Real-time Imaging of Response to Host-Induced Stress in Pseudomonas aeruginosa Aggregates

Summary

Pseudomonas aeruginosa aggregates demonstrate increased tolerance to antibiotics and immune cells. The role of membrane integrity in aggregate survival is currently underexplored. This study demonstrates a method for quantifying changes in cell membrane dynamics within P. aeruginosa aggregates to understand how cellular integrity can impact overall aggregate viability.

Abstract

Chronic infections such as those in cystic fibrosis (CF) are sustained by small, highly tolerant Pseudomonas aeruginosa aggregates that persist despite immune and therapeutic pressures. Unlike classical biofilms, these aggregates represent a distinct pathogenic unit - microscale, spatially organized communities that maintain structural integrity and physiological homeostasis under host-induced stress. However, the mechanisms that enable aggregates to remain intact under these conditions, and whether these homeostatic processes can be selectively disrupted, remain poorly defined. A key barrier to addressing this gap has been the lack of tools capable of capturing the dynamic, spatially resolved processes that link physiological stress to structural stability and collapse in real time. This study presents an integrated imaging and analytical workflow to quantify aggregate responses to host-relevant stressors. Using the voltage-sensitive dye DiBAC₄(5), membrane depolarization was monitored as an early indicator of physiological disruption within aggregates formed in synthetic cystic fibrosis sputum medium (SCFM2). High-resolution time-lapse confocal microscopy enables visualization of aggregates in both stable and stress-induced states, while image segmentation and voxel-based analysis provide quantitative mapping of spatial heterogeneity in membrane integrity and aggregate disassembly at single-cell resolution. This workflow establishes a reproducible and adaptable platform for linking physiological stress to structural outcomes in multicellular bacterial aggregates. By enabling quantitative dissection of the processes that preserve - or compromise - aggregate integrity, this approach provides a critical foundation for identifying and targeting the homeostatic mechanisms that underpin aggregate resilience, advancing new strategies to disrupt this clinically significant mode of bacterial persistence.

Introduction

Chronic bacterial infections are often sustained not by large, surface-attached biofilms, but by small, multicellular aggregates that form within host-associated environments. In cystic fibrosis (CF) and other chronic airway diseases, Pseudomonas aeruginosa commonly exists as suspended aggregates embedded within mucus or sputum rather than as classical biofilms attached to epithelial surfaces. These aggregates represent a distinct mode of growth, characterized by microscale spatial organization, pronounced physiological heterogeneity, and exceptional tolerance to immune and antimicrobial stressors^1,2<....

Protocol

All work with P. aeruginosa must be conducted under appropriate BSL-2 containment conditions and institutional biosafety approvals. The reagents and the equipment used in this study are listed in the Table of Materials.

1. Generation of Pseudomonas aeruginosa aggregates in synthetic cystic fibrosis sputum medium (SCFM2)

NOTE: This step describes preparation of P. aeruginosa cultures and formation of suspended multicellular aggregates under physiologically relevant conditions that mimic the CF airway environment.

1. Preparation of bacterial cult....

Results

Provided in the protocol described above is a method to track changes in the membrane dynamics of aggregates of P. aeruginosa modeled in a cystic fibrosis lung environment (Figure 1). Using a wild-type PAO1 strain carrying a pMRP9-1 (GFP) plasmid (Figure 1A), HNE to simulate immune stress, and DiBAC₄(5) membrane dye (Figure 1C), changes in membrane depolarization, reflecting disruption of membran.......

Discussion

Membrane potential serves as a sensitive, early readout of cellular stress and disrupted homeostasis. Within multicellular bacterial aggregates, these physiological changes occur prior to, and may drive, subsequent structural destabilization. Here, we present an imaging-based workflow that uses the slow-response, potential-sensitive dye bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC₄(5)) to track depolarization in Pseudomonas aeruginosa aggregates exposed to host-derived stress. Tracking .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(3))	AAT Bioquest	21411	lipophilic, anionic molecule
1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(5))	AAT Bioquest	21410	lipophilic, anionic molecule
Carbenicillin	Research products international	C46000-5.0	used for selection of GFP expressing strains of PAO1
Cellvis 4-chamber 35mm glass bottom dish	Fisher	NC0600518	glass bottom chamber dish allows for maitinance of suspended aggregates in growth media while imaging
Centrifuge	Beckman Coulter Life Sciences	B06320	paired with Beckman Coulter Life Sciences SX4400 swinging bucket rotor (part no. B01425)
Confocal laser scanning microscope (CLSM)	Zeiss	LSM 880	the model of CLSM used for the experiments described in this protocol is no longer available but the LSM 900 is an updated equivalent option
Culture Tubes	Genesee	21-130	sterile; polypropylene
DAPI	Thermo Fisher	62248	used for proposed future applications staining neutrophils with DiBAC4(5)
Disposable cuvettes	Brand Tech	759086D	1.5mL, semi-micro
DMSO	Millipore-Sigma	276855-100ML	used for the reconstitution of DiBAC4(3,5)
DRAQ5	Thermo Fisher	65-0880-92	used for proposed future applications staining neutrophils with DiBAC4(3)
Environmental Chamber for CLSM	PECON	LSM 880	this system was made exclusively for Zeiss products and is generally referred to as the "Incubation System S" and all components can be ordered through Zeiss as an addition to the LSM 880 (or similar) system
Gentamycin sulfate	Fisher	BP918-1	used for selection of RFP expressing strains of PA1633
Glycerol	Genesee	18-205	used to freeze strains for long-term storage at -80 °C
Human Neutrophil Elastase (HNE)	Millipore-Sigma	324681-50UG	once reconstituted it is stable for 1 month at 4 °C or 1 year at 20 °C
Imaris x64 software	Oxford Instruments		Version 10.2.0
Immersion Oil	Electron Microscopy Scienes	16919-12	non-drying; standardized at 37 °C
Incubator	Benchmark Scientific	H1001-M	capable of shaking
Lysogeny Broth (LB)	Genesee	11-118	miller mix
mCherry plasmid			obtained from a collaboorator
PA1633 transposon mutant strain			obtained from a collaboorator
Phosphate Buffered Saline (PBS)	Thermo Fisher	3002	pH 7.0
Sodium Acetate	Thermo Fisher	AM9740	3M; pH 5.5; RNase-free
Sodium Chloride	Fisher	S271-500	used for the reconstiutuion of HNE
Spectrophotometer	VWR	634-0882	used to measure cell density for inoculation of PAO1/PA1633 into SCFM2
Sterile .22µm Filter	Genesee	25-244	used to purify reconstituted HNE
Sterile Microcentrifuge Tubes	Genesee	24-272LR	0.6mL
Sterile Syringe	Fisher	14955459	used to purify reconstituted HNE
SX4400 swinging bucket rotor	Beckman Coulter Life Sciences	B01425	paired with Beckman Coulter Life Sciences centrifuge (part no. B06320)
Synthetic Cystic Fibrosis Sputum Medium (SCFM2)	SyntheBiome	10002-500	Batch #: 001-CF2-0724
Ultra Pure Water	Genesee	18-195	used for the reconstitution of HNE
Wild type Pseudomonas aeruginosa PAO1 with pMRP9-1 Plasmid			This pMRP9-1 plasmid contains GFP and expresses carbenicillin resistance
ZEN Black Edition 2.3 Zeiss Imaging software	Zeiss		compatible with the LSM 880