Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Summary

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Abstract

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type¹. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines². Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.

Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism^3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation⁴. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines^5,6,7. A link between IL-1β release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes⁸. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Protocol

1. Purification of Streptolysin O (SLO)

1. Inoculate 20 ml overnight culture of BL21 GOLD cells containing pBADgIII-SLOhis plasmid⁹ into 0.5 L LB broth and add 500 μl 50 mg/ml ampicillin. Shake culture at 225 rpm at 37 ° C until OD₆₀₀ = 0.6, usually ~1.5 hr. Centrifuge 1 ml bacteria (considered T₀) at 10,000 x g 5 min, dissolve pellet in 140 μl 1x SDS sample buffer/1 OD and sonicate to shear DNA for protein purity analysis.
2. Induce bacteria with the addition of 5 ml 20% arabinose to culture, shake at 225 rpm at room temp for 3 hr. Collect 1 ml of bacteria, centrifuge and dissolve in 1x SDS sample buffer as a....

Results

Typically 10⁷-10⁸ U/ml SLO can be obtained with a protein concentration of 4 mg/ml. The amount of toxin required for cell lysis varies by cell type, but is usually 125-500 U/ml SLO (Figure 2B). Cell types like macrophages can be more resistant (4000 U/ml) though others (especially T cell lines) are more sensitive. These sensitivities correspond with commercially available SLO. Toxin activity decreases roughly 2-fold with each freeze-thaw, so hemolytic assays with each batch or thaw .......

Discussion

The techniques described here allow the examination of the responses of immune cells to bacterial toxins. The most critical step is the handling and dosing of the toxin. Toxin activity can be extremely variable, even between different aliquots of the same preparation, due to its fragility. This necessitates either testing each aliquot of toxin against a reference cell line or RBCs or using toxin gradients. Toxin gradients, as delivered by micropipette, allow the full spectrum of toxin-induced activities to be observed in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ni-NTA agarose	Qiagen	30210
polymixin-agarose	Sigma	P1411-5ML
Zeba Desalt Spin col	Fisher	PI-89891
sheep RBCs	Fisher	50-415-688
pBADgIII-SLO	N/A	N/A	see ref9
Cy5 monoreactive dye	GE Healthcare	PA25001
Fura2-AM	Life Technologies	F1221
Calcein AM/ Ethidium homodimer	Life Technologies	L3224
Anti-CD11c-APC	BD Biosciences	550261
collagen-coated glass-bottom dish	Mattek	P35GCol-1.5-10-C
femto-tip II	Fisher	E5242957000
Microloader	Fisher	E5242956003
dextran Alexa 555	Life Technologies	D34679
Injectman NI 2	Eppendorf	920000029
FemtoJet	Eppendorf	5247 000.013
Table 1. List and source of specific reagents and equipment needed. Specific equipment and reagents used in this protocol, along with company and catalogue number are listed.
Buffer Composition Step Used Lyse/Wash 50 mM NaH2PO4 300 mM NaCl 10 mM imidazole, pH 8.0 1.4 Tris/salt 50 mM Tris, pH 8.0 300 mM NaCl 1.7 Elution buffer 50 mM Tris, pH 8.0 300 mM NaCl 250 mM imidazole 1.8 RBC Assay buffer 0.3% BSA 2 mM CaCl2 10 mM HEPES, pH 7.4 2.1 buffer R/B RPMI cell culture medium 2 mM CaCl2 0.5% BSA 3.1
Table 2. List of buffers used in this protocol. The buffers used, their composition and the first step at which they are used in the protocol are listed.