Plasmodium falciparum: Stage-specific lactate production in synchronized cultures

EXPERIMENTAL PARASITOLOGY 54,391-396

Plasmodium

(1982)

falciparum: Stage-Specific Lactate Synchronized Cultures

Production

in

M.A. PFALLER, D.J. KROGSTAD,~AND A.R. PARQUETTE Divisions

of Laboratory Medicine and Infectious Disease, Departments of Medicine and Pathology, Washington University School of Medicine, Saint Louis, Missouri 63110, U.S.A.

AND

P. NGUYEN-DINH Division

of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Department Health and Human Services, U.S. Public Health Service, Atlanta, Georgia 30333, U.S.A.

of

(Accepted for publication 20 June 1982) PFALLER, M.A., KROGSTAD, D.J., PARQUETTE, A.R., AND NGUYEN-DINH, P. 1982. Plasmodium falciparum: Stage-specific lactate production in synchronized cultures. Experimental Parasitology 54, 391-396. Sequential treatment with gelatin followed by sorbitol-produced Plasmodium falciparum cultures that remained synchronous for 3 144hr.

The schizont stage was associated with more lactate production than the other asexual erythrocytic stages of the parasite (11-21 vs 4-7 neq/hr-106 parasitized red blood cells [PRBC]). Lactate production in asynchronous cultures was relatively constant at 8-11 neq/hr-106 PRBC and was directly proportional to parasitemia (P < 0.001). INDEX DESCRIPTORS: Plasmodium falciparum; Protozoa, parasitic; Malaria; Culture; Malaria stages, separation; Gelatin; Sorbitol; Metabolism, stage-specific Lactate production.

treatment of asynchronous cultures with gelatin followed by sorbitol to examine the Because cultures of Plasmodium falstage-specific proteins and glycoproteins of ciparum are typically asynchronous, we P. falciparum (Kilejian 1980), no data were have been interested in the use of synchroprovided on the degree and duration of synnous cultures as an aid in defining the stagechrony achieved. The studies reported here specific metabolism of P. falciparum in demonstrate the feasibility of producing vitro. The techniques previously available for tightly synchronized cultures by sequential producing synchronous cultures (physiogel, treatment with gelatin and sorbitol, and the gelatin, and sorbitol treatments [Reese et use of these cultures in examining bioal. 1979; Jensen 1978; Lambros and Van- chemical fluctuations that take place in derberg 19791) have been only moderately vitro during the erythrocytic cycle of P. falciparum. successful. The degree of synchrony achieved and the period of time over which MATERIALS AND METHODS these cultures remained synchronous have Cultures. Strain Honduras YCDC of Plasmodium been too variable and too short to permit falciparum was grown in vitro in a 9% erythrocyte stage-specific biochemical studies. Al- suspension using the method of Trager and Jensen though Kilejian recently used sequential (1976) with O-positive 4-week-old erythrocytes (JenI Reprint requests to: Microbiology Laboratory, Barnes Hospital, St. Louis, MO 63110, U.S.A.

sen and Trager 1977)and 10% human serum. The cultures were incubated at 37 C in an air-tight modular incubation chamber (Billups-Rothenberg, Inc., Del 391 00144894/82/060391-06$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

392

PFALLER

ET

Mar, CA, USA) which was gassed for 5 min with a mixture of 3% O,, 3% CO,, and 94% N, (Scheibelet ul. 1979b)after each medium change. In these experiments, medium changes and thin smears (for Giemsa staining) were performed at 8-hr intervals. A minimum of 1000 erythrocytes were counted per slide, and parasitemias were expressed as the percentage of parasitized red blood cells (PRBC). The asexual erythrocytic stages of the parasite were identified according to the criteria of Trigg (1969). Control suspensions of uninfected erythrocytes were treated in the same fashion to determine the amount of lactate produced by uninfected red blood cells. Synchronization with gelatin alone. Asynchronous cultures grown to parasitemias of 6- 10% were pooled and treated with gelatin (Type 1, Sigma, St. Louis, MO, USA), according to the method of Jensen (1978) using both 0.5% and 0.75% gelatin. The upper phase (containing trophozoites and schizonts) was collected after sedimentation for 30 min at 37 C and used to inoculate fresh erythrocytes. Starting parasitemias were l-2%. Synchronization with sorbitol alone. Asynchronous cultures were pooled as described above, and treated with a 5% sorbitol solution for 10 min at room temperature (Lambros and Vanderberg 1979). The resulting

AL.

material (consisting of uninfected erythrocytes and erythrocytes infected with ring forms) was washed twice with RPM1 1640 containing 25 n&I Hepes and 0.2% NaHCO,, added to fresh erythrocytes, and adjusted to a 9% hematocrit. Starting parasitemias were 0.5-1.0%. Synchronization

with

gelatin

followed

by sorbitol.

After treatment with gelatin, the material from the upper phase was placed into culture with fresh uninfected erythrocytes and allowed to incubate for 6-8 hr at 37 C (Fig. 1). This allowed some of the schizonts present to mature to merozoites and invade new erythrocytes. Following this incubation period, the culture was treated (as above) with 5% sorbitol, thus destroying all stages except the ring stage. This material was then washed (as above) and rediluted in fresh erythrocytes, resulting in starting parasitemias of 0.5- 1.O%. Sumple processing. Spent medium was collected by aspiration, centrifuged for 10 min at room temperature and 17OOgto remove cellular debris, and frozen immediately at -20 C. Aliquots (3 ml) of the 5-ml medium samples were deproteinized with 10% perchloric acid (0.6 ml of 70% perchloric acid, Mallinckrodt, St. Louis, MO, USA) and clarified by centrifugation at 4 C and 4000g for 10 min. The supemates were neutralized to pH 7 with an approximately equal volume of 2 M

0 0 0 ‘$‘,

Schizonts

*.) Trophorottes

,.;,

Asynchrohous cultures of I! fakiparum contain all stages of the parasite.

( 37’C

FIG.

cells

X 30 minutes)

Treatment with gelatin concentrates schizonts and trophozoites In the supernatant

x 6-8 hours )

After centrifugation of the supernatant, the pellet is replaced into culture with fresh red cells.

young

(37’C

Rings, uninfected

After 6-8 hours of incubation some schizonts have matured to merozoites, and produced new rings.

Sorbitol treatment lyres the schizonts and trophozoites, producing a tightly synchronirad culture of young rings

1. Sequential treatment with gelatin and sorbitol produced tightly synchronized cultures of falciparum ring forms in vitro.

Plasmodium

Plasmodium fulciparum:

STAGE-SPECIFIC

KHCO, (Sigma), and then either frozen at -70 C or assayed for lactate. This protocol resulted in a 1:1 dilution of the original sample. Lactate determinations were performed using a specific enzymatic technique (described below) and were corrected for this dilution factor. Lactate determinations. Lactate was quantitated enzymatically by the method of Drewes (1974) using the reduction of NAD to NADH by L-lactate dehydrogenase (EC 1.1.1.27, Sigma). The absorbance of NADH (which is proportional to L-lactate in this assay) was measured at 340 nm with a Zeiss PM6 spectrophotometer. Lactate production by parasitized red cells was expressed as nanoequivalents of lactate per hour-lo6 PRBC, and was corrected for lactate production by unparasitized red cells, which was determined from uninfected red cell controls and averaged 0.23 neq/hr-106 red cells (range 0.16-0.32). RESULTS

Gelatin alone. Using the gelatin concentration technique (Jensen 1978), Plusmodium fufciparum cultures were virtually asynchronous after one cycle (-48 hr; Fig. 2). Because the lower gelatin concentration (0.5%) required 90- 120 min to produce the same separation achieved within 30 min using 0.75% gelatin, subsequent studies utilized the higher gelatin concentration. Sorbitof alone. Treatment with 5% sor-

LACTATE

PRODUCTION

bitol (Lambros and Vanderberg 1979) resulted in much better synchrony (Fig. 3). However, the results were variable, and synchrony did not persist beyond 96 hr. Gelatin followed by sorbitol. The sequential use of these techniques resulted in much tighter synchrony which was maintained for at least three cycles (~144 hr; Fig. 4). These studies also revealed a significant increase in lactate production associated with the schizont stage of the parasite. Lactate production associated with the schizont stage ranged from 11 to 2 1 neq/hrlo6 PRBC vs 4 to 7 neq/hr-106 PRBC for the other erythrocytic stages of the parasite. The average lactate production calculated from these data was 8-9 neq/hr-106 PRBC and was similar to that observed with asynchronous cultures (8- 11 neq/hr-106 PRBC). The amount of lactate produced by asynchronous cultures increased linearly with increasing parasitemia (Fig. 5). DISCUSSION

As suggested by Trager (1979) and by Kilejian (1980), these results indicate that

GELATIN ALONE

OR 0T 1s

0 8

17 25

33

42 4P 57

393

66 74 81 90 98 105 112 121

HOURS OF INCUBATION

FIG. 2. Plasmodium falciparum cultures treated with gelatin alone were virtually asynchronous within 48 hours. R, right; T, trophozoite; S, schizont, according to the criteria of Trigg (1969).

394

PFALLERET

AL.

SORBITOL ALONE

0

9

18 26

34

A2 50 57 HOURS

FIG. 3. Plasmodium falciparum

67

lb

90 98 105 112 121

139

cultures treated with sorbitol alone were asynchronous

combining the gelatin and sorbitol methods results in better synchrony than using either method alone. Cultures treated with sorbitol alone did not maintain synchrony be-

0 7

74 82

OF INCUBATION

after 96 hr.

yond 96 hr in our hands. Previous reports have suggested that the duration of synchrony with this technique may vary from 72 to 120hr (Mrema et al. 1979;Lambros and

I

I

63 72 78 87 95 102 110 119

IX

HOURS OF lNCU8ATlON

FIG. 4. Plasmodium falciparum cultures treated with gelatin followed by sorbitol remained synchronous for at least three cycles (a144 hr). The lactate production associated with the schizont and trophozoite stages (1 l-21 neq/hr-10” PRBC) was greater than that associated with the other stages of the parasite (4-7 neq/hr-106 PRBC).

Plasmodiumfalciparum:

STAGE-SPECIFIC LACTATE PRODUCTION

FIG. 5. In asynchronous Plasmodium falciparum cultures, lactate production was directly proportional to parasitemia (r = 0.97, P < 0.001) (Colton 1974).

Vanderberg 1979). Thus, although treatment with sorbitol alone can produce synchronous cultures, the duration of this synchrony is variable, and can be improved by combination with the gelatin concentration technique. This combination of techniques, although slightly more cumbersome than either technique alone, allows one to obtain a high degree of synchrony over a longer time period. To the best of our knowledge, there are no previous stage-specific studies of the carbohydrate metabolism of Plasmodium falciparum, although Scheibel er al. (1979a) have clearly demonstrated lactate production by P. falciparum in vitro. Shakespeare et al. (1979) examined stage-specific lactate production by P. knowlesi and found an increase associated with the late trophozoite stage. The increased lactate production we observed associated with the schizont stage of P. falciparum is consistent with the earlier observations in P. knowlesi (Shakespeare et al. 1979). However, in both cases, the data may reflect a relatively constant lactate production (per unit of parasite mass) as the parasite enlarges during its maturation from the ring to the trophozoite and schizont stages. In addition, these data do not exclude the possibility that the para-

395

site might cause an increase in the production of lactate by the parasitized red cell. This would contribute to the total lactate measured and might also vary with the size and maturity of the parasite. In conclusion, we have shown that combining the gelatin and sorbitol techniques results in tighter and longer-lasting synchrony than either technique alone. These results demonstrate the use of synchronized cultures in measuring biochemical fluctuations associated with the in vitro erythrocytic cycle of Plasmodium falciparum, and suggest that this approach may also permit definition of the stagespecific (morphologic and biochemical) effects of antimalarial drugs. Finally, the linear relationship we observed between lactate production and parasitemia suggests that lactate production and other similar measures may be useful markers of parasite growth in asynchronous cultures. ACKNOWLEDGMENTS We thank Richard Kahn for providing the red cells to peform these studies, George S. Kobayashi for the use of his spectrophotometer, and L. William Scheibel for several helpful discussions. Michael Pfaller was supported in part by Training Grant l-T32-AI07172 from the U.S. National Institute of Allergy and Infectious Diseases, and Phuc Nguyen-Dinh in part by a grant from the World BanWUNDP/WHO Special Programme for Research and Training in Tropical Diseases. This material was presented in part at the annual meeting of the American Society for Tropical Medicine and Hygiene in Atlanta, Georgia, U.S.A., November, 1980.

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of Tropical

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JENSEN, J. B., AND TRAGER, W. 1977. Plasmodium falciparum in culture: Use of outdated erythrocytes and description of the candle jar method. Journal of Parasitology

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KILEJIAN, A. 1980, Stage-specitic proteins and glycoIdentification proteins of Plasmodium falciparum: of antigens unique to schizonts and merozoites. Proceedings of the National USA 77, 3695-3699.

Academy

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ET AL.

Tetraethylthiuram disulftde (Antabuse) inhibits the human malaria parasite Plasmodium falciparum. Proceedings of the National USA 76, 5303-5307.

Academy

of Sciences

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