Characterization of permeation pathways appearing in the host membrane of Plasmodium falciparum infected red blood cells

Molecular and Biochemical Parasitology, 14 (1985) 313-322

313

Elsevier MBP 00521

C H A R A C T E R I Z A T I O N O F P E R M E A T I O N P A T H W A Y S A P P E A R I N G IN T H E H O S T M E M B R A N E O F P L A S M O D I U M F A L C I P A R U M I N F E C T E D RED BLOOD CELLS

HAGAI GINSBURG, SHIRLEY KUTNER, MIRIAM KRUGLIAK and Z. IOAV CABANTCHIK

Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel (Received 1 July 1984; accepted 19 October 1984)

The host cell membrane of Plasmodiumfalciparum infected cells becomes permeabilized at the trophozoite stage. A variety of otherwise impermeant substances such as carbohydrates, polyols, amino acids and anions easily gain access to the cytosol of infected cells. Using the isotonic-hemolysismethod or uptake of labeled substances, we characterized the new permeation pathways as pores of approximately 0.7 nm equivalent radius. The pores bear a positivelycharged character which facilitates movement of small anions and excludes cations, so that the ionic composition and osmotic properties of infected cells are not drastically altered. Substances of a molecular size similar to that of disaccharides are fully excluded. Substances of limiting size might be accommodated in the pore, provided they bear a side group of hydrophobic character. The new permeation pathways may provide a vital route for acquisition or release of essential nutrients or catabolites. Key words: Human erythrocytes; Plasmodiumfalciparum; Membrane permeability; Hemolysis

INTRODUCTION The asexual r e p r o d u c t i o n of malarial parasites inside erythrocytes is a c c o m p a n i e d b y changes in c o m p o s i t i o n , structure a n d f u n c t i o n of the host cell m e m b r a n e . Glucose p e r m e a b i l i t y m a r k e d l y increases as Plasmodium berghei infected m o u s e erythrocytes [1,2], Plasmodium lophurae infected duck erythrocytes [3] a n d Plasmodiumfalciparum infected erythrocytes show a b n o r m a l l y high p e r m e a b i l i t y t o w a r d s a m i n o acids [5,6], polyols [4], cations [7-9] a n d a n i o n s [10,11]. A m a j o r e n h a n c e m e n t o f parasite m e t a b o l i s m was also observed at the t r o p h o z o i t e stage [12-14], a n d a possible f u n c t i o n a l relationship between m e m b r a n e p e r m e a b i l i t y a n d m e t a b o l i s m might be invoked.

Abbreviations: PBS, phosphate-buffered saline; Tris, Tris-(hydroxymethyl)-aminomethane; TBS, Tris-buffered saline; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. 0166-6851/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

314 The alleged permeability changes can be attributed to two main factors associated with the host cell membrane. One is related to alterations in the chemical and physical properties of membrane lipids which, in turn, might affect membrane transport systems originally present in the erythrocyte. This might explain the relatively small increase [1.5-3-fold) in monovalent cation transport [7,8] and amino acid transport [6] observed both in infected and non-infected cells exposed to serum, either from infected animals [7] or from infected cultures [6]. The second factor is associated with the appearance in the host cell membrane of new permeation pathways [4,10,11], which display anion selectivity and discrimination ofpermeants according to size. In the present study we examined the properties of the parasite-induced transport pathways, using carbohydrates and amino acids as molecular probes. These two classes of compounds were selected because of their alleged role in intra-erythrocytic parasite growth and because of their chemical properties which facilitate the biophysical definition of the new pathway(s). The results of this work indicate that the new permeability pathway, induced at the trophozoite stage, displays pore-like properties: the pore has an equivalent diameter of 0.7 nm and is probably positively charged; penetrating solutes interact with the pore, possibly through hydrogen bond and/or electrostatic forces, so that the pore might play an essential role in parasite development. MATERIALSAND METHODS

Cultivation of parasites. Cultures of P. falciparum (Gambian strain, kindly supplied by Dr. W.H.G. Richards, and FCR3Tc strain, obtained from Dr. J.B. Jensen) were grown in culture flasks (Nunc) containing growth medium (RMPI-1640 from GIBCO, supplemented with 25 mM N-2-hydroxyethyipiperazine-N'-2-ethane sulfonic acid (Hepes), 1 mM inosine, 32 mM NaHCO3 and 10% v/v, heat-inactivated AB ÷plasma and O ÷ or A÷washed human erythrocytes (2.5% hematocrit). Flasks were gassed with a mixture of 5% 02 and 5% CO2 in N2. Cultures were grown for 4-5 days with daily changes of medium and gassing, until the parasitemia (percentage of parasite infected erythrocytes, as determined microscopically on thin blood smears stained with Giemsa) reached 15-25% [10]. Cultures from several flasks were pooled, centrifuged and washed once with Tris- or phosphate-buffered saline, pH 7.4 (TBS or PBS). Kinetics of hemolysis of infected erythrocytes. The procedures used to assess the permeability of infected erythrocytes to various solutes were previously described in detail [4]. Briefly, a pellet of cells from an infected culture was suspended to 10% final hematocrit in solutions containing 200 mM solute, 50 mM NaC! and 10 mM sodiumphosphate, pH 7.4. Osmolarity of all solutions was brought to 310 mOs (checked by means of a Wescor vapor pressure osmometer). The time course of hemoglobin release was followed at room temperature (22 :t: I°C) by measuring absorbance of sample supernates at 540 nm (A540). Hemolysis is expressed as hemoglobin in super-

315 nate relative to total hemoglobin present in the cell suspension. It has been previously shown that in the presence of iso-osmotic solutions of solutes which penetrate very slowly into normal cells, cell hemolysis is associated exclusively with trophozoite- and schizont-containing erythrocytes [4]. Experiments involving large anions such as gluconate, aspartate, etc., were performed using the ammonium salts of these anions, since for net uptake of solute a permeable cation is required. Although the ammonium is most probably impermeant, net permeation of the salt can rapidly occur via the neutral derivative ammonia. In the case of large cations, the corresponding chloride salts were used due to the large permeability of erythrocyte membranes to chloride anions [15].

Uptake of ~4C-radiolabeled solutes. These experiments were performed as previously described [6]. Triplicates of 20-~tl packed (approx. 70% hematocrit), washed cultured cells were dispensed into 15-ml glass test tubes kept at 20°C. Solutions (180 p.l) containing various concentrations of radiolabeled (1 laCi ml -~) solutes were prewarmed at 20°C and jetted into the tubes, vortexed and incubated at 20°C. At the desired time intervals, 8 ml of ice-cold PBS, containing 0.25 mM phloretin, were injected into each tube and the suspension was rapidly centrifuged (3 500 × g, 2 min, 0°C). The supernate was aspirated, the cell pellet was washed twice in the same buffer and finally lysed in 2 ml distilled water. Of the lysate, 450 ttl were mixed with 50 ttl 100% (w/v) trichloroacetic acid, centrifuged and 400 ~tl of the supernate were taken for scintillation counting in a Packard Instrument equipped with a dpm calculating module. The rest of the lysate was diluted with 2 ml distilled water and the hemoglobin absorbance was determined at 410 nm in a Gilford 240 spectrophotometer. Results are expressed as dpm A-~ and were normalized to the radioactivity of the loading solution. An alternative method which gave essentially identical results consisted of mixing cells with medium containing labeled solutes and withdrawing aliquots into ice-cold PBS containing 250 ttM phloretin. All chemicals were of the best available grade; except for phloretin, which was from K & K Chemicals, all other chemicals were from Sigma Chemical Co. ~4C-labeled materials (~alanine, sucrose and I>-sorbitol) were from the Radiochemical Centre (Amersham). RESULTS

Permeation into infected cells as reflected in solute-induced lysis. Normal erythrocytes suspended at room temperature (22 -I- loC) in isotonic solutions of carbohydrates containing four or more carbon atoms, do not lyse over a period of 1 h. However, a culture of infected cells containing all stages of parasite growth, when suspended in the same conditions, displays a selective hemolysis of trophozoites and schizonts (Fig. 1). The amount of hemoglobin released from a given culture is a function of the percentage of trophozoites and schizonts present in the culture [4]. The lysis of these infected cells

316

7 . 0 ~

es

._1

I 0

I0

20

30

40

50

60

Time ( rain ) Fig. 1. Lysis of malaria infected erythrocytes exposed to iso-osmotic solutions of different polyols and sugars. Asynchronous cultures of P. falciparum in human erythrocytes were harvested after 4-5 days of cultivation, and washed once with TBS or PBS. At time 0, cells were suspended in iso-osmotic solutions of the indicated substance (300 mM in 10 mM Tris-Cl, pH 7.4, 22 + I°C). The time course of hemolysis was followed by measuring the hemoglobin released into the medium, and was expressed as percentage of the total hemoglobin present in the cell suspension, o-Arabitol (*), D-ribose (4-), sorbitol (e), D-glucose (A), Lglucose (ll), myo-inositol (a), sedoheptulose (A), maltose (o).

was shown to result from two properties associated with the host cell membrane: high permeability to osmotically active, uncharged and anionic solutes as well as a relatively low permeability to cations [10,11,4]. The time course of hemolysis induced by various carbohydrates is given in Fig. 1 in terms of hemoglobin release. The half time of hemolysis, th, that is, the time required to obtain the half maximal level of carbohydrate-induced hemolysis, was calculated after subtraction of background hemolysis observed in isotonic salt solutions under identical experimental conditions. The reciprocal of th, provides a measure of the permeation rate of the solute [2_2]. Data given in Table I show the following order of apparent permeabilities, Ph: pentitols > pentoses > hexitols > hexoses > hexuronates. However, there are substantial differences in permeation rates .for congeners of each class of permeants, such as h . . . xylitol >Ph sorbitol >Ph mannitol >Ph dulcitol > > P h myo-inositol. This indicates that sieving of substrates according to their molecular volume is not the only factor involved in the admittance of these solutes into infected cells. A similar finding is obtained with hexoses in that Ph 2-deoxyglucose >~h L-rhamnose >Ph D-glucose > > P h sedoheptulose >P--h myo-inositol > > Ph disaccharides, oligosaccharides. A most conspicuous example of the contribution of steric factors is seen in the marked difference of Ph values displayed by hexuronates and hexonates. Moreover, the relative permeability to hexuronates is also relatively low. Di- and oligosaccharides failed to produce any lysis in infected cells. Similar to the effects obtained with carbohydrates, amino acids failed to induce hemolysis in normal cells but produced a dramatic hemolytic effect on trophozoites and schizonts (Fig. 2). The computed t h and ~ffhvalues presented in Table II indicate that for neutral amino acids the molecular size of the solute was not the only crucial

eh'

317 TABLE I Parameters of permeation of polyols into malaria infected cells Solute

/~h (min-~)22oc

Number of carbons and structure

L-Arabitol D-Arabitol Xylitol D-Sorbitol v-Mannitol Duicitol myo-Inositol L-Arabinos¢ o-Ribose D-Glucose L-Glucose u-Deoxyglucose L-Rhamnose Sedoheptulose Sucrose, maltose Maltotriose, stachyose Gluconate Galacturonate

0.25 0.25 0.14 0.09 0.06 0.05 <0.003 0.20 0.25 0.05 0.03 0.09 0.05 0.02 <0.001 <0.001 0.03 <0.001

5, open chain 5, open chain 6, open chain 6, open chain 6, open chain 6, open chain 6, cyclohexane 5, pyranose 5, furanose 6, pyranose 6, pyranose 6, pyranose 6, pyranose 7, pyranose 12, pyranose-pyranose 18, 24 6, pyranose 6, pyranose

Values of half time of hemolysis (th)~C were determined from hemolysis profiles such as those shown in Fig. 1. Ph is given as the reciprocal of t h. The S.D. of the means were always smaller than 15%, and in each experiment a control of sorbitol was included to verify reproducibility. A second control in isotonic PBS was also included to account for non-specific lysis. This control was invariably smaller than 15% of the maximal lysis, and was subtracted from the latter value when estimating t h.

7.0

i i Amino Acids

i

i

310

40

I

I

3.5

'0

210

I

I

50

I

60

Time (rain)

Fig. 2. Lysis of malaria infected erythrocytes exposed to iso-osmotic solutions of different amino acids. Experimental details are given in the legend to Fig. 1. Solutions were made of 200 mM amino acid in 50 mM NaCI; 10 mM Tris-Cl, pH 7.4, 22 + I°C. Valine (e), isoleucine (o), alanine (*), serine (~), threonine (=), lysine (n), arginine (+).

318 TABLE II Parameters of permeation of amino acids into malaria infected cells Solute

/~ (min-I)~c

Glycine ¢t-Alanine ~-Alanine Vaiine Isoleucine Cysteine Serine Threonine Aspartate Glutamate Asparagine Glutamine Histidine Lysine Arginine

0.09 0.13 0.11 0.10 0.13 0.08 0.03 0.02 0.02 0.04 0.02 0.01 0.04 <0.001 <0.001

Values of ..(P'h)~ocwere determined from profilesof hemolysissuch as those shown in Fig. 2 and as described in the legend to Table 1. For further details, see legend to Table I. factor of admittance into the cell. In fact, as the apolar moiety of the amino acid increased in size, the rate of penetration increased commensurately (e.g.,/~h of glycine v s . / ~ of valine). Substitution of the methyl group for an O H group in the side chain, substantially decreased the rate of penetration (e.g., compare Ph alanine with Ph serine and Ph valine with Ph threonine). The dicarboxylic aspartate penetrated less readily than its carboxamide asparagine, while glutamate was more permeant than glutamine. O f the diamino carboxylic acids, only histidine produced detectable levels of hemolysis. Tris is a weak base which at 20°C has a p K of 8.3. When prepared as isotonic Tris-Cl solutions at p H 6.4 and 7.4, 1.2% and 11% of the solute appeared in the non-protohated (neutral) form. Hemolysis studies conducted with these solutions indicate that when more of the solute was in the free base form, hemolysis was fast (t h : 9.5 min), whereas if more of it was in the cationic form, hemolysis was considerably reduced (t h = 100 min). The alternative explanation that p H affected the membrane permeability properties, was tentatively ruled out on the basis that the course of hexitol-induced hemolysis was essentially indifferent to p H changes (not shown). These experiments indicate that most likely Tris is admitted by the host cell membrane only in the neutral form.

Permeation into infected cells as reflected in uptake of labeled substrates. In order to corroborate the above studies, we conducted uptake measurements of three selected

319

substrates: 15-[14C]alanine, D-['4C]sorbitol and [~4C]sucrose. Stopping at given periods of time was accomplished by quick washing of the suspensions with cold buffer, containing 200 pM of phloretin, and centrifugation. As seen in Fig. 3, both ~alanine and D-sorbitol gained rapid access into infected cells. Sucrose, on the other hand, was impermeant to both infected and uninfected cells. Interestingly, sorbitol showed significant penetration also into uninfected cells, as previously demonstrated [16]. However, permeation into both classes of cells could be efficiently inhibited by addition of phloretin, as demonstrated before with the method of solute-induced hemolysis [4]. The extent of uptake was reasonably constant in a given experiment, although it varied enormously with the age of the culture and the variable susceptibility of cells subjected to centrifugal stresses. Therefore, the results presented in Fig. 3 provide only a semiquantitative picture of the uptake profiles of the various substrates and their susceptibility to the inhibitor phloretin. Our previous observations that disaccharides, while apparently impermeant to infected cells provided substantial protection from sorbitol-induced hemolysis [4], were also reassessed in this work. Using radiolabeled sucrose, we corroborated the impermeability of the host cell membrane to disaccharides (Fig. 3). However, no transport inhibitory effect was exerted by disaccharides such as sucrose or maltose present during uptake of [14C]sorbitol or [~4C]alanine (not shown). These studies indicate that protection from sorbitol-induced hemolysis was probably afforded by an osmotic and/or membrane protective effect, rather than by blocking of permeation routes as previously suggested [4].

A

B

;~,0

,°° I 80 I o 60

I0

/

40

/ V N~rb N~.ala_~phI

T~-ala÷phl C~"~°I

$o~.b+phi

20 5

15

0

30

Time

I i I I I I

5

15

30

( rain )

Fig.3. Uptake of [14Clsorbitol (sorb), I]-['C]alanine (13-ala), and [14C]sucrose (suet) into P. falciparum infected human red cells (I) and uninfected human red cells (N). Uptake of labeled substrates into cells was performed as described in Materials and Methods, and is given in terms o f d p m (A,~o)-L Note the difference in scale between the left and right graphs. Uptake was measured either in the absence or presence of phloretin (phi) (250 pM). 1.2 dpm (A,~0)-t is equivalent to 10 taM in infected erythroeytes.

320 DISCUSSION

Transport studies performed with malaria infected erythrocytes of rodents and birds [17] showed a significant increase in the permeability of the membrane of the host cell relative to that of normal erythrocytes. In some cases permeation across the host cell membrane was shown to be taken over by diffusion-like mechanisms [3]. Recently we have demonstrated the existence of new permeation pathways for anions and hexitols in human erythrocytes infected with P. falciparum in in vitro cultures [4,10,11]. The pathways displayed pore-like properties with selective admittance of anions and exclusion of cations. They appeared at the trophozoite stage and remained throughout schizogony [10]. In the present study we attempted the biophysical definition of the pores and extended the list of solutes to include substances of metabolic relevance to the growing parasite, such as hexitols for lipid synthesis [18] and amino acids for protein synthesis [191. On the basis of transport studies using a series of carbohydrates, we concluded that the parasite-induced permeability pathways discriminate between penetrating solutes, according to their molecular size. This is manifested in the apparent permeability constants which decrease with increasing size of congener molecule, both in neutral and in carboxylated carbohydrates. Such permselectivity behavior is characteristic of pores that have an apparent diameter which is only slightly larger than that of the permeating solutes. Based on the fact that disaccharides are not admitted by infected cells, we suggest a limiting pore diameter of approximately 0.7 nm. The differential permeation rates within each molecular size group probably reflect steric variations and, in turn, suggest a role for chemical interactions between the translocating solute and the pore. Such interactions could involve hydrogen bonding and electrostatic forces, thus implying the proteinaceous character of the putative pore. This tentative conclusion is compatible with, but does not prove, the idea that parasites introduce new antigenic determinants into the membrane of their host cell

[20]. The relative permeability rates of the neutral amino acids indicate that these solutes are not sieved by the pore solely according to their molecular volume since hydrophobic side chains facilitate their translocation, thus, the hydrophobicity of the substance compensates for the apparent increase in size. Such a pattern of selectivity is rather unexpected for a hydrophilic channel and suggests that the pore might be lined with hydrophobic domains that provide a parallel path for the hydrophobic moiety of the penetrating solute and/or increase the partitioning or accessibility of the solute to the pore. An examination of the apparent permeability constants, Ph, of amino acids indicates that OH substitution reduces the permeability by a factor of 4-5 (compare alanine with serine and valine with threonine). Such differences could be compatible with a hydrophobic effect on partitioning of the solute into the pore as well as with an

321

energy requiring dehydration of the hydroxy amino acids prior to their entry into the pore, an energy change which is not fully compensated with hydrogen bonding within the pore. Comparison of the Ph values of the basic amino acids is also informative. Histidine, with a molecular radius very similar to that of lysine, displays a ffh of 0.04 min-1, while neither lysine nor arginine shows any detectable penetration. Since the pI ofhistidine is 7.58, it is suggested that this amino acid is admitted by the pore as zwitterion, while the positively charged lysine and arginine are excluded. This suggestion is in agreement with the pH-dependence of Tris-induced hemolysis, where it is shown that as the concentration of free (uncharged) base is increased, Ph increases in proportional manner. Hence, in agreement with our previous idea [4], we conclude that the pore excludes positively charged solutes and must, therefore, be positively charged. The recently reported increase in cation permeability of infected erythrocytes [8] probably does not involve the pore and can be associated with the general non-specific increase in carrier-mediated transport found in these cells [6]. Such an increase could also provide for the adequate supply of the relatively impermeable basic amino acids. The implications of pore formation to the biology of the intra-erythrocytic parasite are obvious. The permeability changes occur at a stage when parasites enter a state of intense metabolism [12-14] which undoubtedly depends on an ample supply of metabolites and the efficient discharge of waste products. The induced change in permeability is, accordingly, largely non-specific, inasmuch as the new pores need to accommodate a wide variety of solutes. These also include amino acids which need to be imported into malaria infected cells grown in culture conditions, such as isoleucine, cysteine, glutamine and glutamate [21]. Finally, the impermeability of the pores to cations enables the infected erythrocytes to maintain the control of their osmotic volume and salt composition, thus avoiding premature release of the maturing parasites. ACKNOWLEDGEMENTS

This work was supported in part by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, and by a National Institutes of Health grant, No. AI-20342. The technical assistance of Mrs. S. Friedman is gratefully acknowledged. REFERENCES 1 2 3

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