Proc. Natl. Acad. Sci. USAVol. 85, pp. 7602-7606, October 1988Cell Biology

pH homeostasis in Leishmania donovani amastigotesand promastigotes

(intracellular pH)

THERESA A. GLASER*, JOHN E. BAATZt, GEORGE P. KREISHMANt, AND ANTONY J. MUKKADA*Departments of *Biological Sciences and tChemistry, University of Cincinnati, Cincinnati, OH 45221

Communicated by William Trager, July 18, 1988

ABSTRACT Intracellular pH and pH gradients of Leish-mania donovani amastigotes and promastigotes were deter-mined over a broad range of extracellular pH values. Intra-cellular pH was determined by 31p NMR and by equilibriumdistribution studies with 5,5-dimethyloxazolidine-2,4-dione ormethylamine. Promastigotes maintain intracellular pH valuesclose to neutral between extracellular pH values of 5.0 and 7.4.Amastigote intracellular pH is maintained close to neutral atexternal pH values as low as 4.0. Both life stages maintain apositive pH gradient to an extracellular pH of 7.4, which isimportant for active transport of substrates. Treatment withionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclo-hexylcarbodiimide, reduced pH gradients in both stages.Maintenance of intracellular pH in the physiologic range isespecially relevant for the survival ofthe amastigote in its acidicin vivo environment.

Tissue forms (amastigotes) of Leishmania develop and pro-liferate in the acidic niche (pH 4.5-5.5) of phagolysosomes ofmammalian macrophages. This raises several questionsabout the adaptations of the parasite that enable it to survivewithin the acidic in vivo environment. Two questions seemrelevant: (i) Can the organism maintain a physiologicalintracellular pH (pH,) within the acidic surroundings? (ii) Canthe organism use the protons abundantly available in theenvironment for its own benefit? Answers to these questionswill bear importantly on the biology ofthese parasites, whichcause misery for >11 million victims worldwide. Recently wedemonstrated that amastigotes show an acid pH optimum forthe incorporation and catabolism of substrates (1). Subse-quently we found that this hydrogen ion effect is manifestedat the level of membrane transport. Active transport ofsubstrates by promastigotes is driven by an electrochemicalgradient of protons (protonmotive force; PMF) across theparasite membrane (2, 3). A transmembrane pH gradient(,ApH) is one contributing factor of the protonmotive force,and the other is the membrane potential (Aq). Protonmotiveforce is expressed by the equation:

PMF = Ale - 59 ApH,

where 59 is a factor for the conversion of ApH to mV and isequal to (2.3 RT)/F; R, T, and F are the gas constant,absolute temperature, and the Faraday constant, respec-tively (4). Knowledge of the pHi enables the calculation ofApH and ultimately the magnitude of the driving force foractive transport. Such knowledge also provides insight intothe homeostatic mechanisms of these parasites, which en-counter substantial changes of pH in their extracellularenvironments.

Here we report pH, values for Leishmania donovani overa broad range of extracellular pH values (pHe). PromastigotepHi was determined by two methods: the equilibrium distri-bution of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione(DMO) and the chemical shift of intracellular inorganicphosphate as determined by 31P NMR spectrometry. Thehigh density of cells required for NMR prohibited the use ofthis technique for amastigote pHi determination; therefore,amastigote pH1 values were determined by only the DMOmethod.

MATERIALS AND METHODSMaintenance and Preparation of Amastigotes. Amastigotes

of L. donovani Sudan strain IS were maintained by serialpassage in female Syrian golden hamsters. The organism waspassaged by i.p. injection of spleen homogenates containing1 x 107 parasites. Hamsters were sacrificed 8 weeks postin-

fection, and parasites were isolated from liver and spleens by adescribed method (5).Maintenance and Preparation of Promastigotes. Promasti-

gotes of L. donovani IS were maintained at 260C in medium199 (GIBCO) supplemented with 15% fetal bovine serum.Cells were harvested during stationary phase growth bycentrifugation at 1100 x g for 10 min and washed twice in abasal salts solution (6).

Determination of pH, by DMO or Methylamine. The pHi ofL. donovani promastigotes and amastigotes was determinedfrom the distribution of the weak acid DMO or the weak basemethylamine as described by Rottenberg (7), a method basedon the principle that the nondissociated acid is fully perme-able to the membrane but is impermeable in its ionic form.Therefore, as long as the pHi is more alkaline relative to thepHe, the acid will be concentrated by the cell. In cases wherepHi is more acidic than pHe, the amine will be concentrated.Cells were suspended at :e2 mg of cell protein per ml in buffercontaining 10 jM 5,5-dimethyl-[2-14C]oxazolidine-2,4-dione([14C]DMO) (43.2 mCi/mmol; 1 Ci = 37 GBq) or 10 uM[14C]methylamine (49 mCi/mmol) and 3 ACi [3H]water in a1-ml volume. After 2-min incubation at 30'C in a shakingwater bath, three 200-dul samples ofthe reaction mixture weredispensed into microcentrifuge tubes containing 0.1 ml ofsilicone oil. The reaction continued 1 min, and the parasiteswere separated from the medium by centrifugation. (pH,remained constant during incubation from 1-10 min). Fifty-microliter samples of the supernatant were taken, and thebottom of the microcentrifuge tube was cut off to obtain thecell pellet. Both supernatant and pellet were added toseparate tubes containing 1 ml of 1 M HC104 and extractedovernight. After centrifugation, 0.9 ml of supernatant was

Abbreviations: pHi, intracellular pH value; pH,, extracellular pHvalues; ApH, transmembrane pH gradient; DMO, 5,5-dimethyloxa-zolidine-2,4-dione; DCCD, dicyclohexylcarbodiimide; CCCP, car-bonyl cyanide m-chlorophenylhydrazone; [14C]DMO, 5,5-dimethyl-[2-14C]oxazolidine-2,4-dione.

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withdrawn and counted in 10 ml of Aquasol (New EnglandNuclear) in a Packard liquid scintillation spectrometer (Tri-Carb model 460C) programmed for dual-channel counting.

Parallel experiments were performed to determine thepercentage of extracellular water in cell pellets. One milliliterof cells in buffer was added to 1 mCi of [3H]water and 15 mM[14C]inulin and were treated as described above for pH1determination after the 2-min incubation.

Intracellular pH was calculated from the following equa-tion:

pH1 = log [DMOin (10PK + l0PHe) - 10PK]

for DMO experiments or:

- ApH = log ( methylaminei,~methylamineout

for methylamine determinations (7).In experiments where cells were treated with ionophores or

inhibitors, the substance was added 10 min before theaddition ofDMO. These agents were used at concentrationsthat did not affect cell viability.Three buffers were used depending on the external pH

being tested: for pHe from 4.0 to 6.0, 125 mM Tris citrate;from 6.0 to 7.4, basal salts; and from 7.4 to 8.0, 150 mM Trisbase with 67 mM citric acid added to maintain osmolarity.

Determination of pHi by NMR. 31P NMR spectra wereobtained from a Nicolet NT 300FT NMR spectrometer at121.6 MHz with a probe temperature of 12°C and a 10-mmNMR tube modified to allow aeration of the cell suspension.A 900 pulse was applied to the sample at a repetition rate of2 sec. The spectra were signal-averaged over 256 scans. Thechemical shift values of the intracellular inorganic phosphate(Ps) were measured relative to the chemical shift of theinternal standard glycerophosphorylcholine (0.49 ppm).pH1 was calculated using the equation

pHi = PKa + log (OA -,10)

where So is the observed chemical shift of the Pi, 6A is thelimiting chemical shift of Pi in the H2PO state and 6B isthe limiting chemical shift of the HPO2- state of Pi. Valuesfor pKa, 8A, and 8B were determined by the curve resultingfrom the NMR titration of L. donovani lysates.pHe was determined before and after each experiment with

a combined glass electrode. Cells were periodically checkedfor viability as judged by activity under a phase-contrastmicroscope. The buffers used for the lower and upper pHranges were the same as those described for DMO determi-nation. 2-(N-Morpholino)ethanesulfonic acid (193 mM) wasused for the middle range instead of basal salts becauseextracellular phosphate would have interfered with NMRdeterminations.

RESULTSEffect of PHe on pHi and ApH (DMO or Methylamine). As

stated earlier, pH, was monitored by the accumulation ofDMO or methylamine. DMO was concentrated for nearly allpHe values examined. Methylamine was used to measure pHifor those pHe values in which the ratio of [DM0]in/[DMO].u,was 1. As shown in Fig. 1, promastigotes maintain a pH,within physiological range (-6.8-7.4) between the pHr val-

8.01 AMASTIGOTE

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6.0 -

pHi8.0

7.5

7.0 -

6.5 -

6.0

......*......I.

.../. ../.../.

I..

PROM AST IGOTE

0

/

...

:.... /

/

/

/

I , I F

4 5 6 7 8pHe

FIG. 1. Intracellular pH in L. donovani promastigotes and amas-tigotes, as determined by the uptake of ['4C]DMO at different pHevalues. The slashed line is a reference curve indicating pHi = pH.Gray area designates the pH range 6.8-7.4.

ues of 5.0-7.4. At pHe <5.0, pHi rapidly dropped belowphysiological pH and at 4.5, the lowest pHe tested, pHi was-6.0. Measurements of pHi below pHe 4.0 were not at-tempted because the pK of DMO (6.3 at 30°C) makes itsusefulness below this point questionable.Up to a pHe of 7.4, DMO was concentrated from the

medium by promastigotes, showing that the pHi is higher thanthat of the medium. At pHe 7.4, the ratio of [DMO]in to[DMO]ut was equal to 1 and at pHe 7.4, the concentrationratio was reversed (0.93), indicating that the pHi is moreacidic than that of the medium. DMO determinations of pH,above this pH0 would be less meaningful because the chem-ical will not accumulate in the intracellular space that is moreacidic than the outside. Methylamine determinations at pHe7.62 and 8.0 yielded pHi values 7.10 and 7.59, respectively.Amastigotes maintained pHi within physiological range

between the extracellular pH values of4.0 and 7.3. At pH0 7.3the concentration ratio ofDMO was still >1 (1.26) yielding apHi value of 7.4, but at pHe 7.4 and above, determination ofpHi was unsuccessful because the cells rapidly swelled andwould not spin through the silicone oil.

Fig. 2 shows the corresponding curves for ApH. The pHgradient of promastigotes was greatest at pH0 5.0 (2.0).Promastigotes maintain a positive ApH up to pH0 7.4. At 7.4,ApH is reduced to 0 and above 7.4, ApH is negative (- 0.03).The proton gradient across amastigote membranes wasgreatest at pH0 4.0 (2.8) and gradually declined to 0.1 at pH07.3.

Effect ofpHe on pH, and ApH (NMR). 31P NMR can be usedto estimate pHi because the chemical shift of Pi and its NMRis a function of pH (8, 9). The high cell density required todetect intracellular inorganic phosphate byNMR (1.5 g ofwetweight) made this technique impractical with amastigotes.Typically, spleens and livers from three infected hamsters

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8.0 1AMASTIGOTE

75.

,OHi 7.0

0

6.5 -

0

6.0-

PROM AST IGOTE

2.0-

tApH 1.0

0.0-

NMR

*//

....... ..: .....: ......:l:... :~ ~~~. ..:: .::: :: :.~~~~~~~~~~~~~~~~~~~~~~~~~~~~...:: ...l...

.............................../.

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FIG. 2. pH gradient in L. donovani promastigotes and amasti-gotes using the DMO method. ApH was calculated from the data inFig. 1 according to the equation ApH = pH, - pHe. Points below theslashed line represent negative ApH.

yield <0.3 g (wet weight) of purified parasites. We thereforeused NMR solely for promastigote pHi determinations.At pH, values between 5.1 and 7.3, pHi was within

physiological range (Fig. 3). At pH, values >7.6, pH, wasapproximately equal to pHe. Below 5.0, pH, declined rapidlybelow neutral. At the lowest PHe examined (3.85), the pH,was 5.85.The ApH was greatest at the pH, 4.28 (2.02) and gradually

declined to close to 0 at the pHe 7.31 (Fig. 3). At pHe 7.84 andgreater, the ApH was a negative value. This is consistent withthe results from DM0 and methylamine experiments.

Treatments That Affect ApH. The pH gradient was reducedto negative value in promastigotes that were incubated in theabsence of substrate for 1 hr at pH 7.0 (Table 1). Thoseincubated in the presence of 10 mM glucose for 1 hr showeda ApH value 3-fold that of cells tested at time 0.

Nigericin (1 uM) diminished the ApH in promastigotes by=50% (Table 2). Carbonyl cyanide m-chlorophenylhydra-zone (CCCP) (5 ,uM) treatment reduced the pHi of promas-tigotes by 0.4 units as did dicyclohexylcarbodiimide (DCCD).Similar effects were noted in amastigotes. Nigericin (2 ,LM)lowered pHi by 0.25 unit. pH; of amastigotes with 10 uM

Table 1. Effect of glucose on pH; and pH gradient inL. donovani promastigotes

pHe Treatment pHi ApH

7.00 None 7.10 +0.107.00 1-hr incubation

(no substrate) 6.91 -0.097.00 1-hr incubation

(+ 10 mM glucose) 7.34 +0.34

Intracellular pH was determined by the DMO method. ApH = pH;- pHe.

7 8

pHe

FIG. 3. pHi (Upper) and pH gradient (Lower), as determined by31PNMR in L. donovani promastigotes. (Upper) The slashed line andgray area have the same meanings as in Fig. 1. (Lower) Points belowthe slashed line represent negative ApH.

CCCP was decreased by 0.5 pH unit. DCCD (20 /itM) causeda reduction in pH1 by 0.3 unit.

DISCUSSION

The protozoan parasite, L. donovani, encounters vastlydifferent conditions of hydrogen ion concentration during itslife cycle. The promastigote is carried within the gut of thephlebotomine sandfly, which introduces the parasite into amammalian host during a "bloodmeal. " Macrophages of thereticuloendothelial system engulf the promastigote, and theprotozoan transforms to the amastigote and proliferateswithin the phagolysosomal vesicle.

Table 2. Effect of inhibitors of substrate transport on pH, andpH gradient, as determined by DMO in L. donovanipromastigotes and amastigotes

pHr Treatment pHi ApHPromastigotes

6.80 None 7.13 +0.336.80 Nigericin (1 /AM) 6.94 +0.146.80 CCCP (5 1AM) 6.72 -0.086.80 DCCD (10 /LM) 6.76 -0.04

Amastigotes6.0 None 7.25 + 1.256.0 Nigericin (2 juM) 7.00 +1.006.2 None 7.2 +1.006.2 CCCP (10 ,tM) 6.7 +0.56.0 None 7.3 +1.36.0 DCCD (20 /LM) 6.98 +0.98

Promastigotes and amastigotes were suspended at pH valuescloser to their optima.

0

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The recent observation that Leishmania promastigotes usean electrochemical gradient of protons to drive the activetransport of substrates (2, 3) has given new significance to theextremes of pH encountered by the parasite. The highlyacidic environment within the phagolysosome can now beviewed as making a large contribution to the force that drivesthe uphill transport of nutrients into the parasite. Thecontribution made by extracellular hydrogen ions to theprotonmotive force is determined by the difference betweenpHe and pHi. The present study was therefore initiated todetermine pH, values for amastigotes and promastigotes overa broad range ofpH. values and in the presence ofionophoresand inhibitors known to influence substrate transport in bothlife stages of the parasite.

31PNMR of intracellular inorganic phosphate has been usedto determine the pHi of Escherichia coli (10), tumor cells (11),yeast (12), and erythrocytes (13). Although few studies havedirectly compared the values obtained with 31P NMR withother methods, a few indirect comparisons indicate that thereis close agreement with values obtained with microelectrodesand [14C]DMO (14). NMR offers the advantage of determiningpHi in a noninvasive manner. The pK of inorganic phosphatein our system (6.8) makes the chemical shift of Pi a usefulindicator of pH, values between 5.0 and 9.0. In view of thefrequent criticisms of NMR, the cell suspensions used in ourNMR studies were heavily buffered and were maintainedunder fully aerobic conditions to reflect near-normal physio-logic conditions. The equilibrium distribution of the radiola-beled weak acid DMO is probably the most widely usedmethod to determine pHi. It has been used for organelles,single cells, tissues, organs and whole-body studies (7, 14).Direct comparisons of this method to spectroscopic andmicroelectrode methods show close agreement (14). The pK ofDMO (6.3 at 30'C) makes it a useful probe for determining pHiover apHe range of 4.0 to 8.0. Where experiments withDMOindicated that pHi was less than or equal to pHe, the radiola-beled weak base methylamine was used. Methylamine yieldedsomewhat lower pHi values than either NMR or DMO.Several factors might explain this disparity. (i) The cellrepresents a nonhomogeneous space, consisting of intracellu-lar compartments with different pH values. DMO is accumu-lated into the more-alkaline compartments, such as the mito-chondria, whereas methylamine is taken up to a greater extentby acidic compartments. The cytoplasmic pH ofthe cell wouldactually fall between the apparent values obtained with DMOand methylamine (14). (ii) The very high pK of methylamine(11 at 25°C) may reduce the accuracy ofthe pH values obtainedin our system. (iii) It is possible that methylamine is activelytransported by Leishmania as has been reported for certainyeasts and bacteria (15). If this were the case, methylaminewould not be useful.Although results with NMR and DMO closely agree, two

sources of error exist: (i) NMR measures the pH of the bulkintracellular fluid because the highest concentration of inor-ganic phosphate is contained here. DMO provides an averagepH of the entire intracellular content. Because mitochondriagenerally have a pHi somewhat higher than the cytoplasm,accumulation of DMO by the mitochondria could shift thepHi to a more alkaline value. (ii) NMR and DMO determi-nations were performed at 12°C and 30°C, respectively. Thistemperature difference may introduce some error becausepH decreases with increasing temperature in certain systems(13).

Results from both NMR and DMO indicate that L. dono-vani promastigotes maintain a pH, close to neutral and apositive ApH between the pHe values of 5.0 and 7.4.Maintenance of a pHi that is more alkaline than the environ-ment is a feature shared by bacteria (16), yeast (17), andmitochondria (7), in which protonmotive force drives anumber of physiological functions. At pHr >7.5, the pro-

mastigote apparently loses its ability to maintain physiolog-ical pH,, and pHi nearly equals pH, With ApH equal to zero,at pH values >7.5, processes dependent upon the magnitudeof PMF are likely to suffer. In fact, the cells swell and diewithin 2-3 hr in basal salts adjusted to alkaline pH.

This study points out striking differences in pHi ho-meostasis between amastigotes and promastigotes. Promas-tigotes rapidly lose the ability to maintain a pHi withinphysiological range below the pH. of 5.0. However, amas-tigotes maintain a pHi close to neutral at the pHr of 4.0, thelower limit of accuracy for measuring pHi with DMO. Also,attempts to measure the pHi of amastigotes above pH, of 7.4were unsuccessful because cells rapidly lysed and would notspin through the silicone oil. These differences in the behav-ior of promastigotes and amastigotes probably reflect adap-tive mechanisms of the latter to survive within the highlyacidic environment of macrophage phagolysosomes. Both pro-mastigotes and amastigotes were able to maintain a positiveApH at all pH, values examined up to 7.4.

Promastigotes incubated for 1 hr in the presence andabsence ofglucose showed dramatically different ApH values(+ 0.3 and - 0.09, respectively), indicating that maintenanceofpH, is an energy-requiring process. Preincubation with theATPase inhibitor DCCD acidified the cytoplasm in bothstages, further suggesting the participation of proton-extrud-ing ATPase in pHi maintenance. Virtually all microbial cellsand organelles that use protonmotive force contain a DCCD-sensitive proton pump on the surface membrane (18).

Several ionophores that inhibit substrate transport in bothstages of the parasite were examined for their effect on cyto-plasmic pH. The uncoupler CCCP shuttles protons acrossbiological membranes, dissipating a pH gradient (19). Theantibiotic nigericin diminishes the proton gradient through adifferent mechanism. It exchanges a proton for a potassium ion,thus reducingApH without affecting the electrical balance ofthecell (20). Both compounds lowered ApH in amastigotes andpromastigotes at low concentrations.

In line with observations reported for the acidophilicbacterium Coxiella burnetii (4), protonophores, and ATPaseinhibitors reduced ApH in amastigotes, but did not abolish it.This fact suggests that mechanisms in addition to thoseinhibited by DCCD or CCCP may also be involved in con-trolling homeostasis of pHi. The pHi of acidophilic bacteriagenerally ranges from 6.5 to 7.0, whereas that of neutrophilesand yeast exhibits a range of 7.5 to 8.0 (21). That Leishmaniaresembles the acidophiles more closely in this respect is notsurprising in light of its in vivo habitat within the acidicphagolysosomes of infected macrophages. The significance ofthese observations is that membrane potential will most likelyprove the major contributing force to the protonmotive forcefor promastigotes (which grow optimally at neutral pH),whereas the pH gradient can be expected to play the larger rolein substrate transport in the acidophilic amastigotes. Becausemost cellular events in growth and development beginningwith DNA replication require a physiological pH, it is espe-cially important for the amastigotes, developing within anacidic environment, to have an effective means for pHhomeostasis. The results show that they do.

We are grateful to Dr. P. F. Bonventre for helpful comments on themanuscript. This work was supported by U.S. Public Health ServiceGrants Al 17444 and GM 3%22 as well as a University of CincinnatiBiomedical Research Support Grant.

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