Membrane potential of Plasmodium falciparum gametocytes monitored with rhodamine 123

FEMS MicrobiologyLetters 69 (1990) 283-288 Published by Elsevier

283

FEMSLE 04024

Membrane potential of Plasmodium falciparum gametocytes monitored with rhodamine 123 M a y u m i K a t o ~, K a z u y u k i T a n a b e 2 Atsushi Miki ~, K a z u y o l c h i m o r i 3 and Seiji Waki 4 J Department of Medical Zoolo~'. Osaka Ci(v Unice~ity Medical School. Osaka, " LaboratorT of Biology'. Osaka Institute of Technology, Osaka. " Department of Medical " ZooloKv. Teikvo University School of Medicine Tokyo . and J Department of Partt~ltolog~'. Gunma Unir'ersity S~hool of Medicine. Gunma. Japan

Received 15 January 1090 Revision receivedand accepted 23 February 1900 Key words: Membrane potential; Rhodamine 123; P l a s m o d i u m f a l c i p a r u m ;

1. S U M M A R Y The

membrane potential of P l a s m o d i u m gametocytes was monitored with the cationic permeant fluorescent dye rhodamine 123 (R123) as a probe. Epifluorescence microscopy revealed that R123 at 1 p,g/ml rather selectively partitioned into structure resembling large mitochondria. Treatment of R123-1oaded gametocytes with various inhibitors including those of respiration resulted in disappearance of fluorescence from what appeared to be the mitochondria, but not from the cytostfl. These resuits malcate that r. f a l c i p a r u m gametocytes have the mitochondrion maintaining an inside negative membrane potential,

falciparum

2. I N T R O D U C T I O N Protozoan parasites of the genus P l a s m o d i u m , which cause malaria, undergo repetitive cycles of

Correspondence to: Dr. K. Tanabe, Laboratory of Biology. Osaka Institute of Technology,Ohmiya, Asahi-ku, Osaka, 535, Japan.

Malaria; Gametocyte

asexual proliferation in appropriate vertebrate hosts. The asexual forms of P l a s m o d i u m possess an electrogenic proton pump in the parasite plasma membrane inside the host erythrocytes [1-4]. A protonmotive force generated by the proton pump has been ~hown to regulate transmembrane movements of Ca-" ~ and a sugar from the cytosol of infected erythrocytes into the parasite compartment [5,6]. Therefore, it seems that these H÷-de pendent transport systems are important in supporting the intraerythrocytic development of malaria parasites. MeanwmJe, a portton o[ the parasite differentiate into gametocytes within the host erythrocytes. Male and female gametocytes, when ingested by anopheline mosquitoes, transform into male and female gametes, respectively, in the insect alimentary tract, wherein fertilization of female gametes takes place to form zygotes. From a physiological point of view, the gametocyte stage is a critical period, during which the environment surrounding the parasites changes drastically from the host mammalian circulation to the mosquito alimentary tract. It is, then, intriguing to address the issue of mechanism of transport of gametocytes. In contrast to the asexual forms, however, biochemical studies on the sexual forms of P l a s m o d i -

0378-1097/90/$03.50 '~' 1990 Federation of European MicrobiologicalSocieties

284 ton are very limited, largely due to difficulties in obtaining sufficient amounts of materials. No analysis on transport of metabolites at the sexual stages has been conducted to date. A fluorescence microscopy method, using a cationic permeant fluorescent dye rhodamine 123 (R123), has been developed by which one can monitor the membrane potential of living cells [7]. Because of its chemical characteristics, R123 selectively accumulates in subcellular compartments with an inside negative membrane potential like mitochondria [8]. With the R123 staining method, several investigators have shown that the asexual forms of Plasmodium have both the plasma membrane potential [2,3] and mitochondrial membrane potential [4,9]. Since this staining method requires only a small amount of materials, we expected the method would be applicable to gametocytes of P. falciparum. In the present study, we have examined the plasma membrane and mitochondrial membrane potentials of P. falciparum gametocytes. Results show that the gametocytes mitochondria can be visualized by R123 and generate an inside negative membrane potential.

3. MATERIALS A N D M E T H O D S

3.1. Reagents The following reagents and their suppliers were: R123 from Eastman Kodak (Rochester, NY); RPMI 1640 medium from GIBCO (Grand Island, NY); carbonylcyanide m-chlorophenylhydrazone (c'CCP) and a,umycin A from 3~gma Chemical Co. (St. Louis, MO); N-2-hydroxyethylpiperazineN '-2-ethanesulfonic acid (Hepcs) from Boehringer (Mannheim, F.R.G.) and all other chemicals from Wako Pure Chem. (Osaka, Japan).

3.2. Parasite culture The 3D7 strain of P. [alciparum generously provided by Dr. D. Walliker of Edinburgh University was maintained by tile method of Trager and Jensen [10] with a gas mixture of 5% CO 2, 5% 02 and 90% N 2 [11]. The 3D7 strain of P. falciparum has been reported to produce spontaneously mature gametocytes which are infectious

to anopheline mosquitoes I12]. Since P. falciparum tends to lose the ability to form gametocytes during long term passages in culture, a batch of parasites were cryo-preserved to make stabilates. Parasites were then thawed and cultured for the experiments. Human erythrocytes (type A +) were used for culture as described elsewhere [11]. Prior to ust. they were washed 3 times in RPMI 16~0 and resuspended to 5% in complete culture medium ccmsisting of RPMI 1640 plus 25 mM Hepes and 10% human A + serum. Parasites were diluted to, make a parasitemia of 0.2 to 0.3% and cultured for 10 to 15 days. The culture medium was changed daily but was not supplemented with fresh erythrocytes. The asexual forms increased in number for the initial 4 days and thereafter disappeared gradually. Under these culture conditions, stage I11 to V gametocytes [13,14] appeared.

3. 3 Fluorescence microscop.v Because of a low yield of gametocytes (0.4-0.6% of total erythrocytes), erythrocytes collected from culture dishes were fractionated on a 65% Pereoll gradient, as described previously [2]. The erythrocytes from the interface were washed in HBS 2 times and resuspended to 1% in HBS. Gametocytemias of the suspension were approximately 10%. Aliquots of the cell suspension were incubated in HBS at 3 7 ° C for 30 rain in the presence of 0.1, 1 or 10 /~g/ml R123, followed by 3 washings in HBS at 4 ° C . R123-preloaded cells were resuspended to 0.1% in HBS and further incubated in the absence of the dye for 30 rain at 3 7 ° C in [!BS with or without various metabolic inhibitors. Cells were washed 2 times in HBS and subjected to epifluorescence microscopy as described previously to evaluate the effects of inhibitors on the dye-retention by parasites [2,4]. In some cases, the asexual parasites collected from cultures at midlogarithmic phase (i.e. 2 to 3 days cultures) were added to the incubation medium containing gametocytes. This procedure enabled us to monitor the membrane potentials of both the asexual parasites and gametocytes at the same time because almost all the sexual forms die under conditions for gametocyte cultures, Photographs were taken with Kodak Tri-X Pan film [2,15].

4. RESULTS Before starting experiments, it was necessary to remember that the plasma membrane potential of the asexual forms of Plasmodium is detectable with R123 at 10 p,g/ml in incubation medium, at which concentration one can visualise the mitochondriai membrane potential of cult,'red mammalian cells [7,8], whereas the parasite mitochondrial membrane potential is detectable at a very low concentration of R123 (0.1 t t g / m l ) [4,9]. At 10 # g / m l , R123 accumulated intensely in the parasite cytosol, thereby obscuring specific association with the parasite mitochondrion [4,9]. This difference in the concentration dependent R123 accumulation in the parasite cytosol and mitochondria probably reflects a difference in the intensity of the plasma membrane and mitochondrial membrane potentials of the parasite. Although

no data are available as to a value of the mitochondrial membrane potential of Plasmodium, it is estimated that the mitochondria of mouse neuroblastoma have their membrane potential of - 143 mV [16]. The plasma membrane potential of erythrocyte-free P. chabaudi has been calculated to be - 9 0 mV [3]. It is hence likely that the mitochondria of the asexual forms of Plasmodium can actively accumulate R123 even at a low concentration in incubation medium. Since such concentration-dependent differential accumulation might occur in P. falciparum gametocytes, they were incubated with 0.1 to 10 ~ g / m l of R123. At 10 # g / m l , gametocytes exhibited faint fluorescence diffusely in the cytosol (Fig. 1). Occasionally, R123 was densely associated with a restricted region in the cytosol. The intensity of these cytosolic fluorescences varied among gametocytes. Such heterogeneity in R123 staining per-

/ O Fig. 1. Light (A, B) and fluorescent (a, b) micrographs of Plasmodiumfalciparum gametoc)tes and asexual forms. Parasites were preincubated with l0/~g/ml rhodamine 123. followed by treatment with (B, b) or without (A. a) 10/~M CCCP. Note a difference in the intensity of fluorescence in the cytosols of gametocytes and asexual forms (asterisk). and disappearance of fluorescence from asexual forms (arrow).

286

Fig. 2. Light (A, B) and fluorescent'(a, b) micro~aphs of Ptasmodium falciparum gametocytes. Parasites were preincubated with 1 /~g/ml rhodamine 123. Note fluorescence in a structure resembling the mitochondrion (a, b).

O

O Fig, 3. Light (A-F) and fluorescent (a-f) micrographs of Plasmodium falciparum gametocytes. Oametocytes were preloaded with rhodamine 123 at 1/lg/ml and then incubated in the presence of inhibitors: 1/tM CCCP (B, b), 10 ~M CCCP (C, c), 1/~M antimycin A (D, d), 1 mM KCN (E, e), 10 mM NaN3 (F, f) and none (A. a).

287 sisted during culture periods for 7 to 15 days. Gametocytes at different stages of maturation, checked by cell shape, exhibited the staining heterogeneity. By contrast, the accumulation of R123 in the cytosol of the asexual parasites was very intense (Fig. la, asterisk). R123-preioaded parasites were treated with various metabolic inhibitots to see if the dye accumulation reflects the plasma membrane potential. Little fluorescence disappeared from the cytosol of gametocytes after treatment with 10 #M CCCP, a protonophore, whereas CCCP almost abolished the cytosolic fluorescence of the asexual parasites (Fig. lb, arrow) as observed previously [2-4,9]. None of inhibitors of mitochondrial electron transport, antimycin A (1 /~M), KCN (1 mM) and NaN 3 (10 mM), affected the retention of the cytosolic fluorescence by gametocytes (not shown). The dye at 1 /~g/ml partitioned in a structure resembling the mitochondrion but very faintly in the parasite cytosol. The structure consisted of twisted, branched tubules, a n d / o r granular bodies (Fig. 2). By shifting a fine focus of a microscope, it was possible to recognize that those structures were connected to each other. R123 disappeared from these structures after treatment with 1-10 /~M CCCP or 1 # M antimycin A, indicating that the dye-accumulating structure is the gametocyte mitochondrion (Fig. 3b-d). On the other hand, R123 remained accumulated after treatment with 1 mM KCN or 10 mM NaN 3 (Fig. 3e, f). Deprivation of glucose in incubation medium did not affect the mitochondrial dye retention (not shown). At 0.1 /~g/ml of R123, no fluorescence appeared in mitochondrion. From the above results, it is concluded that R123 at 1 / ~ g / m l accumulates in the gametocyte mitochondria but not in the gametocyte cytosol.

5. DISCUSSION The present study has demonstrated that, unlike the asexual forms of P. falciparum, the gametocytes do not generate a high inside negative membrane potential, enough to be detected by fluorescence microscopy, at the plasma membrane, but do possess membrane potential gener-

ating mitochondfia. We have previously noted non-specific binding of R123 to Toxoplasma gondii, which belongs to the same order (Eucoccidiida) as Plasmodium [17]. It is assumed that R123 binds cellular membranes as the dye is rather lipophilic. Relevant to this context is the present observation, that the intensity of R123 fluorescence is heterogeneous in the population of gametocytes. Electron microscopy shows that macrogametocytes are rich in membrane materials such as membranes of the endoplasmic reticulum, in contrast to microgametocytes [18,19]. Also, the heterogeneity of R123 staining did not parallel the maturation of gametocytes. Therefore, it may be that R123 is associated with membraneous materials in gametocytes. Nevertheless, whatever the mechanism for non-specific binding of R123 unrelated to the membrane potential, it is clear that jametocytes differ from the asexual forms with respect to the plasma membrane potential. However, wc do not exclude the possibility that the gametocyte plasma membrane has an apparently weak membrane potential, since fluorescence microscopy using R123 detects relatively strong membrane potentials and may fail to monitor weak membrane potentials. On the other hand, we have detected the mitochondrion in the gametocytes of P. falciparum. The mitochondrion was sensitive to CCCP, a protonophore and antimycin A. Mitochondria of cultured cells [8] are also sensitive to those inhibitors. Thus, this is the first demonstration of membrane potential generating mitochondria of living P. falciparum gametocytes. The shape of the mitochondrion is not uniform: twisted, branched tubules a n d / o r granular bodies. Importantly, these structures are connected to each other, showing a single mitochondrion in the gametocyte. It has been previously reported, based on observations by electron microscopy, that the gametocytes contain many mitochondria. The present finding clearly shows this is not the case for P. falciparum gametocytes. The gametocyte mitochondrion was not sensitive to all inhibitors of the mitochondrial electron transport. The mitochondria of some organisms like Trypanosoma in the mammalian hosts are reported to be insensitive to C N - and N f [20]. In

2~'8

T. bruce;, the CN--insensitive fraction of respiration is mediated by cytochrome o, instead of CN--sensitive cytochrome a + a 3. It would not be surprising if P. falciparum gametocytes modified their electron transport pathways to have alternate terminal electron acceptors. A further study will be necessary to investigate this possibility. In contrast to the mitochondrion of gametocytes, those of the asexual forms are sensitive to both antimycin A and C N - [4,9]. The role of the P. falciparum mitochondrion at the asexual stage remains unknown. It should be, however, stressed in this context that cytochrome systems may not necessarily be associated with respiration but linked to de novo synthesis of pyrimidine instead (see [21,22]). The mitochondrion of the asexual forms of P. falciparum is small and has a few cristae as compared with that of the gametocytes. During gametocytogenesis the mitochondrion undergoes ultrastructural changes to develop the christae [18,19]. These circumstances support the view that the mitochondria of both gametocytes and the asexual forms do not share the same function. In summary, the present study has shown that gametocyte stages apparently differ from the asexual stages in terms of the plasma membrane and mitochondrial membrane potentials.

ACKNOWLEDGMENTS The authors wish to thank Dr. K. Kita of Juntendo University School of Medicine for critical discussion and Professor S. Takada for his encouragement during this study. This work was supported by the 'Grant-in-Aid' from the ministry of Science, Education and Culture (Nos. 61570197 and 61304037).

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