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STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00466-0
10.1016/j.xpro.2024.103301
103301
Protocol
Protocol for examining the T3SS-mediated cytotoxicity of Pseudomonas aeruginosa using the A549 cell line
Huang Jiahui vaney_huang@163.com
14∗
Zhang Yao 1
Sheng Shuo 2
Zhang Lian-Hui lhzhang01@scau.edu.cn
13∗∗
Xu Zeling zelingxu@scau.edu.cn
15∗∗∗
1 Guangdong Province Key Laboratory of Microbial Signals and Disease Control, Integrative Microbiology Research, Centre, South China Agricultural University, Guangzhou, China
2 Key Laboratory of Basic Pharmacology of the Ministry of Education, Joint International Research Laboratory of Ethnomedicine of the Ministry of Education, and Key Laboratory of Basic Pharmacology of Guizhou Province, Zunyi Medical University, Zunyi, Guizhou, China
3 Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
∗ Corresponding author vaney_huang@163.com
∗∗ Corresponding author lhzhang01@scau.edu.cn
∗∗∗ Corresponding author zelingxu@scau.edu.cn
4 Technical contact

5 Lead contact

07 9 2024
20 9 2024
07 9 2024
5 3 103301© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to directly inject effector proteins into host cells, leading to severe acute infections. Here, we present a protocol for detecting the T3SS-mediated cytotoxicity of P. aeruginosa using the A549 cell line. We describe the steps for the preparation of the A549 cell line and P. aeruginosa strains, cell seeding, bacterial culture, infection, and cytotoxicity assay. Additionally, we provide detailed procedures for data analysis.

For complete details on the use and execution of this protocol, please refer to Huang et al.1

Graphical abstract

Highlights

• Steps for culture, passage, and seeding of the A549 cell line

• Guidance for preparation and collection of P. aeruginosa

• Instructions for cytotoxicity assay and data analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to directly inject effector proteins into host cells, leading to severe acute infections. Here, we present a protocol for detecting the T3SS-mediated cytotoxicity of P. aeruginosa using the A549 cell line. We describe the steps for the preparation of the A549 cell line and P. aeruginosa strains, cell seeding, bacterial culture, infection, and cytotoxicity assay. Additionally, we provide detailed procedures for data analysis.

Subject areas

Cell Biology
Cell culture
Microbiology
Molecular Biology
==== Body
pmcBefore you begin

In this protocol, we used the A549 cell line to measure the T3SS-mediated cytotoxicity of P. aeruginosa. The HeLa cells, Chinese hamster ovary (CHO) cells, or other cell lines can also be used for cytotoxicity assay, and procedures using these cell lines can be found elsewhere.2,3,4

Culture and maintenance of A549 cells

Timing: 50 min each day, 4–5 days

The steps below describe the specific procedures for recovering and passaging of A549 cells.1. Preparing culture medium.a. Preheat a water bath to 37°C.

b. Thaw Fetal Bovine Serum (FBS) in the preheated water bath.

c. Incubate Dulbecco’s Modified Eagle’s Medium (DMEM) in the preheated water bath.

d. Add FBS to DMEM (10%; v/v) to obtain the medium for cell culture.

Note: 1% antibiotics such as penicillin and streptomycin can be alternatively added to prevent bacterial contamination.

2. Recovery of A549 cells.a. Take the vial containing A549 cells from the nitrogen tank and thaw the A549 cells at 37°C in a water bath immediately.

b. Transfer all the cell suspension to a 15 mL tube containing 8 mL pre-warmed FBS-containing DMEM.Note: Wipe the vial with 70% EtOH before opening it to prevent contamination. See problem 1 for the contamination solution.

c. Centrifuge at 200 × g for 5 min at room temperature.

d. Discard the supernatant and resuspend the cell pellet in 10 mL pre-warmed FBS-containing DMEM.

e. Transfer 5×105 cells to a 10 cm dish with a total volume of 8 mL.Note: A seeding density of 3 × 103–1×104 cells/cm2 is recommended.

f. Shake the dish gently to ensure the even distribution of cells.

g. Check the cell distribution under the inverted microscope.

h. Culture at 37°C with 5% CO2 and 95% humidity.

3. Passage A549 cells.a. Observe the cell adhesion under the inverted microscope.CRITICAL: Passage cells when confluence reaches 80%–90%. Refreshing the culture medium 24 h prior to passage may be helpful in promoting growth.

Note: See problem 2 for the cell cluster solution.

b. Discard the spent medium and wash cells twice with 3 mL of pre-warmed PBS.

c. Add 3 mL Trypsin-EDTA (0.05%) and incubate 2 min at room temperature.

d. Check dissociation under the inverted microscope.Note: The cells will become completely round after Trypsin-EDTA treatment.

CRITICAL: Complete digestion of cell-cell adhesion is critical. Extend the incubation time if digestion is incomplete, but not exceed 5 min.

e. Discard trypsin and add 5 mL pre-warmed FBS-containing DMEM.

f. Resuspend cells thoroughly by gently pipetting.

g. Add 20 μL cell suspension to Count Star cell counter plate.

h. Determine cell number 3 times using the Count Star automated cell counter.

i. Seed 5×106 cells into a 10 cm dish with a total volume of 8 mL.Note: A seeding density of 6 × 104–7×104 cells/cm2 is recommended. An appropriate cell density would help prevent spontaneous differentiation and promote cell growth. Transfer cells to two or more dishes if necessary.

j. Shake the dish gently and observe under the inverted microscope to ensure cells have spread evenly.

k. Culture cells at 37°C with 5% CO2 and 95% humidity. Refresh the medium every 2 days.CRITICAL: Cells must undergo 2–3 times passages before conducting infection experiments.

Recovery of Pseudomonas aeruginosa strains

Timing: 20 min

4. Prepare LB agar plates.

5. Take the bacterial strain stored at −80°C from the freezer and place it on ice or in liquid nitrogen.

6. Using a sterilized inoculation loop to streak bacterial cells onto LB agar plates containing 50 μg/mL Gentamicin Sulfate from the stored vial.

7. Incubate the plates at 37°C overnight.

Note: Recovered P. aeruginosa cells can be stored at 4°C for 1–3 days. Freshly recovered bacterial cells are recommended for cytotoxicity assays.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Bacterial and virus strains	
	
Pseudomonas aeruginosa PAO1(EV)	Lab collection	Not applicable	
Pseudomonas aeruginosa ΔfleR(EV)	Lab collection	Not applicable	
Pseudomonas aeruginosa ΔfleR(fleR)	Lab collection	Not applicable	
	
Chemicals, peptides, and recombinant proteins	
	
Fetal bovine serum (FBS)	Gibco	REF#10099141C	
Dulbecco’s modified Eagle’s medium (DMEM)	Gibco	REF#C11995500BT	
Trypsin-EDTA (0.05%), phenol red	Gibco	REF#25200056	
PBS	Gibco	REF#10010500BT	
Tryptone	Oxoid	LP0042B	
Yeast extract	Oxoid	LP0021B	
NaCl	Aladdin	C111538	
Gentamicin sulfate	Macklin	G6064	
Penicillin-streptomycin (5,000 U/mL)	Gibco	Cat# 15070063	
	
Critical commercial assays	
	
CytoTox 96 non-radioactive cytotoxicity assay	Promega	REF# G1780	
	
Experimental models: Cell lines	
	
Human lung epithelial A549 cell lines	Lab collection	Not applicable	
	
Software and algorithms	
	
GraphPad Prism 8	GraphPad Software	https://www.graphpad.com	
	
Other	
	
Airstream II, type A2 biosafety cabinet	ESCO	AC2-4S1	
CO2 incubator	Thermo Fisher Scientific	3111	
Portable sputum aspirator	FOLEE	H03-B	
Inverted microscope	Olympus	CKX53SF	
Automated cell counter	Countstar	IC1000	

Materials and equipment

Pseudomonas aeruginosa culture medium

Reagent	Final concentration	Amount	
Tryptone	10 g/L	10 g	
Yeast extract	5 g/L	5 g	
NaCl	10 g/L	10 g	
Milli-Q H2O	-	Add up to 1 L	

Note: 1.5% (w/v) agar is added to prepare LB agar plates. Sterilize the medium by autoclaving at 121°C for 20 min. For liquid medium, store at room temperature. For agar plate, store at 4°C for up to 4 weeks.

A549 cell line culture medium

Reagent	Final concentration	Amount	
Gibco Dulbecco’s Modified Eagle Medium	-	500 mL	
Gibco Fetal Bovine Serum	10%	50 mL	

Alternatives: 1% antibiotics (50 U/mL of penicillin-streptomycin) can be added to prevent contamination.

Note: Store at 4°C for up to 4 weeks.

A549 cells and P. aeruginosa co-culture medium

Reagent	Final concentration	Amount	
Gibco Dulbecco’s Modified Eagle Medium	-	500 mL	
Gibco Fetal Bovine Serum	1%	5 mL	

Note: Do not add antibiotics to the co-culture medium. Store at 4°C for up to 4 weeks.

Step-by-step method details

Seeding A549 cells into 96-well plate

Timing: 50 min

In this section, we describe steps for seeding A549 cells into a 96-well plate, which should be operated 1 day before infection.1. Take out the cell culture dish (Step 3k before you begin) from the incubator and observe the confluence under the inverted microscope.

CRITICAL: Seed cells when confluence reaches 80%–90%. See problem 2 for the cell cluster solution.

2. Remove the spent medium and wash twice with 3 mL pre-warmed PBS.

3. Add 3 mL Trypsin-EDTA (0.05%) and incubate at room temperature for 2 min.

4. Check the digestion under the inverted microscope.

CRITICAL: Complete digestion of cell-cell adhesion is critical. Extend the incubation time if digestion is incomplete, but do not exceed 5 min.

5. Discard trypsin and add 5 mL FBS-containing DMEM.

6. Resuspend cells thoroughly by gently pipetting.

7. Add 20 μL cell suspension to Count Star cell counter plate.

8. Determine the cell number three times on the Count Star automated cell counter.

9. Seed 1.5×104 cells into each well in a total volume of 100 μL.

10. Shake the plate gently, and observe under the microscope to ensure cells have spread evenly.

CRITICAL: Ensure cells are evenly distributed to avoid cell clusters.

11. Culture 24 h at 37°C with 5% CO2 and 95% humidity.

Overnight culture of P. aeruginosa strains

Timing: 20 min

Overnight culture of P. aeruginosa strains should be performed 1 day before infection.12. Inoculate a single colony from the agar plate into 2 mL fresh LB medium containing 50 μg/mL Gentamicin Sulfate.

13. Culture overnight at 37°C with 200 rpm.

Subculture and collection of P. aeruginosa cells

Timing: 4–5 h

In this section, we describe steps for culturing and collecting P. aeruginosa cells. It is necessary to perform a colony-forming unit (CFU) assay to determine the approximate cell number corresponding to the OD600 of 1.0. In this assay, OD600 of 1.0 corresponds to the 1 × 109 CFU/mL cell number.14. Transfer 100 μL overnight bacterial culture into a 50 mL tube containing 10 mL fresh LB medium.

15. Subculture at 37°C with 200 rpm until the OD600 of the culture reaches 1.0.

Note: The OD600 of 1.0 refers to the log phase growth state of P. aeruginosa.

16. Harvest 2 mL bacterial culture and centrifuge at 6,000 × g at 4°C for 5 min.

17. Discard the supernatant and add 2 mL PBS to resuspend the cell pellet.

18. Centrifuge at 6,000 × g at 4°C for 5 min.

19. Discard the supernatant and resuspend the cell pellet with 2 mL DMEM containing 1% FBS.

CRITICAL: The DMEM medium containing 1% FBS for bacterial resuspension is used to maintain osmotic pressure and the growth state of A549 cells and inhibit A549 cell proliferation during infection. A high concentration of FBS may promote cell proliferation, leading to inaccurate results.

20. Dilute the bacterial cell culture to an optimal density and place it on ice for further use.

Note: The dilution depends on the CFU corresponding to an OD600 of 1.0, and the number of A549 cells in the 96-well plate after incubation.

Co-culture A549 cells with P. aeruginosa strains

Timing: 5 h

In this section, we describe steps for the co-culture of A549 cells and P. aeruginosa strains.21. Take out the 96-well plate and observe the cells' distribution under the inverted microscope.

CRITICAL: Wells with cell clusters cannot be further used.

22. Remove the spent medium and wash twice with 100 μL PBS.

23. Co-culture the A549 cells with P. aeruginosa strains at a multiplicity of infection (MOI) of 50 in a total volume of 100 μL. Add 100 μL DMEM containing 1% FBS for positive and negative controls.

Note: It is recommended to perform positive and negative controls. Set up at least triplicate wells treated with 10× Lysis Solution (provided by the Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit) to generate a maximum LDH release as a positive control. Set up at least triplicate wells with untreated cells to serve as a negative control.

24. Culture at 37°C with 5% CO2 and 95% humidity for 4 h.

Note: For the first experiment, co-culture at different time points is recommended. Do not incubate overnight, as cell proliferation may lead to inaccurate results.

Measurement of lactate dehydrogenase (LDH) release

Timing: 1 h

In this section, we describe the measurement of LDH release after co-culture of A549 cells and P. aeruginosa strains using Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (The Kit). Reagents used in this section are provided in the Kit.25. Add 10 μL 10× Lysis Solution (provided by The Kit) to positive control wells 45 min before adding CytoTox 96 Reagent (provided by The Kit).

Note: 10× Lysis Solution is used to generate a maximum LDH release. The CytoTox 96 Reagent is prepared by adding 12 mL Assay Buffer (provided by The Kit) to the Substrate Mix bottle (provided by The Kit). Do not dissolve all the Substrate Mix at once.

26. Thaw the CytoTox 96 Reagent in the dark. Place it on ice or 4°C once thawed.

27. Transfer 50 μL supernatant from the co-culture plate (Step 24) using a multichannel pipettor to a new 96-well plate.

CRITICAL: Do not pipette up and down while transferring. Avoid transferring cells into the measurement 96-well plate.

28. Add 50 μL CytoTox 96 Reagent to each well and incubate for 30 min at room temperature in the dark.

29. Add 50 μL Stop Solution (provided by The Kit) to each well and mix by gently wrapping the plate or shaking on a plate shaker.

30. Pop any large bubbles using a syringe needle.

31. Measure the absorbance at 490 nm using a microplate reader.

Note: The absorbance should be read within 1 h after adding the stop solution. Refer to problem 3 and problem 4 for the solution of abnormal absorbance.

Calculation of results

32. Subtract the average values of the culture medium background from all values of experimental wells.

33. Use the corrected values in the following formula to calculate percent cytotoxicity:

percent cytotoxicity = 100 × mutant A490/wild-type A490.

Note: Refer to problem 5 for a solution of poor sample reproducibility.

Expected outcomes

The A549 cell line has been used as an acute infection model.1,5,6,7 This protocol outlines procedures for measuring LDH release from A549 cells as a readout of T3SS-mediated cytotoxicity. Proper recovery, maintenance, and seeding of A549 cells without cell clusters are critical for the following bacterial infection steps. Besides, accurate calculation of both numbers of the A549 cells and P. aeruginosa cells prior to their co-culture are essential to establish an accurate MOI.

We have recently identified that the response regulator FleR is a repressor of T3SS in P. aeruginosa.1 The data presented in Figure 1A shows the absorbance measured at step 31 for the negative/positive control groups and the A549 cell culture groups after infection by P. aeruginosa PAO1 wild-type, ΔfleR, and ΔfleR(fleR) strains. It was shown that co-culturing of the A549 cells with the ΔfleR mutant significantly increased absorbance at 490 nm compared to the co-culturing of the A549 cells with the wild-type strain PAO1 and complemented expression of fleR in the ΔfleR mutant restored the absorbance to the wild-type level, suggesting the higher cytotoxicity of ΔfleR. The absorbance of the negative and positive control groups represents the background without bacterial treatment and the maximum LDH release treated by 10x lysis solution, respectively. Therefore, the average absorbance of negative controls is subtracted from other wells used for the calculation of LDH release. Relative LDH release is shown in Figure 1B. Higher LDH release of the ΔfleR mutant confirmed the negative regulation of FleR on the expression of T3SS in P. aeruginosa.Figure 1 T3SS-mediated cytotoxicity of P. aeruginosa PAO1 wild-type, ΔfleR and ΔfleR(fleR) strains

(A) The absorbance of the experimental groups and the control groups at A490.

(B) The relative cytotoxicity of ΔfleR and ΔfleR(fleR) strains compared to the PAO1 wild-type strain. Statistical analysis was performed using one-way ANOVA. ∗∗, p < 0.01; ns, no significance compared to the indicated group.

Limitations

This protocol describes a method for examining T3SS-mediated cytotoxicity in P. aeruginosa using the A549 cell line, which is a straightforward evaluation of the T3SS activity. Because the host’s immune response is complex and activated during bacterial infection, T3SS activity is recommended to be further verified in vivo using infection models such as Galleria mellonella, mice, or other animal models.8,9,10

Troubleshooting

Problem 1

Contamination during the culture of A549 cell lines (Preparation one- culture and maintenance of A549 cells and Step-by-step- seeding A549 cells into 96-well plate).

Potential solution

• Sterilize the cell culture room thoroughly. Replace the water in the water bath and heat the water to 100°C for 4–5 h. Wipe the incubator and other devices in the cell culture room with 70% EtOH. Use of ultraviolet to disinfect the cell culture room overnight.

• It is recommended that the bacterial infection assay be performed in a separate area from the cell culture area to avoid cross-contamination.

Problem 2

Clustering of cells during passaging (Preparation one- culture and maintenance of A549 cells and Step-by-step- seeding A549 cells into 96-well plate).

Potential solution

• Gently moving the dishes or wrapping the plate and then observing the cell distribution under the inverted microscope before incubation would help to avoid cell clusters.

• Insufficient time for trypsin treatment may lead to cell clustering. Check the cell digestion with a microscope. Extend the incubation time if the digestion is insufficient, but do not exceed 5 min.

• It is possible to break cell clusters apart by pipetting or pressing during trypsinization. Avoid doing this in 96-well plates, as it may damage cells and cause LDH release.

Problem 3

High absorbance for the background (negative control) and low absorbance for maximum LDH release (positive control) (Step 31).

Potential solution

• A high concentration of serum may lead to a high background absorbance. Use DMEM containing 1% FBS as the co-culture medium. The FBS concentration should not exceed 5%. Besides, High absorbance for the background may also be ascribed to cell death. Avoid vigorous pipetting when seeding or washing cells. Make sure cell density does not exceed 1.5×106 cells/mL. In addition, bubbles in the wells may cause high absorbance. Eliminate bubbles using a syringe needle.

• Ensure the 10× Lysis Solution (provided by The Kit) is added and effective for cell lysis. 10× Lysis Solution (provided by kit) should be added 45 min before adding CytoTox 96 Reagent (provided by The Kit). 10× lysis solution should be stored at 4°C.

Problem 4

Low absorbance after the co-culture of A549 cells with the P. aeruginosa wild-type strain (Step 31).

Potential solution

• Incorrect wavelength of the plate reader may lead to low absorbance. Set the wavelength to 490 nm or 492 nm.

• Researchers may add antibiotics in DMEM during the culture of A549 cells to prevent bacterial contamination. The presence of antibiotics in the co-culture medium can slow down the growth of P. aeruginosa strains, leading to a reduced release level of LDH and, consequently, a lower detected absorbance than expected. To solve this problem, wash the cells twice with PBS before co-culture with P. aeruginosa and do not add antibiotics to the co-culture medium.

• Increase the incubation time of co-culture from 4 h to 6–8 h. Do not incubate for more than 12 h, because cell proliferation may occur. Try different time points for the incubation, which is recommended for the first time.

• Make sure the final concentration of FBS in the co-culture medium is 1%. Higher concentration of FBS in the co-culture medium will cause cell proliferation.

Problem 5

Poor reproducibility of the assay (Step 33).

Potential solution

• Ensure that all the bubbles have been eliminated completely.

• Carefully transfer the supernatant to the LDH measurement plate and avoid transferring cells to the LDH measurement plate. Do not resuspend the culture medium after co-culture.

Resource availability

Lead contact

Further information and requests should be directed to the lead contact, Zeling Xu (zelingxu@scau.edu.cn).

Technical contact

Further technical issues should be directed to the technical contact, Jiahui Huang (vaney_huang@163.com).

Materials availability

This study did not generate new unique reagents. Plasmids and bacterial strains used in this study are available upon request after a Materials Transfer Agreement is completed.

Data and code availability

• The lead contact will share all data reported in this study upon request.

• This study does not report the original code.

• Any additional information required to reanalyze the data reported in this study is available from the lead contact upon request.

Acknowledgments

The icons used in the graph abstract were downloaded from the Bioicons website (https://bioicons.com) with modifications.

This work was supported by the National Natural Science Foundation of China (no. 32370188 ), Undergraduate Training Programs for Innovation of South China Agricultural University (no. 202310564078 ), the Science and Technology Project of Guizhou Province (Qian Ke He Ji Chu – ZK [2022] no. 604), and Zunyi City (Zun Shi Ke He HZ Zi [2023] no. 171).

Author contributions

J.H. wrote the manuscript; J.H. and Y.Z. performed the experiments; S.S. provided research materials; Y.Z., S.S., and Z.X. acquired financial supports; L.-H.Z. and Z.X. revised the manuscript.

Declaration of interests

The authors declare no competing interests.
==== Refs
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