Giardia Potencial Membrana Fluorescencia[1]

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The membrane potential of Giardia intestinalis

Giancarlo A. Biagini a, David Lloyd bY*, Kiaran Kirk c, Michael R. Edwards a

a School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, NSW 2052, Australiab School of Biosciences, University of Wales, Cardi CF1 3TL, UK

c Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra 0200, Australia

Received 27 July 2000; accepted 28 August 2000

Abstract

Giardia intestinalis is a primitive microaerophilic protozoan parasite which colonises the upper intestine of humans. Despite theevolutionary and medical significance of this organism, its physiology is very poorly understood. In this study we have used a novel flow

cytometric technique to make quantitative measurements of the electrical potential across the plasma membrane of G. intestinalis

trophozoites. In media lacking both K and Na, G. intestinalis trophozoites maintained a high negative plasma membrane potential (8m)

of31343 mV. The 8m was unaffected by the addition of Na to the extracellular medium, whereas the addition of K resulted in a

significant membrane depolarisation, consistent with the G. intestinalis trophozoite plasma membrane having a significant (electrophoretic)

permeability to K. The membrane was also depolarised by the H ionophore m-chlorophenylhydrazone and by the H ATPase inhibitors

dicyclohexylcarbodiimide and N-ethylmaleimide. These results are consistent with G. intestinalis trophozoites maintaining a high resting

8m, originating at least in part from an electrogenic H pump acting in concert with a K diffusion pathway. 2000 Federation of

European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Anaerobic protozoa; Bioenergetics; H pumping; K diusion pathway

1. Introduction

Giardiasis is one of the most common water-borne dis-

eases in humans, with an estimated 280 million infections

annually [1]. The organism responsible, Giardia intestina-

lis, is a protozoan parasite which colonises the mucosa of

the gastrointestinal tract of humans. It is an evolutionarily

ancient organism, placed amongst the earliest branching

eukaryotes in the tree of life, the diplomonads [2]. How-

ever, despite the medical and evolutionary signicance of

G. intestinalis, the physiology of this organism is poorly

understood.The plasma membrane of all cells is characterised by a

transmembrane electrical potential (8m), which plays a

central role in the trac of solutes between the intra-

and extracellular solutions [3]. In cells of higher eukary-

otes the 8m arises predominantly from the diusion of

monovalent inorganic ions (principally K, Na and Cl3)

down the concentration gradient established by the action

of `primary' and `secondary' active transporters. In cells of

lower eukaryotes (e.g. fungi and protozoa) there is, in

addition to the contribution of the diusion of the major

ions, a direct and substantial contribution to the 8m from

electrogenic H pumps in the plasma membrane. Elucida-

tion of the mechanisms involved in the generation of 8min G. intestinalis may give key insights into the evolution

of 8m in eukaryotic cells as well as highlight possible

routes for chemotherapy for this pathologically important

organism.A variety of methods have been used for the estimation

of plasma membrane 8m. In large cells, 8m may be mea-

sured directly using microelectrodes. However, for small

cells such as bacteria and protozoa this method is imprac-

tical. One method that has been widely applied to bacteria

(as well as intracellular organelles) entails measuring the

distribution of radiolabelled lipophilic cations such as tet-

raphenylphosphonium. However, these probes are unable

to monitor the heterogeneity of 8m within a cell popula-

tion which often occurs upon treatment with inhibitors or

antibiotics (e.g. [4]).

The development of uorescent 8m indicators such as

0378-1097 / 00 / $20.00 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

PII: S 0 37 8 - 1 0 9 7 ( 0 0 ) 0 0 4 1 8 -3

* Corresponding author. Tel.: +44 (2920) 874 772 ;

Fax: +44 (2920) 874 305; E-mail: [email protected]

FEMS Microbiology Letters 192 (2000) 153^157

www.fems-microbiology.org


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bis-oxonol (bis-1,3-dibutylbarbituric acid trimethine oxo-

nol, DiBaC4(3)), a non-cytotoxic anionic lipophilic dye,

has enabled a number of alternative approaches to the

estimation of 8m including the use of spectrouorimetry,

single cell uorescence imaging and ow cytometry.

In using uorescent indicators to make quantitative es-

timates of 8m the approach most commonly taken is toobtain a calibration curve, relating the uorescence to the

8m. This usually entails measuring the uorescence from

cells in which the 8m is `set' to a range of values by

treating the cells with the ionophore gramicidin D and

then suspending them in media (Na-free) having a range

of dierent K concentrations [5,6]. The 8m is then calcu-

lated using the Nernst equation for K. This approach

rests on the assumptions that the intracellular Na does

not contribute to the 8m, and that the intracellular K

concentration is unaected by the addition of gramicidin.

Both are a potential source of error.

In the present study we have used an alternative ap-proach to make quantitative estimates of the 8m of G.

intestinalis trophozoites, adapting the method of Kraznai

et al. [7], whereby the 8m is calculated directly from the

transmembrane distribution ratio of bis-oxonol. The ori-

gin of the 8m of G. intestinalis was investigated by mon-

itoring the response of 8m to a range of extracellular ion

substitutions and pharmacological agents.

2. Materials and methods

2.1. Organism and culture

G. intestinalis Portland 1 strain were grown as described

previously [8]. For each experiment, cells were grown to

late exponential phase, harvested by centrifugation at

650Ug for 5 min and then resuspended in the appropriate

solution.

2.2. Solutions

Phosphate-buered saline (PBS, pH 7.2) contained 150

mM NaCl, 5 mM K2HPO4 and 1.8 mM KH2PO4. Na-

and K-free medium (NMG Cl solution) contained 140

mM N-methyl-D-glucamine, 0.8 mM Mg2Cl, 1 mM CaCl2,10 mM HEPES and 11 mM glucose, and was pH-adjusted

to 7.4 with HCl. Cl3-free media (Na gluconate solution)

contained 140 mM Na gluconate, 0.8 mM MgPO4, 1 mM

CaPO4, 10 mM HEPES and 11 mM glucose, pH 7.4.

2.3. Fluorescence measurements

Cell uorescence (green emission, 530^540 nm) was

monitored by ow cytometry using a MoFlo cytometer

with excitation at 488 nm from a water-cooled 200-mW

argon-ion laser. Forward light scatter and right angle side

scatter were measured and used for gating the data collec-

tion. Typically, signals from v5U104 cells were acquired

(and averaged) for each sample and analysed using Cy-

clops software. Confocal laser scanning microscopy

(Leitz-Biorad MRC 1024) was used under similar condi-

tions of excitation and emission, to image intracellular

uorescence of bis-oxonol-treated trophozoites.

2.4. Estimation of8m

The 8m of G. intestinalis trophozoites was determined

uorometrically using a method based on that described

by Krasznai et al. [7]. DiBAC4(3) was added to cells sus-

pended in either PBS or in NMG Cl solution and allowed

to equilibrate for 10 min. As a lipophilic anion, the dye

distributes across the plasma membrane in accordance

with the Nernst equation, 8m = RT/Fln(Din/Dout), where

R, T, F, Din and Dout represent the universal gas

constant, the absolute temperature, the Faraday constant

and the intra- and extracellular dye concentrations, respec-tively.

Din was estimated by ow cytometry. A calibration

curve relating cell uorescence intensity to Din was ob-

tained using cells that had been heat-xed at 60C for 15

min in order to eliminate the 8m (heat xation did not

signicantly aect the autouorescence of the cells). Under

these conditions (8m = 0) Din = Dout. The cells were sus-

pended in media having a range of bis-oxonol concentra-

tions (0.01^0.5 WM) and their uorescence measured by

ow cytometry. Cell uorescence was plotted as a function

of Dout ( = Din) and this curve was then used for the esti-

mation of Din (and hence 8m, via the Nernst equation)

from the measured uorescence of live cells suspendedunder dierent ionic conditions and in the presence and

absence of inhibitors.

2.5. Eect of extracellular ions and inhibitors

A range of pharmacological agents were tested for their

eect on the 8m of G. intestinalis trophozoites. Cells were

pre-incubated for 15 min with the inhibitors prior to the

addition of bis-oxonol (added at 0.5 WM). The agents

tested included the protonophore carbonylcyanide m-

chlorophenylhydrazone (CCCP, 10 WM), the carboxyl-

blocking reagent dicyclohexylcarbodiimide (DCCD, 100WM), the sulfhydryl reagent N-ethylmaleimide (NEM,

200 WM), the anion transport inhibitor 4,4-isothiocyana-

to-stilbene-2,2-disulfonic acid disodium salt (DIDS, 500

WM), the V-type ATPase inhibitor balomycin A1 (100

nM) and the P-type ATPase inhibitor orthovanadate

(100 WM). The orthovanadate solution was boiled until

colourless before use to ensure that the vanadate was

present in monomeric form.

The eect of K and Na (on the 8m of cells suspended

in NMG Cl solution) and the eect of Cl3 (on the 8m of

cells suspended in Na gluconate solution) were tested by

the addition of KCl, NaCl and choline Cl, respectively.

G.A. Biagini et al. / FEMS Microbiology Letters 192 (2000) 153^157154


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3. Results

In live, bis-oxonol-stained trophozoites viewed by con-

focal laser scanning microscopy the anionic dye was

largely excluded by the resting 8m of the plasma mem-

brane (Fig. 1a). Cells in which the membrane had been

depolarised by heat-xation, took up more of the dye and

had, as a result, a higher uorescence (Fig. 1b). The lack

of intracellular organelles such as mitochondria and per-

oxisomes facilitates plasma membrane 8m measurements

in G. intestinalis; the uorescence arises homogeneously

from the cell cytosol and the 8m-dependent accumulation

can be attributed wholly to the 8m of the plasma mem-

brane.

Increasing the extracellular dye concentration resultedin an increase in cell uorescence for both live and heat-

xed cells. Using ow cytometry it was possible to mon-

itor these increases accurately (Fig. 2A). A typical calibra-

tion curve relating cell uorescence to the intracellular dye

concentration for heat-xed trophozoites suspended in

NMG Cl solution containing bis-oxonol at a range of

concentrations is shown in Fig. 2B.

With the aid of the calibration curve, shifts in uores-

cence, indicating changes in the plasma membrane 8m,

due to the addition of various ions and inhibitors (Fig.

3), could be monitored and quantitated. Results are sum-

marised in Table 1.The addition of NaCl (40 mM) to cells in NMG Cl

solution (in which 8m was estimated as 31343 mV;

n = 11, S.E.M.) had no signicant eect on 8m. Simi-

larly, the addition of choline Cl (40 mM) to cells in Na

gluconate solution (in which 8m was estimated as

3132 6 mV; n = 3) had no signicant eect on 8m. By

contrast, the addition of KCl to G. intestinalis tropho-

zoites suspended in NMG Cl solution cells, resulted in a

signicant concentration-dependent membrane depolarisa-

tion (Fig. 4).

Of the various pharmacological agents tested, the two

causing the most pronounced eect were the H iono-

phore CCCP and the general H pump inhibitor

DCCD, both of which reduced8

m to almost half its nor-mal value (Table 1). The H/Na/K ionophore gramici-

din also caused a marked depolarisation. The combination

Fig. 1. Uptake of bis-oxonol by G. intestinalis trophozoites. Confocal laser scanning optical sections of live (a) and heat-killed (b) Giardia trophozoites

in PBS, stained with the voltage-sensitive indicator bis-oxonol (0.5 WM).

Fig. 2. Flow cytometric determination of 8m. (A) Flow cytometric his-

tograms of heat-killed G. intestinalis trophozoites suspended in NMG Cl

solution and stained with increasing concentrations of bis-oxonol. (B)

Calibration curve showing intracellular uorescence intensity of heat-

xed trophozoites as a function of extracellular dye concentration. The

data are averaged from three separate experiments and are shown

S.E.M.

G.A. Biagini et al. / FEMS Microbiology Letters 192 (2000) 153^157 155


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of DCCD and 40 mM KCl caused a signicantly greaterdepolarisation than DCCD alone. The sulfhydryl reagent

NEM also caused a signicant depolarisation. However,

neither the P-type ATPase inhibitor vanadate, nor the V-

type ATPase inhibitor balomycin A1 had any eect on

the 8m. Both the balomycin A1 stock and the vanadate

stock used in this study were shown to be active in control

experiments in which they were tested for their eect on

the intracellular pH of the malaria parasite Plasmodium

falciparum [9] and the yeast Saccharomyces cerevisiae

[10], respectively (data not shown).

The anion transport inhibitor DIDS caused a small but

signicant membrane depolarisation (Table 1).

4. Discussion

The plasma membrane potential plays a central role in

the physiology of all cells. In this study, we have adapted a

novel ow cytometric technique to make quantitative es-

timates of the plasma membrane potential of G. intestinalis

trophozoites and to investigate its origin.

The 8m value of3134 mV obtained for trophozoites

suspended in a Na- and K-free buer (Table 1) is higher

than that typically found in cells from higher eukaryotes

( =390 mV) but is of a similar magnitude to that reported

for other parasitic protozoa such as Leishmania major

(3113 mV, [6]) and Trypanosoma cruzi (3110 mV, [11]).

The dierence in 8m values between these organisms and

that ofG. intestinalis may also reect the dierent method-ologies adopted for their estimation.

In experiments in which the extracellular ionic compo-

sition was varied, the 8m of G. intestinalis trophozoites

was largely unaected by either Na or Cl3. However,

it showed a signicant dependence on K, decreasing in

magnitude as the extracellular K concentration was in-

Table 1

Eect of ions and inhibitors (reagents) on 8m of Giardia intestinalis trophozoites

Suspending solution Reagent Concentration n 8m (mV)a Pb

NMG Cl ^ ^ 11 3134 3 ^

NMG Cl NaCl 40 mM 43

122 6 NSNMG Cl KCl 40 mM 4 31073 6 0.001

Na gluconate ^ ^ 3 3132 6 ^

Na gluconate choline Cl 40 mM 3 3130 5 NS

NMG Cl DCCD 100 WM 6 37 5 2 6 0.001

NMG Cl DCCD+KCl 100 WM, 40 mM 3 36 2 7 6 0.001

NMG Cl CCCP 10 WM 8 37 6 4 6 0.001

NMG Cl gramicidin 1 WM 3 38 5 2 6 0.001

NMG Cl NEM 200 WM 3 31013 6 0.001

NMG Cl orthovanadate 100 WM 3 3136 6 NS

NMG Cl balomycin A1 100 nM 3 3138 3 NS

NMG Cl DIDS 500 WM 3 3119 2 0.001

aValues are means S.E.M. of independent experiments.bP values (one tail, paired t-test) for dierences between 8m measured in the presence of the dierent reagents tested and that measured in their ab-

sence (for cells in the same suspending solution). `NS' indicates Ps0.05.

Fig. 4. Eect of K on 8m. KCl was added at varying concentrations

to G. intestinalis trophozoites suspended in NMG Cl solution and the

8m estimated ow cytometrically. The data are averaged from three

separate experiments and are shown S.E.M. The large error bars in

the gure arose predominantly as a result of the variation between ex-

periments of absolute 8m values, rather than variation of the K depen-dence within each experiment.

Fig. 3. Eect of inhibitors on 8m. Flow cytometric histogram of G. in-

testinalis trophozoites (suspended in NMG Cl solution) showing in-

creases in uorescence intensity on treatment of the cells with NEM

(200 WM) and CCCP (10 WM).

G.A. Biagini et al. / FEMS Microbiology Letters 192 (2000) 153^157156


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creased (Fig. 4). A K-dependence of the 8m is character-

istic of many cells of higher vertebrates and is consistent

with the G. intestinalis trophozoite plasma membrane hav-

ing a signicant (electrophoretic) permeability to K.

However, if the 8m in Giardia were to have arisen pre-

dominantly from the K diusion (as in many vertebrate

cell-types), it can be estimated from the Nernst equation(assuming an intracellular K concentration of 92 mM

[12]) that cells suspended in a solution containing 40

mM K would have a 8m of 321 mV. The measured

value of3107 mV is substantially higher than this, indi-

cating the presence of additional component(s) contribut-

ing to the 8m.

The marked depolarisation that occurred on addition of

the protonophore CCCP (Table 1), is consistent with a

role for active H extrusion in the maintenance of the

resting 8m. In other protozoa (e.g. Leishmania, Trypana-

soma) an electrogenic H pump makes a signicant con-

tribution to8

m. Such pumps are inhibited by DCCD andNEM, both of which caused a signicant depolarisation of

the G. intestinalis trophozoite plasma membrane. The fail-

ure of the specic V-type ATPase inhibitor balomycin A1to have a signicant eect on 8m argues against a signi-

cant role for a H pump of this type in the generation of

8m. Similarly, the lack of eect of vanadate on 8m might

be taken as evidence against the involvement of a P-type

ATPase; however, vanadate is highly charged and its lack

of eect on 8m may be due to an inability to enter the

cells, rather than to a lack of involvement of a P-type

ATPase.

In this study the greatest depolarisation was observed in

cells treated with a combination of DCCD and K, how-ever no manoeuvre (other than a prolonged incubation of

cells at high O2 tensions, data not shown) was found to

induce complete (8m = 0 mV) depolarisation. Under all

of the conditions tested in this study the cells appeared

viable (i.e. cells showed normal agellar movement and

attachment as observed by light microscopy) and would

therefore be expected to have retained some degree of

polarisation. It is noteworthy, that previous studies dem-

onstrating H-ATPase activity in protozoa also failed to

induce complete depolarisation by the addition of ATPase

inhibitors [6,11,13,14].

In L. major, Cl3

conductance was shown to support H

pumping by partially dissipating 8m [6]. Blocking Cl3

conductance by DIDS, resulted in the hyperpolarisation

of the 8m. In the present study, substitution of the large

organic anion gluconate for Cl3 in the suspending solu-

tion had no signicant eect on the 8m and the (non-

specic) anion transport blocker DIDS caused only a mi-

nor perturbation of 8m. It therefore appears that Cl3

conductance has little if any role to play in the generation

of 8m in G. intestinalis.

In summary, in this study, a novel ow cytometric tech-

nique has provided quantitative estimates of the 8m of

G. intestinalis trophozoites. The technique should be ap-

plicable to a range of other unicellular organisms, the

physiology of which is, in general, poorly understood.

Ion substitution experiments implicate a role for K and

results obtained with a range of pharmacological agents

are consistent with a role for electrogenic H pumping in

the maintenance of a high (inwardly negative) resting 8m.The identity of the membrane transport pathways in-

volved, and their role in G. intestinalis physiology remain

to be claried.

Acknowledgements

Mr. R. Wadley is thanked for technical assistance. This

work was supported by the Australian Research Council

and by the Royal Society (D.L.).

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