钟。
    11. 弃去上清液,将沉淀在-20℃下储存过夜(注8)。
    12. 将沉淀在室温下解冻,重悬于3ml ?的Milli-Q水,并且一半分成两个预先称重的2ml管。
    13. 将悬浮液以真空速度冻干过夜
    14. 测定含有细胞的管的重量并计算干重
    15. 将细菌底物细胞在Milli-Q中彻底重悬浮 水,终浓度为50 mg/ml,储存于-20°C(注9)。

  3. 通过凝胶酶谱法分析粗自溶提取物
    1. 将底物细胞解冻,并保证完全重悬 ?在室温下搅拌过夜,使用磁力搅拌器 最大速度(注10)。
    2. 两种拆分丙烯酰胺/0.2% 制备双丙烯酰胺SDS-PAGE凝胶(Laemmli,1970),其包含a 最终浓度为2mg/ml的来自金黄色葡萄球菌的底物细胞(10% 凝胶)和M。叶黄素(8%凝胶)(注释11和图1)。
    3. 将缺少底物细胞的标准堆积凝胶放置在分辨凝胶的顶部(Laemmli,1970)。
    4. 10μg(对于金黄色葡萄球菌凝胶)和15μg(金黄色葡萄球菌凝胶) 粗自动酶提取物(注12)与Laemmli混合 加载缓冲液(Laemmli,1970),终浓度为2x(样品 不加热,见配方)。
    5. 所用的电泳条件如下:
      1. 温度:室温。
      2. 电压常数:70V(每凝胶)。
      3. Runnning缓冲液:Tris-甘氨酸-SDS缓冲液。
    6. 电泳后,将凝胶用Milli-Q水漂洗一次。
    7. 凝胶用Milli-Q在室温下轻轻搅拌洗涤3次,每次15分钟。
    8. 将凝胶在37℃下在复性缓冲液(注13)中温和搅拌温育过夜
    9. 将酶谱在亚甲基蓝溶液中染色1小时 在水中脱色,直到条带清晰为止(注14和图2)

      图1:10%酶谱凝胶和SDS-PAGE凝胶。 左面板显示a 酶谱凝胶,其略微不透明。右侧面板显示一个常规 SDS-PAGE凝胶,由于缺乏底物而完全透明 ?细胞

      图2. A。粗自溶的凝胶酶谱分析 酶提取物。 aureus 。 M。 luteus 和 S。 aureus zymogram gels are ?用于分析从中收获的自分解酶提取物 父母。 aureus NCTC8325-4菌株和缺乏的突变体 不同的自溶素(NCTCΔ atl ,其缺乏主要的ΔI自溶素; NCTCΔ lytM ,其缺少甘氨酰 - 甘氨酸内肽酶LytM; NCTCΔ lytN ?其缺乏推定的细胞壁水解酶LytN和NCTCΔ sle1 ,其 缺乏N' - 乙酰基胞壁酰基-L-丙氨酸酰胺酶Sle1)。五种不同的形式 ?的主要自溶素Atl与底物细胞。 但是,只有 s。金黄色葡萄球菌底物细胞允许检测Sle1 活性,因此显示Sys的底物特异性。金黄色葡萄糖聚糖。 B.翻译后裂解的代表 (黑色箭头)的em1 atl 编码的蛋白质(左)。加工的酰胺酶 (AM)通过切割PGN之间的键从PGN释放茎肽 茎肽和Nε - 乙酰胞壁酸残基。葡糖胺酶 (GL)通过切割聚糖链的Nε - 乙酰胞壁酸和Nε - 乙酰葡萄糖胺之间的糖苷键而释放出神经肽。 Sle1的表示(右),其包括三个LysM结构域 (负责与PGN的交互)和一个CHAP域(关联 ?具有不同PGN水解酶的酰胺酶活性)。


  1. 工作区域应该是在火焰附近工作的安全场所,即 ,没有气流,远离爆炸物,易燃材料和溶剂。对于该协议,靠近火焰的工作可以通过在生物安全柜中工作来代替。严格遵守所有建议的安全指南和您的主办机构建议的废物处置规定。
  2. 菌株的电镀应该从甘油储备液到新鲜培养基平板上以确保细菌培养物的活力和防止抑制突变体的选择。生长。 aureus 菌株不受光照/黑暗条件的影响
  3. 将细菌培养物孵育至OD 600±0.3确保了在指数生长期期间收获细菌。细菌快速转移到冰/乙醇浴中将降低自溶素的活性,其将修饰存在于底物细胞表面的PGN,并防止细菌裂解,这将导致特定PGN水解酶损失到生长培养基。
  4. 需要该步骤来提取与细菌细胞表面相关的蛋白质,而不诱导细菌的裂解。
  5. 将提取物以不同等分试样分开将防止将导致某些PGN水解酶的活性丧失的连续解冻和冷冻步骤(3个解冻和冷冻循环不导致Atl和Sle1的细胞壁溶解活性的丧失) br />
  6. 收获 S。 aureus 和 M.luteus ,请考虑他们的不同增长率。
  7. 该步骤确保该细胞悬浮液中的酶丧失其活性,因此在凝胶酶谱分析期间不引起底物细胞的裂解。
  8. 当在冷冻干燥步骤之前将高压灭菌的细胞储存在-20℃时,我们获得了更好的结果。我们假设冷冻步骤可以帮助底物的再悬浮
  9. 在凝胶酶谱分析中使用不同的细菌作为底物细胞可能导致在酶谱中观察到的条带的强度和条带数量不同。
  10. 这是一个关键的步骤,如果底物细胞没有很好地重悬,所得到的酶谱将是颗粒状的,降低了凝胶酶谱分析的灵敏度。
  11. M。叶黄素细胞壁对PGN裂解酶比对SX敏感得多。 aureus 细胞。因此,当 M。使用藤黄细胞,在酶谱中检测到的条带的强度和条带数目增加。当使用时,优选使用8%的凝胶。藤黄烯作为底物细胞,使得高分子量具有更好的分离。在金黄色葡萄球菌凝胶中,优选运行更浓缩的凝胶以检测接近37kDa蛋白质标准的Sle1的活性。
  12. 还由于M的高易感性。 luteus细胞对PGN水解酶的最佳提取物蛋白质的最佳量为5-10μg,而对于金黄色葡萄球菌凝胶,最小提取物蛋白质的量应为15μg。
  13. 需要该步骤以恢复在用SDS溶液处理期间损失的PGN水解酶的活性。
  14. 在该步骤中,由于存在自溶素而观察到清晰的条带。完整的底物细胞将保留染料,而在底物细菌细胞已裂解的情况下将观察不到颜色。
    凝胶与 S。金黄色葡萄球菌细胞将需要至少三次15分钟的洗涤之前,乐队开始看到。我们通常在三次洗涤后拍照,然后在约3h-5h的第四次或第五次洗涤之后。建议最后一夜洗涤以尽可能多地去除凝胶,以便更好地观察到活性较低的溶解蛋白(例如,GL活性)。


  1. 70%乙醇
  2. 500mM Tris-HCl(pH7.5) 向30ml Milli-Q水中加入30.3g Trizma底物 让它搅拌直到溶解
    用1M HCl将pH调节至7.5 用Mili-Q水填充至500毫升
  3. 洗涤缓冲液
    50mM Tris-HCl(pH7.5)/150mM NaCl 向4.4g NaCl中加入50ml 500mM Tris-HCl(pH7.5)和400ml Milli-Q水
  4. 4%SDS(w/v)
    将4g SDS溶于80ml Milli-Q水中 SDS是一种刺激剂
    用Mili-Q水填充至100ml 过滤灭菌(0.2μm)
  5. 10%SDS(w/v)
    重量10g,通过搅拌溶解在80ml Milli-Q水中 SDS是一种刺激剂
  6. 1.5 M Tris-HCl(pH 8.8)
    将18.2g Trizma底加入到60ml Milli-Q水中 让它搅拌直到溶解
    用1M HCl将pH调节至8.8 用Milli-Q水填充至100ml,
    保持其作为无菌储备溶液 - 使用等分试样(?20毫升)作为工作溶液
  7. 0.5M Tris-HCl(pH 6.8)
    向6.0ml的Milli-Q水中加入6.06g的Trizma底物 让它搅拌直到溶解
    用1M HCl将pH调节至6.8 用Mili-Q水填充至100ml 确认pH和过滤灭菌(0.2um)
    保持它作为无菌储备溶液 - 使用等分试样(约20毫升)作为工作溶液。
  8. 10%APS(w/v)
    重量1克,溶解在10ml Milli-Q水中
  9. Zymogram SDS-PAGE凝胶

  10. 5x Laemmli加载缓冲区
    重量0.77g DTT,4.3g甘油,2mg溴酚蓝
    加入5ml 0.5M Tris(pH6.8),并用Milli-Q水填充至10ml -20°C保存在50μl等分试样
  11. Tris-甘氨酸-SDS缓冲液
    在1000ml水中溶解30.0g Tris碱,144.0g甘氨酸和10.0g SDS,
  12. 复性缓冲区
    50mM Tris-HCl(pH7.5),0.1%(v/v)Triton X-100,10mM CaCl 2,10mM MgCl 2:
    重量0.74g二水合氯化钙和1.02g氯化镁六水合物 取50ml 500mM Tris-HCl(pH7.5)和500μlTriton X-100 搅拌至溶解
  13. 亚甲基蓝溶液
    0.1%(w/v)亚甲基蓝在0.01%氢氧化钾中的溶液 将溶于500ml Milli-Q水中的0.5g亚甲基蓝和0.05g氢氧化钾在室温贮存


该方案由Grilo等人改编或修改。 (2014)和Yokoi等人(2008)已被用于描述自溶素可以阻止在细菌细胞表面检测PGN(Atilano等人,/em>,2014)。这项工作得到了SFRH/BD/78748/2011研究金的支持,以及来自Funda??opara aCiênciae Tecnologia的SRF项目PTDC/BIA-PLA/3432/2012的支持。我们感谢InêsGrilo的建议和Teresa Baptista da Silva的技术支持。


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Copyright Vaz and Filipe. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Vaz, F. and Filipe, S. R. (2015). Preparation and Analysis of Crude Autolytic Enzyme Extracts from Staphylococcus aureus. Bio-protocol 5(24): e1687. DOI: 10.21769/BioProtoc.1687.
  2. Atilano, M. L., Pereira, P. M., Vaz, F., Catalao, M. J., Reed, P., Grilo, I. R., Sobral, R. G., Ligoxygakis, P., Pinho, M. G. and Filipe, S. R. (2014). Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system. Elife 3: e02277.