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Why Escherichia coli -hemolysin induces calcium oscillations in mammalian cells–the pore is on its own

Andreas Koschinski^*,1, Holger Repp^*,1,2, Baris Ünver^*, Florian Dreyer^*, Dierk Brockmeier^*, Angela Valeva, Sucharit Bhakdi and Iwan Walev^,2

^* Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University, Giessen, Germany; and

Institute of Medical Microbiology and Hygiene, Johannes-Gutenberg-University, Mainz, Germany

²Correspondence: Frankfurter Str. 107, Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University, 35392 Giessen, Germany. Email: Holger.Repp{at}pharma.med.uni-giessen.de; and Obere Zahlbacher Str. 67, Institute of Medical Microbiology and Hygiene, Johannes-Gutenberg-University, 55101 Mainz, Germany. Email: walev{at}mail.uni-mainz.de

SPECIFIC AIMS

The hemolysin of Escherichia coli (HlyA) is prototypic of a large family of pore-forming toxins that are produced by many medically important gram-negative pathogens. It was reported that HlyA deregulates membrane Ca²⁺ channels, thus inducing periodic low-frequency Ca²⁺ oscillations that trigger transcriptional processes in mammalian cells. Our aim in this study was to delineate the mechanism underlying the Ca²⁺ oscillations that are induced by HlyA.

PRINCIPAL FINDINGS
1. HlyA induces nonperiodic Ca²⁺oscillations in renal cells
Within 30 s after application of HlyA (5 ng/ml) to human kidney tubule epithelial (IHKE) cells, intracellular Ca²⁺ concentration ([Ca²⁺]_i) increased and exhibited oscillations during further monitoring. It was previously reported that HlyA provoked periodic Ca²⁺ oscillations in primary rat proximal tubule (RPT) cells, with a constant periodicity of Ca²⁺ peaks of 12 min. However, we found no constant periodicities but instead found large variations of oscillation kinetics and peak-to-peak spans. Examples are shown in Fig. 1 A–D for Ca²⁺ tracings and in Fig. 1E for false-color images of [Ca²⁺]_i. No Ca²⁺ oscillations were induced by the nonhemolytic HlyA mutant S177C/K564R/K690R, which is fully bindable to the plasma membrane but unable to form membrane pores (Fig. 1G ). Statistical analysis of time spans between directly neighboring Ca²⁺ peaks in Fig. 1A-D and 48 other Ca²⁺ tracings yielded a most frequent peak-to-peak span (not to be confused with peak periodicity) of 70 s, whereas spans above 10 min were very rare. Peak-to-peak span distribution was dependent on HlyA concentration, whereupon higher concentrations led to shorter time spans.

View larger version (43K): [in this window] [in a new window]   Figure 1. Ca2+ oscillations induced by HlyA in IHKE cells loaded with the fluorescent Ca2+ indicator Fura-2/acetoxymethyl ester. A–D) Cells were exposed to HlyA (5 ng/ml) 3 min after start of recording. Traces represent examples of time courses of [Ca2+]i of 4 single cells. E) Single frames from an image series of [Ca2+]i. Frames 1–4 correspond to numbers 1–4 above trace in A. Traces in A and B were recorded from cells that are marked with A and B. F) Superposition of Ca2+ responses of 49 IHKE cells in the presence of nifedipine (100 µM). Application of HlyA (5 ng/ml) is indicated by arrow. Gap represents time when field of view was changed to an area that had not been UV irradiated before. G) Ca2+ tracing of a single IHKE cell on consecutive exposure to a nonhemolytic HlyA mutant (=nh HlyA, 25 ng/ml) and wild-type HlyA (2.5 ng/ml).

2. A periodicity of Ca²⁺ peaks is pretended by a power spectrum analysis
We subjected the Ca²⁺ data sets to a power spectrum analysis, which was previously used to identify HlyA-induced periodic Ca²⁺ oscillations of low frequency. Despite marked heterogeneity of the original Ca²⁺ tracings and rare occurrence of peak-to-peak spans above 10 min, this analysis yielded a periodicity of Ca²⁺ peaks with a dominant wavelength of 12.8 min, which was in remarkable agreement with the previously reported value of 12.0 min. Surprisingly, in Ca²⁺ tracings monitored in the absence of HlyA and devoid of Ca²⁺ elevations, a periodicity of 12 min was again computed. Furthermore, analysis of a signal consisting only of white noise superimposed to a discretely drifting baseline yielded the same periodicity. In conclusion, power spectrum analysis appeared as an inadequate mathematical model for a proper interpretation of the Ca²⁺ data sets.

3. Nifedipine does not inhibit Ca²⁺ oscillations
When voltage-gated L-type Ca²⁺ channels in IHKE cells were blocked by nifedipine, HlyA still led to Ca²⁺ oscillations (Fig. 1F ). This contrasted with previous data of RPT cells where nifedipine abolished the Ca²⁺ response to HlyA. A striking phenomenon that we observed during the Ca²⁺ measurements can account for this difference. Nifedipine-treated cells that were in the microscopic field of view for several minutes during Ca²⁺ imaging, i.e., irradiated by ultraviolet (UV) light, did indeed not show Ca²⁺ oscillations after application of HlyA, whereas Ca²⁺ oscillations were observed in cells that had not "seen" UV light (Fig. 1F ). Thus, the previously reported suppressive effect of nifedipine on HlyA-induced Ca²⁺ oscillations was uncovered as experimental artifact. Additionally, HlyA still led to Ca²⁺ oscillations in HEK293 cells when a combination of blockers of voltage-gated and receptor-operated Ca²⁺ channels was present, including nifedipine (100 µM), Cd²⁺ (2 mM), and SK&F 96365 (25 µM).

4. Ca²⁺ oscillations and pore formation cease rapidly on removal of HlyA
The effect of HlyA on [Ca²⁺]_i is essentially dependent on the presence of toxin in extracellular medium. Instantaneously after the start of a washout in HlyA-treated IHKE cells, [Ca²⁺]_i decreased and reached initial concentration after 3 min. Reapplication of HlyA again led to Ca²⁺ oscillations, which disappeared after the restart of washout. After the start of washout, disappearance of Ca²⁺ oscillations and HlyA pores followed the same time course. This synchronism was discovered by subjecting single IHKE cells exposed to HlyA to simultaneous measurement of [Ca²⁺]_i and pore formation, using at the same time Ca²⁺ imaging and electrophysiological patch-clamp recording. A few seconds after the start of washout the number of open pores decreased and went to zero within 4 min. A second HlyA application again led to pore formation. The short life span of HlyA pores was confirmed by propidium iodide influx measurements. Cells became permeable to dye during incubation with HlyA, but membranes returned to the impermeable state shortly after toxin removal.

5. Ca²⁺ elevations result from Ca²⁺ influx through HlyA pores
By simultaneous monitoring of pore formation and [Ca²⁺]_i in IHKE cells exposed to HlyA, we detected an increase in [Ca²⁺]_i directly after pore opening and a good temporal correlation between formation and closure of HlyA pores and Ca²⁺ elevations. We hypothesize that the decrease after an elevation reflects the restoration of cellular Ca²⁺ homeostasis by physiological Ca²⁺ redistribution after HlyA pore closure (Fig. 2 ).

View larger version (54K): [in this window] [in a new window]   Figure 2. Schematic overview. Ca2+ oscillations induced by HlyA occur only in the presence of the toxin and are not blocked by nifedipine. After washout of HlyA, Ca2+ oscillations cease rapidly. Readdition of the toxin again leads to Ca2+ oscillations. We assume that the decrease after a Ca2+ elevation is managed by plasma membrane and sarco/endoplasmic reticulum Ca2+ pumps (illustrated by circles with blades).

CONCLUSIONS AND SIGNIFICANCE
The results of this study lead us to a new explanation for Ca²⁺ oscillations that occur in mammalian cells treated with HlyA. Contrary to a previous report, we showed that the ability of HlyA to induce Ca²⁺ oscillations does not rely on endogenous L-type Ca²⁺ channels. Furthermore, we found that the effect of HlyA on [Ca²⁺]_i is not dependent on the activity of any voltage-gated or receptor-operated membrane Ca²⁺ channels. Instead, Ca²⁺ oscillations result from pulsed influxes of Ca²⁺ through short-lived HlyA pores, which are rapidly closed or removed from the membrane. The causal connection was directly seen in simultaneous patch-clamp measurements of pore formation and Ca²⁺ measurements, where Ca²⁺ elevations occurred as a consequence of opening of HlyA pores.

Pore formation by HlyA happens at random, which explains our observation that the Ca²⁺ peaks in individual cells occur in a stochastic sequence. This contrasts a previous claim on the existence of periodic low-frequency Ca²⁺ oscillations induced by HlyA. However, the mathematical model that this claim was based on turned out as inadequate for a proper interpretation of our measured Ca²⁺ data. Moreover, we found that the mean time span between two Ca²⁺ elevations was dependent on the concentration of HlyA, whereupon higher concentrations led to shorter time spans. This concentration dependence definitely rules out the idea that HlyA molecules may switch on in the target cell a periodic "Ca²⁺ oscillation run" with one defined frequency.

Washout experiments showed that pore formation, propidium iodide influx, and Ca²⁺ oscillations only occurred as long as HlyA molecules were present at the extracellular side. The observation that the Ca²⁺ oscillations ceased with the start of washout demonstrates further that the cell has no lasting memory on the encounter with HlyA regarding [Ca²⁺]_i.

Whensoever Ca²⁺ oscillations are initiated by HlyA, the pore is on its own. Nonperiodic ("chaotic") Ca²⁺ oscillations resulting from the interplay of formation and disappearance of the pores along with cellular Ca²⁺ redistribution will obviously vary depending on toxin concentration and susceptibility and may provide the starting point of myriad reactions, but on a very individual basis, in cells attacked by pore-forming toxins such as HlyA.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4561fje

¹ These authors contributed equally to this work.

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4561fjev1 20/7/973    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Koschinski, A. Articles by Walev, I. Search for Related Content PubMed PubMed Citation Articles by Koschinski, A. Articles by Walev, I.