Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Summary

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Abstract

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC₄(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca²⁺, K⁺, and H⁺, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Introduction

Our lab has identified a protein-lipid complex from human milk named HAMLET (for Human Alpha-lactalbumin Made LEthal to Tumor cells) that induces apoptosis in tumor cells, but is also able to kill a variety of bacterial species^1,2. The species that were found to be particularly sensitive were those that target the respiratory tract, with Streptococcus pneumoniae (the pneumococcus) displaying the greatest sensitivity and an apoptosis-like phenotype of death^2,3. Membrane depolarization and specific ion transport events are well-described and crucial events during apoptosis in eukaryotic cells, particular in the mitochondria, where radioact....

Protocol

1. Preparing Bacterial Cultures

1. Growing culture for use in experiments
   1. In a 37 °C heating block, thaw a frozen stock-vial of S. pneumoniae and add its content to 9 ml of fresh, prewarmed Todd-Hewitt broth with 0.5% yeast extract (THY) for a total volume of 10 ml in a glass culture tube.
   2. Incubate statically at 37 °C until the culture reaches mid-log phase (Abs_600nm ≈0.5-0.6).

2. Detecting Membrane Depolarization and Rupture

1. Preparing reagents
   1. Prepare a 50 µM stock of DiBAC₄....

Results

For all experiments, there is one sample and set of conditions present in each well. Thus, each tracing represents the fluorescence intensity of an entire population of bacteria over time. The results should be easily interpretable, with a clear distinction between the fluorescence of the treated samples and that of the untreated controls. The kinetics and degree of an observed change in fluorescence could provide information about the possible mechanism and extent of the event being monitored.

Discussion

Despite the limitation that size presents for using classic electrophysiology methods to detect changes in polarity and integrity of the membrane and changes in ion concentrations within bacteria, we have described a way to measure these events in S. pneumoniae using fluorescent dyes. Our protocols are the first of their kind described for the pneumococcus and one of the few described for bacterial species in general. By using a fluorescence detection plate reader, these events can be measured in small, 200 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Todd-Hewitt broth	Bacto, BD Diagnostics	249240
Yeast Extract	Bacto, BD Diagnostics	212750
Phosphate Buffered Saline (PBS; pH 7.2)	Invitrogen (GIBCO)
Dimethyl sulfoxide	Sigma-Aldrich	D5879	DMSO
DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol)	Molecular Probes	B-438
Propidium Iodide	Sigma-Aldrich	P4170	Make up in deionized water
D-(+)-Glucose	Sigma-Aldrich
PowerLoad	Molecular Probes	P10020	100x concentrate
Probenecid	Molecular Probes	P36400	Make 100x stock by adding 1 ml of PBS to one 77 mg vial
Fura-2/AM	Molecular Probes	F1221	Special packaging (50 µg aliquots)
PBFI/AM	Molecular Probes	P1267	Special packaging (50 µg aliquots)
Nigericin	Sigma-Aldrich	N7143
KH2PO4	JT Baker	3246-01	monobasic
NaOH	JT Baker	5565-01
K2HPO4	JT Baker	4012-01	dibasic
BCECF/AM	Molecular Probes	B1170	Special packaging (50 µg aliquots)
CCCP	Sigma-Aldrich		Protonophore that causes an influx of H+ into the cytoplasm, dissipating the electrical potential and the H+ gradient.
Culture tube	VWR	53283-802	Fits the Spectronic spectrophotometer; borosilicate glass
Spectrophotometer	Thermo Scientific		Spectronic 20D+
15 ml Plastic conical tube	Corning	430790
Clear 96-well polystyrene microtiter plate	Fisher Scientific	12-565-501
Plate reader	BioTek	Synergy 2 Multi-Mode
Gen5 software	BioTek	Gen5™ Software