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 potentia....

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 ....

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