©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Membrane Depolarization Prevents Cell Invasion by Bordetella pertussis Adenylate Cyclase Toxin(*)

Angela S. Otero (1)(§), Xiao B. Yi (1), Mary C. Gray (2), Gabor Szabo (1), Erik L. Hewlett (2) (3)

From the (1) Departments of Molecular Physiology and Biological Physics, (2) Medicine, and (3) Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Adenylate cyclase toxin from Bordetella pertussis is a 177-kDa calmodulin-activated enzyme that has the ability to enter eukaryotic cells and convert endogenous ATP into cAMP. Little is known, however, about the mechanism of cell entry. We now demonstrate that intoxication of cardiac myocytes by adenylate cyclase toxin is driven and controlled by the electrical potential across the plasma membrane. The steepness of the voltage dependence of intoxication is comparable with that previously observed for the activation of Kand Nachannels of excitable membranes. The voltage-sensitive process is downstream from toxin binding to the cell surface and appears to correspond to the translocation of the catalytic domain across the membrane.


INTRODUCTION

AC() toxin is an important virulence factor for Bordetella pertussis, the causative agent of whooping cough (1, 2, 3, 4, 5) . In vitro this potent toxin induces massive increases in the intracellular levels of cAMP in phagocytes and is postulated to use this mechanism to disarm the host's response to infection (6, 7) . Productive interaction between AC toxin and target cells requires calcium ions and a post-translational modification that is dependent upon the product of another gene, cyaC (8, 9, 10, 11, 12, 13, 14) . A specific cell surface receptor for the toxin has never been identified; rather, there is evidence that the first step in the intoxication process may involve an interaction with negatively charged lipids (15, 16, 17) . As observed with several other bacterial toxins (18, 19, 20) , AC toxin was recently shown to form an ion-conductive pathway in artificial lipid bilayers in a voltage-dependent manner (17) . This finding raises the possibility that in vivo the membrane potential might control the translocation of AC toxin across the plasma membrane. Although studies using ionophores or agents that collapse the mitochondrial electrochemical gradient have suggested that the movement of proteins into or across intracellular membranes might be affected by membrane potential, the complexity of the eukaryotic transport systems and the limitations inherent in the methods employed have precluded direct evaluation of this hypothesis (21, 22, 23, 24) . In contrast, the effects of membrane voltage on entry of AC toxin into cells can be studied directly in single cardiac myocytes by monitoring cAMP-stimulated calcium currents with the patch clamp technique (13) . This approach allows full control of the potential across the plasma membrane, while the development of intoxication is measured with high sensitivity and time resolution. We report here the results of experiments demonstrating that intoxication of atrial cells by AC toxin is highly voltage-dependent, being abolished by membrane depolarization.


EXPERIMENTAL PROCEDURES

Purification of Adenylate Cyclase Toxin

Wild type B. pertussis (strain BP338) and a mutant in which Lys-58 was replaced with a methionine residue (strain BPLM58IE) were grown as described previously (25, 26) . AC toxin was purified by urea extraction, phenyl-Sepharose chromatography, sucrose gradient centrifugation, and calmodulin-Sepharose chromatography (13) . Each toxin batch was dialyzed against external solution immediately prior to experiments and then diluted to the desired concentration with external solution. Enzyme activity was measured by the conversion of [-P]ATP to [P]cAMP in a cell-free assay (13) . The cAMP formed was isolated as described by Salomon et al. (27) . Enzyme specific activities for wild type toxin, BP338, ranged from 0.8 to 1.2 mmol of cAMP/min/mg of toxin. Toxin activity of wild type toxin, as determined by quantitation of intracellular cAMP in Jurkat cells exposed to AC toxin for 30 min at 37 °C (13) , was 12-15 µmol of cAMP/mg of Jurkat cell protein/mg of toxin. The mutant toxin, strain BPLM58IE, exhibited a 1000-fold reduction in enzymatic activity relative to wild type toxin and was unable to increase cAMP in target cells. Electrophysiologic Studies-Myocytes were enzymatically dissociated from bullfrog atria as described previously (28) . Membrane currents were recorded at 22-24 °C in the whole-cell configuration of the gigaseal patch clamp technique (29) . The internal solution contained 120 mM CsCl (to block Kselective channels), 1 mM EGTA, 2.5 mM MgATP, 0.2 mM LiGTP, 5 mM HEPES, adjusted to pH 7.4 with KOH, while the external solution contained 85 mM NaCl, 20 mM CsCl, 2 mM MgCl, 2 mM CaCl, 20 mM HEPES (pH 7.4 with NaOH), and 5 µM tetrodotoxin, a Nachannel blocker. The standard pulse protocol consisted of 0.5-s steps from a holding potential of 85 mV to 135, 75, 45, 15, 15, and 45 mV. The cell was kept at 85 mV for 2 s between pulses. The amplitude of Iwas measured as the difference between the peak inward current and the steady state current averaged during the last 10 ms of the pulse.


RESULTS AND DISCUSSION

AC toxin, at concentrations of 3.5-4.5 µg/ml, elicits a large increase (11.7-fold ± 4.4; n = 16) in the amplitude of Cacurrents ( I) of frog atrial myocytes (13) (Fig. 1), comparable in magnitude to the stimulation of Iinduced by -adrenergic agonists (30) . This effect of AC toxin is characterized by a short latency period (0.59 ± 0.17 min; n = 6) followed by a slow increase of Cacurrents, with a maximum being reached within 3.5-9 min. An enzyme-deficient AC toxin, in which lysine 58, a residue involved in catalysis, is replaced by a methionine (26) has no effect on I(Fig. 1). Thus, the increase in the amplitude of the Cacurrent is directly related to intracellular cAMP production, which is an intrinsic property of the wild type toxin and leads to activation of protein kinase A and phosphorylation of L-type Cachannels (31) .


Figure 1: Time course of stimulation of calcium currents by AC toxin. Peak inward currents elicited by 0.5-s steps from a holding potential of 85 to 15 mV are plotted as a function of time; cells were superfused with wild type AC toxin (4 µg/ml, ) or with mutant toxin, BPLM58IE (5.2 µg/ml, ) for the interval indicated by the horizontal bar.



To determine the voltage dependence of intoxication, we applied AC toxin to cells for a fixed period while the membrane potential was clamped at the values specified in the figure legends. Calcium currents were measured at the beginning of the experiment, for determination of basal levels, and following the holding period. We found that the increase of Iis virtually complete following 5 min of toxin application to myocytes held at 85 mV (Fig. 2 A). In contrast, there is no increase in Cacurrents when the membrane potential is kept at +45 mV during the first 5 min of AC toxin application (Fig. 2 B); upon return to the standard pulse protocol, Iis still at the basal level and only then starts to increase, following a time course similar to that shown in Fig. 1.


Figure 2: Effect of AC toxin application on cells held at constant voltages. After the establishment of the whole-cell recording mode, I() was elicited by 0.5-s voltage steps to 15 mV from a holding potential of 85 mV, and basal currents were measured. The cell was then clamped at 85 mV ( A) or +45 mV ( B) for the period indicated by dotted lines. AC toxin (4 µg/ml) was applied for the period indicated by the horizontal bar. After 5 min of toxin exposure at a constant voltage, the pulse protocol was resumed directly and Iwas measured again.



Fig. 3 shows the percent increase in calcium currents observed for cells held at various potentials as a function of the membrane potential. The results indicate that there is essentially no stimulation of Iwhile cells are held in the range of 0 to +45 mV; as the holding potential becomes negative, the toxin-induced increases in Irise steeply, reaching a maximum around 45 mV. The solid line is a fit of the data to the Boltzmann equation (1) ,

 

On-line formulae not verified for accuracy


Figure 3: Voltage dependence of Istimulation. Experiments followed the protocol illustrated in Fig. 2. During the first 5 min of exposure to AC toxin (4 µg/ml) the voltage was kept at the values shown. Calcium currents recorded immediately after the pulse protocol was resumed were normalized to the maximal Iamplitude in each cell (). Plotted are results obtained with individual cells; symbols with error bars are means ± S.E. from three separate experiments. Also shown are the results of experiments following the same protocol, but using 0.2 µM isoproterenol () instead of AC toxin to stimulate I.



To verify that the observed voltage dependence is a property of AC toxin, and not a characteristic of one or more components of the phosphorylation cascade triggered by cAMP, we performed experiments in which Iwas stimulated not by AC toxin but by isoproterenol, a -adrenergic receptor agonist, which increases cAMP production through stimulation of the endogenous adenylate cyclase. As seen in Fig. 3, enhancement of cardiac Cacurrents by isoproterenol shows little influence of the membrane potential, confirming that the striking voltage dependence observed with AC toxin is a characteristic of the intoxication process.

Experimental evidence indicates that B. pertussis AC toxin invades target cells directly, rather than depending on an endocytic pathway such as the one utilized by Bacillus anthracis toxin, the only other invasive bacterial toxin known to have adenylate cyclase activity (15, 16, 33) . Therefore, a basic model of AC toxin entry into cells involves a sequence of at least two steps: 1) binding to the outer surface of the membrane and 2) insertion into the membrane with translocation of the catalytic portion into the cell interior (5) . In principle, the membrane potential could affect either or both these processes. To better define the step affected by the voltage, we performed experiments similar to the one in Fig. 2B, applying toxin for 5 min while holding the cell at +45 mV. Before the pulse protocol was resumed, however, the toxin-containing solution was replaced with standard external solution and the cell was washed for 2-3 min. Were the toxin not able to bind to the membrane at positive potentials, this washout period should completely remove it from the cell surface as well as the bathing solution, yielding no increase in Iafter pulsing is resumed. In contrast, if binding occurs normally at positive potentials and only the translocation step is prevented, Ishould increase at the end of the holding period even if the toxin is no longer present in the bath. Indeed, Fig. 4 shows that even when the toxin is removed from the bath, Iincreases upon return to the standard pulse protocol, indicating that during the period at +45 mV toxin molecules attached themselves to the plasma membrane in a manner that was resistant to subsequent washout. The results obtained under these conditions ( n = 3) are qualitatively identical to those in Fig. 2 B, establishing that the voltage-sensitive step in toxin delivery is downstream from binding and is likely to represent translocation of the catalytic site to the cytoplasm.


Figure 4: Effect of AC toxin application and washout at 45 mV. The experimental protocol and symbols are the same as in Fig. 2, except for the duration of toxin application and the inclusion of a washout period as indicated by the horizontal bar.



Our results demonstrate that the delivery of AC toxin to its site of action is driven and controlled by the electric field across the plasma membrane. Intoxication of atrial cells is readily observed within minutes of exposure to adenylate cyclase toxin when myocytes are held at negative potentials but hindered at membrane potentials positive to 45 mV and is totally prevented when the membrane is kept at positive potentials. This steep voltage dependence of intoxication (5.5 mV per e-fold increase in intoxication) is comparable with that of the voltage-gated Kand Nachannels of squid axon (4.8 and 3.9 mV per e-fold increase in conductance, respectively (Ref.32 and references therein)) and places AC toxin delivery among the most voltage-sensitive biological processes known. In our studies, the ability to follow the time course of intoxication of single cells by AC toxin has allowed direct demonstration of a role for membrane potential in toxin delivery. This novel experimental system should help expand our present understanding of the molecular mechanisms underlying membrane translocation of proteins in general and of bacterial toxins in particular.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL 48726 (to A. S. O.), HL 37127 (to G. S.), and AI 18000 (to E. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Physiology and Biological Physics, Jordan Hall, Box 449, University of Virginia School of Medicine, Charlottesville, VA 22908. Tel.: 804-982-1896; Fax: 804-982-1616; E-mail: ado2t@virginia.edu.

The abbreviation used is: AC, adenylate cyclase.


ACKNOWLEDGEMENTS

We thank Sheila Van Cuyk for assistance with toxin purification.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.