Establishment and Comparison of Fluorescence-Based T6SS Activity Detection Methods in Acinetobacter baumannii

Summary

Here, we propose a quantitative detection method based on fluorescent labeling (especially Luciferase) to efficiently and accurately assess the activity of bacterial T6SS, which is suitable for high-throughput analysis of clinical strains.

Abstract

The Type VI Secretion System (T6SS) is a crucial mechanism mediating intercellular interactions in Gram-negative bacteria, particularly in pathogenic species such as Acinetobacter baumannii. Previous studies have shown that the large plasmid pAB3 in the A. baumannii ATCC 17978 strain encodes a TetR-like protein that inhibits the expression of core T6SS genes. In contrast, the WT^R- strain, which lacks pAB3, can stably express and secrete the T6SS effector protein Hcp and exhibits the ability to kill E. coli. The tssM gene, one of the core genes of T6SS, is essential for its activity; its deletion directly leads to the inactivation of T6SS. However, traditional T6SS activity detection methods, such as killing assays, suffer from low throughput and insufficient sensitivity. To address these limitations, we have developed quantitative detection methods based on fluorescent labeling.

To improve T6SS activity detection, we developed three fluorescent labeling methods: (1) A quantitative detection method based on Luciferase labeling, which is characterized by high specificity, sensitivity, and reproducibility, making it suitable for high-throughput analysis; (2) A detection method based on green fluorescent protein (GFP) labeling, which, despite being susceptible to environmental interference, offers the advantage of high throughput; (3) Flow cytometry detection, which can quantitatively assess bacterial viability but is operationally complex and costly. After a comprehensive comparison, the Luciferase-based labeling method proved to be the most accurate, sensitive, and user-friendly. When applied to 20 clinical isolates of A. baumannii, this method was confirmed to rapidly and accurately evaluate T6SS activity.

Introduction

The Type VI Secretion System (T6SS) is an important protein secretion system in Gram-negative bacteria, widely involved in bacterial competition, antagonism against eukaryotic hosts, and regulation of host immune responses^1,2. By injecting toxic effector proteins into neighboring bacteria or eukaryotic cells, T6SS helps bacteria maintain a competitive advantage in complex environments. In recent years, the significant role of T6SS in bacterial adaptability, pathogenicity, and microbial interactions has made it a research hotspot, especially in multidrug-resistant pathogens, where its functions provide potential targets for the development of new antibacterial strategies^3,4,5.

Acinetobacter baumannii (Ab) is a Gram-negative opportunistic pathogen widely distributed in hospital settings. Due to its high drug resistance and strong adaptability, it has become an important nosocomial pathogen worldwide^6,7,8. The survival of A. baumannii in complex microbial environments relies on multiple secretion systems, among which T6SS plays a key role in its competition with other microbes and interactions with the host. With its highly conserved T6SS gene cluster and well-defined functions, A. baumannii has become an ideal model for studying T6SS mechanisms. Moreover, the unique structure of the T6SS in A. baumannii provides a distinctive perspective for further revealing the diversity and functions of T6SS.

The T6SS gene cluster of A. baumannii contains 12 core protein genes (such as tssA-tssM, lacking tssJ) and several genes with unknown functions (such as tagX, tagN, tagF, etc.)^2,9,10. Among them, the hemolysin coregulated protein (Hcp) is an important component of T6SS, and its secretion is considered a hallmark of functional T6SS. Studies have shown that the large plasmid pAB3 in A. baumannii inhibits T6SS expression by encoding a TetR-like regulatory protein. In contrast, strains that have lost pAB3 (such as WT^R-) can stably express and secrete Hcp, exhibiting significant T6SS activity¹¹. The tssM gene is one of the core genes of T6SS, and its deletion directly leads to the inactivation of T6SS.

However, traditional T6SS activity detection methods (such as killing assays) have limitations such as low throughput, poor reproducibility, and insufficient sensitivity, which severely restrict the in-depth study and widespread application of T6SS⁵. Therefore, the development of efficient and accurate T6SS activity detection methods has become an urgent need in current research. This study aims to establish and compare T6SS activity detection methods based on fluorescent labeling to overcome the shortcomings of traditional methods. Through fluorescent labeling technology, we have achieved real-time, quantitative monitoring of T6SS activity, providing a more precise and reliable tool for the study of bacterial interactions. This study has validated the effectiveness of fluorescent labeling methods and, by comparing the advantages and disadvantages of different fluorescent labeling strategies, has provided new perspectives and methodological support for future research. It not only offers new technical means for T6SS research but also lays a scientific foundation for understanding the complex mechanisms of bacterial interactions and developing new antibacterial therapeutic strategies.

Protocol

1. Sources of strains and plasmids

1. Use A. baumannii standard strain ATCC 17978, E. coli (pZY01), and the clinical strain of A. baumannii isolated from the Department of Laboratory Medicine at the First Hospital of Jilin University.
2. Use the plasmid pZY01-GFP (carrying a kanamycin resistance gene, capable of stable expression in A. baumannii), which was modified and preserved in the laboratory, and the commercially purchased plasmid pGEN-luxCDABE (carrying an ampicillin resistance gene).
3. Culture A. baumannii and E. coli in Luria-Bertani (LB) medium or a minimal medium containing: 50 mM potassium phosphate buffer (pH 7.4), 15 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.2% (w/v) sodium succinate as the sole carbon source, and 0.01% (w/v) yeast extract¹². Add antibiotics at the following concentrations when necessary: E. coli: ampicillin (100 µg/mL) and kanamycin (50 µg/mL); A. baumannii: streptomycin (100 µg/mL).
   NOTE: E. coli (pZY01) and E. coli (pZY01-GFP) express kanamycin resistance. E. coli (pZY01) does not have a fluorescent marker so it can be referred to as E. coli in the following text.

2. Preparation of bacterial strains before the experiment

1. Retrieve the bacterial strains from the -80 °C freezer 2 days prior to the experiment. Inoculate the bacterial strains onto LB agar plates containing the corresponding antibiotics. Incubate the plates overnight at 37 °C.
   NOTE: Ensure that all cells retain the plasmids at the start of the experiment.
2. Pick two independent single colonies of each bacterial strain 1 day before the experiment. Inoculate each colony separately into two individual tubes containing 5 mL of LB liquid medium. Incubate the cultures overnight at 37 °C with shaking at 200 rpm. Use these cultures for subsequent experiments.
   NOTE: Optimize the incubation time based on the bacterial growth rate to ensure an adequate amount of bacterial biomass is obtained.

3. Preparation of E. coli (GFP)/ E. coli (Luciferase) strains

1. Transformation
   1. Add 50 ng of plasmid DNA (pZY01-GFP or pGEN-luxCDABE, quantified via NanoDrop at A260/A280) into 100 µL of E. coli DH5α competent cells, respectively, and mix gently.
   2. Incubate on ice for 30 min, heat-shock at 42 °C for 60 s, and immediately place on ice for 30 s.
   3. Add 700 µL of antibiotic-free LB broth and incubate at 37 °C with shaking at 180 rpm for 2 h.
2. Plating and culturing
   1. Centrifuge at 420 × g for 2 min, discard the supernatant, and resuspend the cell pellet in 100 µL of antibiotic-free LB broth.
   2. Spread the pZY01 (GFP) transformation product onto LB agar plates containing 50 µg/mL kanamycin.
   3. Spread the pGEN-luxCDABE transformation product onto LB agar plates containing 100 µg/mL ampicillin. Incubate at 37 °C overnight.
3. Verification of positive clones
   1. Pick single colonies and extract DNA as polymerase chain reaction (PCR) templates the next day. Perform PCR amplification and verify by agarose gel electrophoresis.
   2. Confirm positive strains and label them as E. coli (GFP) and E. coli (Luciferase).
      NOTE: Ensure the quality of competent cells is high for efficient transformation. Avoid excessive bacterial liquid during plating to prevent interference with single colony isolation.

4. Western blotting

1. Sample preparation
   1. Collect bacterial cultures and centrifuge (1500 × g, 10 min, 4 °C) to separate the cell pellet and supernatant.
   2. Add an appropriate amount of loading buffer to both the cell pellet and supernatant. Boil at 100 °C in a metal bath for 10 min. Centrifuge (1500 g, 10 min, 4 °C) and collect the supernatants as protein samples.
   3. Measure protein concentration using the bicinchoninic acid (BCA) or Bradford method and adjust to the same concentration.
2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
   1. Prepare SDS-PAGE gels of appropriate concentrations (e.g., 12% separating gel, 5% stacking gel)¹³.
   2. Load 20 µg protein samples into the gel wells along with a 1-2 µL pre-stained protein marker. Perform electrophoresis at 100-120 V until the bromophenol blue indicator reaches the bottom of the gel.
      NOTE: Running Buffer: Tris 25 mM, Glycine 192 mM, SDS 0.1% (w/v), distilled water to 1 L.
3. Transfer (Western Blotting)
   1. Remove the gel from the glass plates and soak it in transfer buffer. Prepare a polyvinylidene difluoride (PVDF) or nitro cellulose (NC) membrane, activate the PVDF membrane with methanol, and then soak it in transfer buffer.
      ​NOTE: Transfer Buffer: 25 mM Tris, 192 mM Glycine, 20% Methanol
   2. Assemble the transfer apparatus in a "sandwich" structure (filter paper-gel-membrane-filter paper). Transfer at 100 V for 60-90 min (adjust time based on protein molecular weight; Host cell proteins (HCP) has a molecular weight of approximately 17 kDa).
      NOTE: Place the transfer tank in ice water and add ice packs to maintain a low temperature. If the transfer time is long, replace the ice packs midway.
4. Blocking
   1. After transfer, place the membrane in 5% skim milk-blocking solution. Block at room temperature (RT) on a shaker for 1 h or overnight at 4 °C.
5. Primary antibody incubation
   1. Wash the membrane three times with Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) buffer, 5 min each time. Incubate the membrane with diluted primary antibodies (mouse anti-Hcp antibody, 1:1000 dilution; mouse anti-isocitrate dehydrogenase (ICDH) antibody, 1:1000 dilution) at 4 °C overnight or RT for 2 h.
6. Secondary antibody incubation
   1. Wash the membrane with TBST buffer 3 times, 5 min each. Incubate the membrane with diluted secondary antibody (Horseradish peroxidase [HRP]-conjugated anti-mouse IgG, 1:3000 dilution) at RT for 2 h.
7. Detection
   1. Wash the membrane with TBST buffer 3 times, 5 min each. Prepare ECL chemiluminescent substrate and evenly apply it to the membrane.
   2. Detect signals using a chemiluminescence imaging system and capture images (HCP protein has a molecular weight of approximately 17 kDa, ICDH is a cytoplasmic metabolic enzyme with an expected molecular weight of approximately 45 kDa in A. baumannii).
      NOTE: ICDH is an important quality control marker that can only be detected in the cell pellet fraction (containing intact bacteria) and not in the supernatant fraction (containing secreted proteins), thereby demonstrating that no cell lysis occurred during sample preparation. Hcp secretion (17 kDa) should only be visible in supernatants from T6SS-active strains.

5. Preparation of 96-well plates with LB agar medium

1. Quickly add autoclaved antibiotic-free LB agar medium, pre-warmed, into a sterile 96-well plate, 50 µL per well. Allow the plate to sit at RT for at least 1 h to ensure complete solidification of the medium.
2. Use black-walled plates with a transparent bottom for detecting GFP fluorescence.
3. Use light-proof 96-well plates for detecting Luciferase fluorescence to avoid cross-well fluorescence interference.
   NOTE: Avoid generating bubbles when adding the medium to ensure uniform gel thickness, which promotes optimal bacterial contact and growth.

6. A. baumannii killing assay

1. Standard killing assay
   1. Culture WT^R- and ΔtssM mutant strains overnight at 37 °C with shaking in LB medium supplemented with 100 µg/mL streptomycin, grow E. coli separately in LB medium containing 50 µg/mL kanamycin under identical conditions.
   2. Wash the bacterial cultures twice with sterile PBS and resuspend them in fresh antibiotic-free LB medium. Adjust the OD₆₀₀ to 1.0 for standardization.
   3. Mix WT^R- and ΔtssM mutant strains with E. coli at a 1:1 ratio.
   4. Spot 10 µL of the mixed bacterial suspension onto antibiotic-free LB agar plates and incubate at 37 °C for 6 h.
   5. Scrape the bacterial spots and resuspend them in 1 mL of PBS. Perform five 10-fold serial dilutions.
   6. Spot 10 µL of the diluted bacterial suspensions onto LB plates containing kanamycin and incubate overnight at 37 °C.
   7. Analyze the T6SS-dependent antibacterial activity of WT^R- and ΔtssM mutant strains by colony counting.
      NOTE: Ensure thorough mixing of bacterial suspensions to avoid experimental errors. Perform at least three independent replicates for each sample to ensure data reliability.
2. Quantitative detection of T6SS activity using luciferase labeling
   1. Standardize the OD₆₀₀ of E. coli (Luciferase), WT^R-, ΔtssM mutants, and other strains (e.g., ATCC 17978, E. coli, Ab#40, Ab#170, Ab#180) to 1.0. Add 5 µL of bacterial suspension to a 96-well plate containing antibiotic-free LB agar. After air-drying in the dark, measure luciferase-based bioluminescence to verify the luminescence specificity of E. coli (Luciferase).
   2. Perform a 5-fold serial dilution of E. coli (Luciferase). Spot 10 µL of the diluted bacterial suspension onto a 96-well plate with LB agar and an empty 96-well plate (both light-proof). Measure luciferase-based bioluminescence to evaluate the sensitivity of the luciferase-based detection method and confirm whether LB agar interferes with the luminescence signal.
   3. Mix E. coli (Luciferase) with WT^R-, ΔtssM mutants, and 20 clinical strains (OD₆₀₀ standardized to 1.0) at a 1:1 ratio. Dilute 10-fold, add 5 µL to a 96-well plate, and set up three technical replicates.
   4. After air-drying in the dark, measure bioluminescence. Incubate at 37 °C in the dark for 4-6 h, then measure bioluminescence again (emission wavelength: 562 nm).
   5. Quantitatively assess T6SS-dependent antibacterial activity by comparing bioluminescence values before and after co-culture.
      NOTE: Ensure thorough mixing of bacterial suspensions to avoid experimental errors. Perform at least three independent replicates for each sample to ensure data reliability. Use Student's t-test for statistical analysis.
3. Detection of T6SS activity using GFP labeling
   1. Follow the same procedure as the Luciferase labeling experiment (Excitation wavelength: 480 nm, emission wavelength: 509 nm).
   2. Mix E. coli (GFP) with WT^R- and ΔtssM mutants (OD₆₀₀ standardized to 1.0) at a 1:1 ratio. Spot 10 µL onto antibiotic-free LB agar plates. After air-drying at RT, capture images using the GFP fluorescence channel (Alex488 program, excitation: 488 nm, emission: 509 nm) of a multifunctional imaging system.
   3. Incubate at 37 °C in the dark for 4-6 h, then capture images again. Assess T6SS activity based on changes in fluorescence intensity of bacterial spots.
      NOTE: Perform all steps in the dark to avoid interference with fluorescence signals. Ensure thorough mixing of bacterial suspensions to avoid experimental errors. Perform at least three independent replicates for each sample to ensure data reliability.
4. Flow cytometry detection
   1. Mix WT^R- with E. coli (GFP) and ΔtssM mutants with E. coli (GFP) at a 1:1 ratio. Take an appropriate amount of the mixed bacterial suspension and dilute it to a suitable concentration (typically OD₆₀₀ of 0.1-0.5) using sterile PBS or 1× buffer.
      ​NOTE: 1× buffer refers to Flow Cytometry Staining Buffer (1×), the ingredients include: 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4), supplemented with: 2-5% (v/v) heat-inactivated FBS (or 1% BSA as alternative), 2 mM EDTA.
   2. Centrifuge the diluted mixed bacterial suspension at 300 x g for 2 min and discard the supernatant. Resuspend the pellet in 1 mL of sterile PBS or 1× buffer and mix gently. Filter the resuspended solution through a 35 µm cell strainer to remove aggregates and ensure a single-cell suspension.
   3. Turn on the flow cytometer, preheat the laser, and calibrate the instrument.
      1. Set the detection parameters: use a 488 nm laser to excite GFP. Detection channel: FL1 (typically a 530/30 nm filter for GFP fluorescence detection). Set forward scatter (FSC) and side scatter (SSC) parameters to distinguish bacterial cells.
      2. Use unlabeled E. coli as a negative control, adjust the voltage and gain, and ensure the GFP-negative population is at the baseline of the fluorescence signal.
   4. Gently mix the filtered sample and transfer 100 µL to a flow cytometry tube.
      1. Analyze the samples using the following instrument settings: Set the FSC-H threshold at 19,000 to exclude debris, with gain values of 100 (FSC), 110 (SSC), and 80 (FITC).
      2. For gating, initially exclude debris and non-target signals in the FSC-A vs. SSC-A scatter plot, followed by population analysis in the FITC channel. Acquire a minimum of 10,000 events from the target population (defined by FSC/SSC characteristics) for each sample.
   5. Analyze the data using flow cytometry software (e.g., FlowJo, BD FACSDiva).
      1. Plot FSC vs. SSC scatter plots to gate the target bacterial population. Plot FL1 fluorescence histograms to distinguish GFP-positive and GFP-negative populations.
      2. Calculate the percentage of GFP-positive cells and the mean fluorescence intensity (MFI).
   6. Centrifuge the remaining mixed bacterial suspension at 300 g for 2 min and discard the supernatant. Resuspend the pellet in 10 µL of sterile water and spot it onto antibiotic-free LB agar plates.
   7. Incubate at 37 °C in the dark for 5 h, then scrape the bacterial spots and resuspend them in 500 µL of 1× PBS. Take 100 µL of the resuspended solution, dilute appropriately, and perform flow cytometry detection, repeating steps 6.4.4-6.4.5.
   8. Compare the percentage of GFP-positive cells and fluorescence intensity before and after co-culture. Evaluate the killing effect of WT^R- and ΔtssM mutants on E. coli (GFP).
      NOTE: Ensure a single-cell suspension to avoid cell aggregation affecting detection results. Calibrate the instrument before detection to ensure the accuracy and reproducibility of fluorescence signals. Avoid exposing samples to strong light during the experiment to prevent GFP fluorescence quenching. Perform at least three replicates for each sample to ensure data reliability.

Results

Traditional methods for detecting T6SS activity typically involve co-culturing predator and target strains on antibiotic-free LB agar plates for 5 h, followed by scraping the bacterial spots, resuspending them, and performing serial dilutions. The diluted bacterial suspension is then spotted onto selective agar plates. By counting the colonies of the target strain on the plates the next day, T6SS activity can be qualitatively assessed. However, this method has limitations, including cumbersome procedures, reliance on man...

Discussion

The Type VI Secretion System (T6SS) is a complex multi-protein machinery whose functions are primarily realized through the secretion of effector proteins. These effector proteins mediate interbacterial competition by killing or inhibiting other bacterial species, thereby providing the host bacterium with a competitive advantage in microbial communities¹⁵. Beyond its role in competition, studies have shown that T6SS is involved in various cellular processes, including bacterial colonization, envir...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10%, 15% SDS-PAGE Gel Preparation Kit	Epizyme	PG112
-20 °C Freezer	Haier	HYCD-290
37 °C Incubator	Bluepard	181254254
4 °C Refrigerator	Haier	HYC390
-80 °C Freezer	Haier	DW-86L726G(726L)
96-well plate	JET	TCP010096
Agar	Biofrox	8211KG001
Ammonium Persulfate (APS)	Thermo	7727-54-0
Biological Safety Cabinet	ESCO	AC2-4S1
Electronic Balance	Sartorius AG	BSA124S-CW
Electrophoresis apparatus	BIO-RAD	POWER PAC1000
Flake Ice Machine	GRANT	XB70
Flow Cytometer	BECKMAN COULTER	AW38143
Flow Cytometry Staining Buffer	proteintech	PF00018
Gel Imaging System	BIO-RAD	Gel Doc 2000
Glycine	Zike	ZK-L2577
High-speed Centrifuge	Eppendorf	5405IN106358
HRP-conjugated Rabbit/Mouse Secondary Antibody	proteintech	SA00001-2
LB Broth	Solarbio	L8291
Low Temperature High Speed Centrifuge	Thermo	17R
Methanol	Thermo	R40121
Micro UV-Vis Spectrophotometer	Thermo	Nanodrop one
Microcentrifuge	allsheng	Mini-6k
Microplate Reader	Bio-Tek	H1M
NuPAGE LDS Sample Buffer	Thermo	NP0007
Phosphate Buffer Solution (PBS)	Zike	ZK-L1649
Precision Plus Protein Dual Color Standards	Bio-Rad	1610374
SDS Sample Loading Buffer	Beyotime	poo15L
Skim Milk Powder	Thermo	LP0033B
Sodium Dodecyl Sulfate (SDS)	Zike	zk6885
Thermostatic Water Bath	JingHong	DK-420S
Transfer Apparatus	Bio-Rad	PowerPac HC
Tris(hydroxymethyl)aminomethane (Tris)	Zike	ZK-L2557
Vertical Electrophoresis Apparatus	Bio-Rad	Mii-PROTEAN Tetra