Legionella Phospholipases Implicated in Infection: Determination of Enzymatic Activities
The intracellularly replicating lung pathogen Legionella pneumophila expresses a multitude of different phospholipases which are important virulence tools during host cell infection. To study the lipolytic properties including substrate specificities of potential L. pneumophila phospholipases A (PLA), we used different assays to monitor lipid hydrolysis. Here we describe methods for quantitative analysis of liberated fatty acids via a photometric assay and for identification of specific lipids which are generated by PLA action by means of lipid extraction and thin-layer chromatography. The latter approach also identifies glycerophospholipid:cholesterol acyltransferase activity which may be associated with PLA activity and is responsible for the transfer of fatty acids derived from a phospholipid to an acceptor molecule, such as cholesterol. These methods applied for specific L. pneumophila enzyme knockout mutants compared to the wild type or for recombinantly expressed protein allow to conclude on substrate specificity and/or contribution of a specific enzyme to the total lipolytic activity. Further, via analysis of separated cellular fractions, such as culture supernatants and cell lysates, information on the localization of the enzymes will be obtained.
Key wordsLegionella Phospholipase A (PLA) Lysophospholipase A (LPLA) Glycerophospholipid:cholesterol acyltransferase (GCAT) Lipid hydrolysis Lipid extraction Thin-layer chromatography
L. pneumophila is an intracellularly replicating bacterial pathogen causing a severe pneumonia termed Legionnnaires’ disease (7, 8). Bacterial phospholipases may support pathogenesis by a multitude of mechanisms, including acquisition of nutrients, prevention of phagosome maturation via modification of signal transduction or remodeling of membranes, bacterial egress from the phagosome/host cell, attraction of phagocytic cells to the site of infection, and lung destruction (4, 9, 10, 11, 12, 13).
Genome analysis of L. pneumophila so far uncovered at least 15 different PLA-encoding genes which are divided into three main groups (4, 5). The first group consists of the PLAs/LPLAs PlaA, PlaC, and PlaD belonging to the GDSL-family of lipases (4, 5, 6, 14, 15). Within this group PlaA displays the major secreted LPLA activity which is important for the detoxification of cytotoxic lysophospholipids (15). In addition to its PLA and LPLA activities, PlaC additionally transfers fatty acids from lipids to cholesterol, a major component of eukaryotic membranes and therefore comprises GCAT activity (6). L. pneumophila also exhibits a cell-associated PLA/LPLA, PlaB, which possesses the highest phospholipolytic activity described to date for this bacterium (16). PlaB also showed hemolytic activity against human red blood cells and supports bacterial growth and dissemination in a guinea-pig infection model (16, 17, 18). The third group are the patatin-like proteins (PLPs) and the genome of sequenced L. pneumophila strains encodes 10–11 PLPs (4, 5). Enzymatic PLA/LPLA activity has so far been shown for L. pneumophila PatD (patatin-like protein D), a cell-associated PLP together with the adjacently encoded 3-hydroxybutyrate dehydrogenase essential for intracellular bacterial replication (19). Other PLPs such as VipD/PatA, VpdA/PatC, and VpdB/PatG are injected into the host cell via the Icm/Dot type IVB secretion system. VipD/PatA (vacuole protein sorting inhibitor protein A/patatin-like protein A) causes trafficking defects of the late secretory pathway when expressed in Saccharomyces cerevisiae (20, 21).
In summary this armory of different phospholipases of L. pneumophila contributes to the establishment and maintaining of a life inside a host and therefore it is worth to study their lipolytic activities and the different substrate specificities by using the methods described in this chapter.
All solutions and buffers should be prepared using ultrapure sterilized water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ cm at 25°C) and analytical grade reagents.
2.1 Reagents for Preparation of Bacterial Products
Sodium azide: 300 mM NaN3 stock solution. Fill in about 50 mL water to a beaker glass for better resolving of the reagent and add 1.95 g NaN3 and water to a volume of 100 mL (see Note 1).
Tris–HCl buffer: 40 mM Tris–HCl, pH 7.5. Fill in about 100 mL water to a beaker glass for better resolving of the reagent and add 6.3 g Tris–HCl and water to a volume of 900 mL. When reagents are completely dissolved, adjust pH at 25°C with HCl and make up to 1 L with water.
Lysozyme/Triton X-100 solution: Dissolve 10 mg lysozyme in 1 mL 40 mM Tris–HCl, pH 7.5 buffer by intensive vortexing and add 1 μL Triton X-100.
2.2 Reagents for Lipid Hydrolysis and Free Fatty Acids Assay
Sodium azide, Triton X-100, ethanol.
Materials/Kits: NEFA-HR kit (Wako Chemicals, USA) consistent of NEFA-HR(2) R1 Set (Code No. 434-91795), NEFA-HR(2) R2 Set (Code No. 436-91995), and NEFA Standard (Code No. 270-77902).
Tris–HCl buffer: see Subheading 2.1.
- 4.Lipid substrates: Use the following lipid substrates (Avanti Polar Lipids/Sigma-Aldrich/Fluka) in a final concentration of 13.4 mM in Tris–HCl buffer (Table 1). For example, weigh 10 mg of DPPG into a round-bottom tube and add 1 mL Tris–HCl buffer (see Note 2). Cholesterol (Sigma) was used for GCAT determination. Further resuspension steps are described in Subheading 3.2.Table 1
Examples of net weights for 1 mL of 2× phospholipid mix
Lipid (all palmitoyl)
2.3 Reagents for Lipid Extraction and Thin-Layer Chromatography
Methanol, chloroform, n-hexane, diethylether, glacial acetic acid, petroleum ether, NaCl, Naphtol Blue Black.
Materials/Kits: Silica gel thin-layer chromatography (TLC) plates (Merck), filter paper.
3.1 Preparation of L. pneumophila Cell Lysates and Culture Supernatants
Take a volume of 1 mL bacterial culture, for example at the end of exponential growth phase (i.e., at an OD660 of 2.0 to 2.1), and spin for 5 min in a tabletop centrifuge at 5,000 × g.
Carefully remove about 900 μL supernatant and add a final concentration of 3 mM sodium azide to the supernatant by adding 9 μL of the 300 mM NaN3 stock solution (see Note 3) and preserve for subsequent analysis.
Remove and discard as much residual supernatant as possible without disturbing the bacterial pellet. Lyse the cell pellet by adding 50 μL lysozyme/Triton X-100 solution. Incubate the samples for 30 min at 37°C with shaking. Homogenize samples by repeated passing five- to tenfold through a 24-gauge needle. Rinse the needle and syringe with a volume of 950 μL Tris–HCl buffer and fill up the samples with that liquid to the original culture volume and preserve for subsequent analysis.
3.2 Digestion of Lipid Substrates
Prepare lipid substrates at a final concentration of 13.4 mM in Tris–HCl buffer (Table 1). PE, 1,2-DG and TG should first be dissolved into 50 μL ethanol prior to adding 950 μL of Tris–HCl. Further add sodium azide at a final concentration of 6 mM and 1% v/v Triton X-100. After intensive vortexing incubate the substrate for 10 min at room temperature (no shaking) and afterwards at 37°C with continuous agitation at 250 rpm (see Note 4). When culture supernatants or cell lysates are assessed for GCAT activity, 50 μL/mL of cholesterol in ethanol (10 mg/mL) was added to a DPPG mixture prior to sonication.
Sonicate the lipid substrate three times, for 15 s each time, at cycle 4 with an intensity of 65%.
Incubate the lipid substrate with bacterial products at a volume ratio of 1 + 1. For example for photometric detection of fatty acid release, fill in 25 μL of lipid substrate (see Subheading 3.2) into a 96-well microtiter plate and add 25 μL of bacterial cell lysate or culture supernatant (see Subheading 3.1). Seal the plate with adhesive film. For TLC analysis, add 50 μL of lipid substrate (see Subheading 3.2) into 2 mL plastic tubes and add 50 μL of bacterial cell lysate or culture supernatant (see Subheading 3.1). For photometric detection of free fatty acids (FFA), incubation of L. pneumophila preparations is typically performed at 37°C for about 4–6 h (for lower activities up to 16 h) detecting secreted activities and about 1 h or less for cell-associated activities with continuous agitation at 150 rpm (see Note 4). A 1:20 dilution of the cell lysates is recommended and those preparations may be incubated for 4–6 h. For detection of lipids via TLC, incubation of L. pneumophila preparations is typically performed at 37°C for about 16 h detecting secreted activities and about 16 h or less for cell-associated activities with continuous agitation at 150 rpm (see Note 5).
The final concentration of the components of the reaction are 6.7 mM phospholipid (or 3.35 mM for the above-mentioned exceptions), 3 mM sodium azide, 0.5% v/v Triton X-100, and half of the original culture concentration of supernatant or cell lysate.
Depending on the nature of the experiment, BYE broth, LB broth, or Tris–HCl buffer was incubated, treated like the cultures, and subsequently used as a negative control.
3.3 Measurement of Free Fatty Acids
Transfer 10 μL of the incubated mixture of lipid substrates with bacterial products and the negative control into a new 96-well microtiter plate (see Note 6).
The kit also provides an oleic acid standard (1 mM) that is diluted to prepare a panel of standard concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM FFA. Pipette 10 μL of the prepared standard solutions in duplicate per well (see Note 7).
Freshly prepare solution R1 after the manufacturer’s recommendations (addition of reagent R1a to solution R1) and add 100 μL per well and incubate the plate for 5 min at room temperature with shaking.
Afterwards prepare solution R2 after the manufacturer’s recommendations (addition of reagent R2a to solution R2) and add 50 μL per well and agitate the plate packed in aluminum foil for another 5 min at room temperature (see Note 8).
Purple color develops depending on the amount of released fatty acids. Read out the plates with an ELISA reader at 550 nm.
3.4 Lipid Extraction and Thin-Layer Chromatography
Add 400 μL of methanol and 200 μL of chloroform to the 2 mL tube containing 100 μL of the reaction mixture and incubate for 30 min at 37°C. Subsequently add 280 μL of bidest. water and 200 μL of chloroform. Shake the samples for 10 min at room temperature using a rotator. Afterwards centrifuge the tubes at 2,000 × g for 5 min to get a well-defined phase separation.
Gently pipette out and throw away the upper aqueous phase. The remaining chloroform phase contains the lipids. Carefully evaporate the chloroform in a speed vac for approximately 50 min at 30°C.
Resuspend the lipid film in 25 μL chloroform:methanol at a ratio of 2:1. Use 10 μL for TLC of lipids.
For precise sample addition mark places with a pencil on a silica gel TLC plate (see Note 9).
- 5.Prepare the solvents (A), (B) (23), and (C) (24) dependent on the properties of the investigated lipids after the following instruction:
Apolar lipids (focus on detection of FFA and mono- or diglycerides): n-hexane:diethylether:glacial acetic acid, v:v:v, 70:30:4.
Polar lipids (focus on detection of phospholipids): Chloroform:methanol:water, v:v:v, 65:25:4.
Acyltransferase (cholesterolester) detection: Petroleum ether:diethylether:glacial acetic acid, v:v:v, 90:10:1.
Fill in about 100 mL of the prepared solvents into the TLC chamber and add filter paper for sufficient saturation. After that close the chamber.
- 7.Carefully add max. 10 μL samples per spot to the TLC plate (see Note 10). Depending on the application the following standards should be used:
Apolar lipids: FFA, 1-MPG, 1,2-DG, and TG.
Polar lipids: MPLPC, MPLPG, DPPC, DPPG, and DPPE.
Acyltransferase: FFA, 1-MPG, 1,2-DG, cholesterol, TG, and cholesterol ester.
Add 0.5–2 μL of a standard stock solution with a concentration of 10 mg/mL in chloroform:methanol (2:1).
Put the loaded plate slightly diagonal into the solvent tank, close the tank, and check that the solvent front runs evenly.
When the solvent front is 2–5 cm away from the top of the plate, the run has been completed. Take out the plate and let it dry in the chemical hood.
- 10.To stain the plate (25) soak it a couple of seconds in bidest. water. Dye it in 0.2% (w/v) of Naphtol Blue Black in 1 M NaCl for 5–10 min until the spots are visible (see Note 11). Rapidly wash the plate in 1 M NaCl. Subsequently dry the plate for a couple of minutes and take a photo or scan it (see Note 12) (example see Fig. 2).
Sodium azide is very toxic and dangerous for the environment. For this reason wear gloves and a mask to prepare the stock solution.
You should prepare a minimal amount of 2 mL of the resuspended lipid substrates, because there will be foam formation after vortexing and sonication. Pipetting then is much easier with a larger amount of dissolved lipids.
Prepared cell lysates and culture supernatants should be used immediately or can be stored overnight at 4°C. To avoid contamination sodium azide is added. Longer storage might be possible, but loss or reduction of hydrolytic activity might be the result and in such cases the use of protease inhibitors may be taken into consideration.
Prepared lipid substrates can be stored at −20°C and used once again after thawing at 37°C, continuous shaking, and optional sonication.
After the incubation step, the assay can be interrupted and the microtiter plate may be stored at −20°C.
After incubation at 37°C, there will be water condensation at the top of the microtiter plate under the adhesive film. Hence you should spin down the plates in a microtiter plate centrifuge for a couple of seconds at 800 × g. Then the plates should be gently shaken for a couple of seconds.
Dilute the provided oleic acid standard with Tris buffer to the concentrations chosen for the standard and store it at 4°C.
Both reagents R1 and R2 could be stored for 1 month at most. To check whether the solutions are still working, we recommend a preliminary test done by pipetting just the oleic acid standard and look for getting appropriate photometric reads.
Mark places for sample addition 1–2 cm from the bottom of the plate and from the edges so that the samples do not get in direct contact with the solvent in the tanks.
For precise sample addition we recommend the use of 10 μL glass capillaries.
Pay attention that you do not get any undissolved material onto the plate.
You can also scan the plate when it is still wet. In doing so, the spots might be better visible.
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