Leishmania amazonensis:Cultivation and Characterization of Axenic Amastigote-like Organisms

JOBNAME: JEP 83#1 96 PAGE: 1 SESS: 18 OUTPUT: Thu Jun 20 16:27:06 1996/xypage/worksmart/tsp000/70152f/15

Leishmania amazonensis: Cultivation and Characterization of AxenicAmastigote-like Organisms

V. H. HODGKINSON,1 LYNN SOONG, S. MONROEDUBOISE,2 AND DIANE MCMAHON-PRATT

Department of Epidemiology and Public Health, Yale University School of Medicine,New Haven, Connecticut 06520-8034, U.S.A.

HODGKINSON, V. H., SOONG, L., DUBOISE, S. M., AND MCMAHON-PRATT, D. 1996.Leishmaniaamazonensis:Cultivation and characterization of axenic amastigote-like organisms.ExperimentalParasitology83, 94–105. Extracellular amastigote-like forms ofLeishmania amazonensiscan bemaintained in axenic culture at 32°C, pH 4.6, with a generation time of approximately 17 hr. Thisspecies ofLeishmaniais of particular interest since it has been associated with cutaneous, diffusecutaneous, and mucocutaneous forms of the disease. Immunofluorescence, Western and Northern blotanalyses, and immunoprecipitation have been used to estimate the expression levels of amastigote orpromastigote antigens in axenically cultured amastigotes. In these analyses, monoclonal antibodies(mAbs) specific for either the amastigote (A-1, A-2, P-2, P-4, P-5, P-8) or promastigote (M-2, P-9,and F-4) and a DNA probe that was specific for the amastigote gene encoding the protein reactivewith mAb P-4 have been employed. The amastigote-like organisms were infective for peritoneal andJ774.G8 macrophages and BALB/c mice. While amastigote-like forms maintained at pH 4.6, 32°Ctransformed to promastigotes when transferred to pH 7.3, 24°C, transformation of promastigotes toamastigote-like organisms required a period of growth at pH 4.6 24°C prior to transfer to 32°C. Thisis the first report of the axenic cultivation ofL. amazonensisamastigote-like organisms. This speciesgrows in continual culture at a lower pH than any other species characterized to date. These organ-isms will prove useful in further studies of the biochemistry, immunology, developmental biology,and molecular biology of this parasite.© 1996 Academic Press, Inc.

INDEX DESCRIPTORS ANDABBREVIATIONS: Leishmania amazonensis;protozoa, parasitic; axenicamastigote; immunofluorescence; Western blot; Northern blot; morphology; FCS, fetal calf serum;PBS, phosphate-buffered saline; IgG, immunoglobulin G; bp, base pair; mRNA, messenger ribo-nucleic acid; kDa, kilodalton; gp, glycoprotein; SDS–PAGE, sodium dodecyl sulfate–polyacrylamidegel electrophoresis; mAb, monoclonal antibody; IMDM, Iscove’s modified Dulbecco medium.

INTRODUCTION

Leishmaniaspp. have been identified in allcontinents except Australia and Antartica (Pe-ters and Killick-Kendrick 1987) and present awide range of clinical disease manifestations.Although with any species ofLeishmania,thehost immune response influences the course ofthe disease (Sher and Coffman 1992),L. ama-zonensisinfection is associated with the devas-tating diffuse cutaneous leishmaniasis and has

also been reported to be a causative agent ofmucocutaneous and cutaneous leishmaniasis. Itis thus critical thatLeishmaniacontrol measuresbe effective against this species.It is the intracellular, amastigote stage of

Leishmaniaspp. that is associated with the ver-tebrate pathology. Survival of this stage withinphagolysosomes was first demonstrated by Al-exander and Vickerman (1975) forL. mexicanaand by Chang and Dwyer (1976) forL. dono-vani. The promastigote is the flagellated stagethat exists in the insect vector and grows inaxenic culture at 24°C, pH 7.3. Successfullycombating the pathology that is produced byinfection is dependent upon elucidating and un-derstanding the characteristics of the amastigotestage. Until recently this has been hampered bythe dependence on parasites obtained from in-

1 To whom correspondence should be addressedat Biology Department, Fairfield University, Fair-f ie ld, CT 06430. Fax: (203)254-4253. E-mai l :[email protected].

2 Present address: Harvard Medical School, Departmentof Microbiology, New England Regional Primate ResearchCenter, 1 Pinehill Drive, P. O. Box 9102, Southborough,MA 01772.

EXPERIMENTAL PARASITOLOGY83, 94–105 (1996)ARTICLE NO. 0053

940014-4894/96 $18.00Copyright © 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.


fected animals. SeveralLeishmaniaspecieshave been cultured axenically as amastigote-like organisms (Bates 1994; Castillaet al.1995;Eperon and McMahon-Pratt 1989a; Joshiet al.1993; Pan 1984; Pralet al.1993; for review seePanet al. 1993), but there has been extensivevariation in the number and type of criteria thathave been used to justify the designation “amas-tigote-like,” the culture conditions employed,and the reporting of inoculated culture mediumpH. Studies have indicated that parasite cultureconditions vary for each species and that strin-gent evaluation of each extracellularly culturedamastigote strain is critical.Morphology and reproductive rate differ-

ences between the promastigote and amastigotecan be used to identify potential amastigote-likepopulations, but documentation of additionalamastigote characteristics and the absence ofpromastigote characteristics is necessary to es-tablish the relationship of axenic culture amas-tigotes to amastigotes reproducing in the verte-brate host. Stage-specific mAbs have provided aparticularly sensitive tool for identifying mul-tiple amastigote and promastigote specific mol-ecules (Eperon and McMahon-Pratt 1989b;Jaffe and Rachamin 1989; Pan and McMahon-Pratt 1988), thus expanding the characterizationof axenic amastigote-like organisms beyondmorphological or single enzymatic analyses.Using a DNA probe and mAbs that were spe-cific for amastigote or promastigote molecules,we have analyzed molecular and immunologi-cal characteristics ofL. amazonensisamasti-gote-like organisms. The present report docu-ments for the first time the production of amas-tigote molecules byL. amazonensisthat can bemaintained continually as amastigote-like or-ganisms in axenic culture. Each one of thesemolecules represents a potential vaccine candi-date and/or a target for parasite control.

MATERIALS AND METHODS

Parasite cultures.The strain ofL. amazonensis(MHOM/BR/77/LTB0016, obtained originally from Dr. P. Marsden)was cultured as promastigotes at 24°C in Schneider’sDro-sophila medium, pH 7.3 (GIBCO, Grand Island, NY),supplemented with 20% FCS (heat inactivated at 56°C for75 min) and gentamicin (50mg/ml). The same medium was

used for culturing amastigotes at 32°C except that the pHwas adjusted to 4.6 using HCl and gentamicin was presentat either 12.5 or 25mg/ml. Promastigotes recently trans-formed from lesion-derived amastigotes were used as thestarting population for adaptation to axenic amastigotes.The feasibility of beginning with potentially attenuatedlong-term culture promastigotes has not been investigated.Log phase cultures were used for experimental purposes.Fetal calf serum was screened prior to use to establish suit-ability for growth and maintenance ofL. amazonensis.Complete medium was stored at 4°C for no longer than 2–4weeks. In order to maintain amastigote morphology in>95% of the organisms it was necessary to passage thesecultures once they reached a concentration of 1 × 107–2 ×107/ml. Unless indicated otherwise, all results presentedwere from amastigotes growing at 32°C, pH 4.6.Amastigote infectivity was monitored and maintained by

periodic infection of J774.G8 macrophage cell line (Chang1980b; Eperon and McMahon-Pratt 1989a), BALB/c peri-toneal macrophages, and/or subcutaneous injection ofBALB/c mice in the foot (5 × 105–5 × 106 axenic amasti-gotes per mouse). Macrophages were collected followingintraperitoneal injection of 5 ml of 3% FCS-PBS andwashed. Cells were seeded in 8-well Lab-Tek ChamberSlides (Nunc, Inc., Naperville, IL) and incubated for 3 hr at37°C in 5% CO2 in IMDM (GIBCO) supplemented with10% FCS, 5 × 10−5M 2-mercaptoethanol (Sigma ChemicalCo., St. Louis, MO), 50mg/ml gentamicin, 100 U/ml peni-cillin, and 100mg/ml streptomycin sulfate. After 3 hr, cul-tures were inoculated with axenic amastigotes at a 5:1 ratioat 32°C. Free parasites were removed by washing after 5–6hr of incubation. Infected cultures were incubated at 37°C.Chamber slides were washed, air dried, fixed with metha-nol, and stained with Giemsa (Sigma Chemical Co.).Growth kinetics.Complete Schneider’s medium was in-

oculated with either promastigotes or amastigotes (10 ml/T25 flask, in triplicate) at an initial concentration of 4 ×105/ml. Viability was determined from motility for promas-tigotes and with 0.4% erythrosin B in PBS for amastigotes(Hodgkinsonet al. 1980). The density of the cultures wasdetermined from hemocytometer counts and the pH of themedium was determined after the organisms were removedby centrifugation at 8160g.Monoclonal antibodies.Preparation of the following

mAbs has been reported previously: P-2, P-4, P-5, P-8, P-9(Pan and McMahon-Pratt 1988), M-2 (Kahl and McMahon-Pratt 1987), A-2 (Traub-Csekoet al.1993), and F-4 (Ismachet al.1989). Table I summarizes the characterization of themolecules recognized by these antibodies. The mAb A-1(C3A1A8) was produced as previously described (Pan andMcMahon-Pratt 1988) by the fusion of NS-1 mouse plas-macytoma cells with the spleen cells of a BALB/c mouse,immunized with a membrane preparation ofL. amazonensislesion-derived amastigotes. The supernatant from culturesof NS-1 cells (the parental myeloma cell line) was em-ployed as the negative control.Immunofluorescence.Organisms were washed with 5%

Leishmania amazonensis:AXENIC AMASTIGOTE-LIKE ORGANISMS 95


FCS in PBS (pH 7.0 for promastigotes, pH 4.6 for amasti-gotes) and checked for viability using erythrosin B. Para-sites were then dispensed on 12-well slides (Cel-Line As-sociates, Newfield, NJ), rapidly air-dried, and evaluatedwithin 2–3 days. Organisms that were to be assayed withmAb M-2 were fixed with absolute methanol for 5 min priorto incubation with the antibody. The reactions with all othermAbs were assessed on air-dried, unfixed organisms. In allexperiments, cells were preincubated for 1 hr at 37°C with5% FCS in PBS, pH 7.0 (also used for subsequent wash-ings). Cells were incubated with dilutions of specific mAbsfor 45 min at 24°C, washed, incubated with dilutions ofrhodamine-labeled anti-mouse IgG (Jackson Immuno-Research Lab, West Grove, PA) for 45 min at 24°C,washed, and mounted in mowiol (Calbiochem Corp,LaJolla, CA; Heimer and Taylor 1974). Slides were exam-ined with a Leitz Orthoplan microscope (Wetzler, Ger-many). A minimum of 200 organisms were evaluated.Negative controls were incubated with NS-1 culture super-natant and/or the second antibody alone.Western blot analysis.Proteins were electrophoretically

transferred from 12% SDS–PAGE gels (Laemmli 1970) tonitrocellulose (0.45mm) or Immobilon-P (0.45mm, Milli-pore Corp., Bedford, MA) and visualized according to themethod of Towbinet al. (1979) as modified by Pan andMcMahon-Pratt (1988). Alternatively, antigens were visu-alized using a modification of the procedure described by

Ey and Ashman (1986). Strips were incubated for 2 hr at24°C in anti-mouse IgG (1:1000) conjugated to alkalinephosphatase (Bio-Rad Laboratories, Hercules, CA), fol-lowed by a 5- to 10-min incubation in 150 mM veronalbuffer, pH 9.5. The protein bands were developed using analkaline phosphatase substrate (BCIP/NBT, Kirkegaard andPerry Laboratories Inc., Gaithersburg, MD) and the reactionwas stopped by rinsing in distilled water.Northern blot analysis.Total RNA was isolated from 2 ×

107 promastigotes or axenic amastigotes using a MicroRNA isolation kit (Stratagene, La Jolla, CA). RNA samples(20mg per lane) were loaded onto a 1.2% agarose gel con-taining 1.1M formaldehyde. Electrophoresis, transfer, andhybridization were carried out as described previously(Campos-Netoet al.1995). The membrane was probed witha 32P-labeled 540-bp DNA fragment that was specific for agene encoding the protein recognized by mAb P-4 (Soongetal., unpublished data). To verify the concentration ofmRNA loaded in each lane, the same membrane wasstripped and reprobed with a32P-labeled Ld p23 peptide/gene, 640-bp DNA fragment that recognizes a gene encod-ing a surface protein that is expressed in both amastigotesand promastigotes and conserved among differentLeish-maniaspecies (Campos-Netoet al. 1995).Immunoprecipitation.Promastigotes and axenic amasti-

gotes were metabolically radiolabeled with [35S]-methionine (Amersham, Arlington Heights, IL) using me-

TABLE ISummary of Characterized Molecules Associated with Stage-SpecificLeishmaniaEpitopes Recognized by mAbs

Employed in This Study

mAb Stage Species kDa Reference

A-1 Amastigote L. amazonensisa 36, 40 Duboise 1994Promastigote L. amazonensis 36,b 40b Duboise 1994

A-2 Amastigote L. amazonensisa 27 Duboise 1994L. pifanoia,c 27 Panet al. 1993

Duboiseet al. 1994P-2 Amastigote L. pifanoia,c 40, 45 Panet al. 1993

Duboiseet al. 1994P-5 Amastigote L. amazonensisa 36, 40 Duboise 1994

L. pifanoia,c 34, 43 Pan and McMahon-Pratt 1988Promastigote L. amazonensis 19.5, 36,b 40b Duboise 1994

L. pifanoi 34,b 43b Pan and McMahon-Pratt 1988P-8 Amastigote L. amazonensisa 36, 40 Duboise 1994

L. pifanoia,c 34, 43 Pan and McMahon-Pratt 1988Promastigote L. amazonensis 19.5, 36,b 40b Duboise 1994

L. pifanoi 21, 34,b 43b Pan and McMahon-Pratt 1988P-9 Promastigote L. amazonensis >100 Duboise 1994

L. pifanoi >100 Pan and McMahon-Pratt 1988M-2 Promastigote L. amazonensis 46 Kahl and McMahon-Pratt 1987F-4 Promastigote L. amazonensis 70 Ismachet al. 1989

a Lesion and/or J774.G8 macrophage-derived amastigotes.b Significantly reduced in comparison to amastigotes.c Axenic amastigotes.

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thionine-free Schneider’s medium supplemented with dia-lyzed heat-inactivated FCS. The radiolabeled cells wereimmunoprecipitated using ascitic fluid containing mAb A-2and processed as previously described (Kahl and McMa-hon-Pratt 1987) except that protein G, 4 fast flow resin(Pharmacia LKB, Uppsala, Sweden) was employed.

RESULTS

Culture. During the adaptation process, in-creased expression of amastigote-specific anti-gens recognized by mAbs A-1 and A-2 paral-leled inoculation of cultures into increasinglyacidic media. Table II summarizes results frompopulations growing at pH 5.5 to pH 7.0 thatwere amastigote-like as viewed by phase mi-croscopy. In the first three populations the num-ber of flagellated organisms increased with ad-ditional subpassages. Stable amastigote-likepopulations were obtained only at 32°C, pH 4.6.This was achieved by initial temperature incre-ments of 2°C followed by decreases of 0.5 pHunits from pH 7.0 to pH 5.0 and then 0.2 pHunits to pH 4.6.Culture density was always maintained be-

low 2 × 107/ml since during the adaptation pro-cess, populations of axenic amastigote-like or-ganisms were observed with up to 29% flagel-lated organisms at a density of 3.6 × 107/ml orhigher. This increase in flagellated forms waseliminated if the organisms were centrifugedand resuspended in fresh medium, suggesting astringent nutrient requirement for the mainte-nance of amastigotes. Amastigote-like popula-tions are presently being maintained that exhibit

no more than 7% flagellated forms at concen-trations as high as 8 × 107/ml.Cultures proved sensitive to gentamicin at 50

mg/ml as previously reported for axenic amas-tigotes ofL. braziliensisandL. panamensis(Ep-eron and McMahon-Pratt 1989a), but 12.5mg/ml was never inhibitory and some populationswere maintained at 25mg/ml. The ability ofaxenic amastigotes to transform into promasti-gotes was assessed by periodic transfer to 24°C,pH 7.3. Amastigote cultures were reestablishedby shifting the promastigote culture back to32°C, pH 4.6 following at least one passage at24°C, pH 4.6. To date, it has not been possibleto maintain organisms isolated directly from alesion as axenic amastigotes.Although Bates (1994) described induction

of metacyclic forms following a decrease in pHof L. mexicanacultures, morphological metacy-clic forms were not identified in pH 4.6, 24°Ccultures ofL. amazonensis(strain MHOM/BR/77/LTB0016). In addition, while low pH sta-tionary phase metacyclic cultures ofL. mexi-cana converted to amastigote-like organismswhen shifted to a higher temperature (Bates1994), a greater percentage of morphologicallyheterogeneous log phase, not stationary phaseL. amazonensisorganisms, survived the in-crease to 32°C and converted to amastigotes.Recently, this laboratory obtained axenic amas-tigotes by shifting a previously adapted culturedirectly from pH7, 24°C to pH 4.6, 32°C; how-ever these organisms were not used in the pre-sent study. The new serum lot appears to beresponsible for this, emphasizing the impor-tance of possible growth and nutritional factorsin this transformation process.Since the first successful report of continuous

culture of axenic amastigotes employed mediacontaining hemin (Pan 1984) this componentwas included in the initial adaptation stages us-ing L. amazonensis.However, no difference inparasite growth rate or the percentage of non-flagellated forms was detected in medium withor without hemin. In an attempt to provide acomponent from the host cell that might be re-quired to attain and maintain amastigote mor-phology, extracts of J774.G8 macrophages were

TABLE IIImmunofluorescence of Nonflagellated Organisms

Growing at 32°C in Media pH 4.6–7.0, Detected by Reac-tion with Amastigote-Specific mAbs A-1 and A-2

pH(No. of passages)

mAb

A-1a A-2a

7.0 (13) 34 26.0 (3) 32 175.5 (1) 50 184.6 (6) 85 92

a Percentage of fluorescent organisms based on the evalu-ation of at least 200 cells.



added toL. amazonensiscultures, but no effectof macrophage extracts was observed.Morphology and growth.As shown in Fig. 1,

the morphology ofL. amazonensisaxenic amas-tigotes was clearly distinct from the promasti-gotes. The growth of axenic amastigotes asround doublets and short chains was observedby phase microscopy and has also been reportedas an amastigote characteristic for other species(Eperon and McMahon-Pratt 1989a).In addition to morphology, the growth curve

of axenic amastigotes was distinct from that ofpromastigotes. Log phase promastigotes exhib-ited a generation time of 8 hr. while the genera-tion time for log phase axenic amastigotes was17 hr. During the late log phase of promastigotegrowth the pH of the culture medium decreasedwhile in comparable amastigote cultures the pHwas increasing (Fig. 2). The increase in pH dur-ing growth has not been reported for other ax-enic amastigotes, but has been described for theprotistsNaegleria (Weik and John 1977) andTetrahymena(Seaman 1955), growing at pH6.5–6.7.

Antigenic characterization.The amastigote-specific antigens recognized by mAbs A-1, A-2,P-2, P-4, P-5, and P-8 (Table I) were detectedby indirect immunofluorescence inL. ama-zonensisaxenic amastigotes maintained in con-tinuous culture, but not in promastigotes. On theother hand, mAbs M-2, F-4, and P-9 that rec-ognized promastigote antigens usually exhib-ited no reaction with axenic amastigotes; how-ever, occasionally a small subpopulation wasreactive with mAb P-9. Representative reac-tions are seen in Fig. 3.To confirm the molecular specificity of the

indirect immunofluorescence, Western blotanalysis and immunoprecipitation experimentswere performed (Figs. 4 and 5). All moleculesrecognized by the stage-specific mAbs were inthe molecular weight range of those previouslyidentified inL. amazonensislesion and macro-phage-derived amastigotes andL. pifanoimac-rophage-derived or axenic amastigotes (TableI). In Fig. 4 a broad band at 36 kDa was recog-nized in theL. amazonensisaxenic amastigotepopulation by mAbs A-1, P-5, and P-8. This

FIG. 1. Morphology of Giemsa stainedL. amazonensis.(a) Promastigotes at 24°C, pH 7.3. (b) Axenic amastigote-likeorganisms at 32°C, pH 4.6. Bar, 5mm.

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band was resolved in one experiment usingmAb A-1 into bands at 35 and 37 kDa (resultsnot shown). The same band was absent orbarely visible in promastigotes. Using mAb P-8,two bands in the axenic amastigotes, at 20 and18 kDa were distinct from the 19.5-kDa bandfound in the promastigotes (Fig. 4; Duboise

1994). The previous observation of an addi-tional band at 40–43 kDa with mAbs A-1, P-5,and P-8 in lesion and macrophage-derivedamastigotes (Table I) was interpreted as the re-sult of loading differences. However, differ-ences due to degradation and/or processing byaxenic amastigotes cannot be ruled out.

FIG. 2. L. amazonensisgrowth curve and growth medium pH. Each point represents averaged counts or pH determina-tions from three cultures, initially inoculated with 4 × 105 organisms/ml into Schneider’s medium. (a) Promastigotes wereinoculated at pH 7.3 and incubated at 24°C. (b) Axenic amastigote-like organisms were inoculated at pH 4.6 and incubatedat 32°C.



Intermediate and precursor molecules (41and 45 kDa) of theLpcys2cysteine proteinase(Duboiseet al. 1994) recognized by mAb P-2were present inL. amazonensisaxenic amasti-gotes, but not promastigotes (Fig. 4). This an-

tibody was previously shown to recognize anepitope associated with the COOH-terminal ex-tension of the cysteine proteinase, which wasremoved during intracellular processing (Du-boiseet al. 1994). Further, the 27-kDa mature

FIG. 3. Indirect immunofluorescence ofL. amazonensisaxenic amastigote-like organisms and promastigotes. Axenicamastigotes (a, c, e, and g) or promastigotes (b, d, f, and h) were incubated with amastigote specific mAbs: A-1 (a and b);P-4 (c and d); or P-8 (e and f); or the promastigote specific mAb M-2 (g and h). The second antibody was rhodamineconjugated anti-mouse IgG. Bar, 15mm.

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proteinase was selectively identified inL. ama-zonensisaxenic amastigotes in immunoprecipi-tation experiments using mAb A-2 (Fig. 5).The molecules recognized by mAbs F-4,

M-2, and P-9 were detected by immunofluores-cence in all of the promastigotes examined. Inthe population of axenic amastigotes evaluated

by Western blot analysis in Fig. 4, none of theorganisms appeared to react with mAbs F-4 orM-2 by immunofluorescence, but a small sub-population (<1%) was reactive with mAb P-9.Consequently, it is likely that the very low levelof expression of both the M-2 and P-9 proteinsdetected in the Western blot analysis was due tothis subpopulation.Northern blot analysis of P-4 mRNA.The

protein (35 kDa) recognized by mAb P-4 waspreviously demonstrated inL. amazonensisandL. pifanoi macrophage-derived, lesion amasti-gotes and inL. pifanoiaxenic amastigotes (Du-boise 1994; Panet al. 1993). Since the geneencoding the P-4 protein has been cloned(Soonget al.,unpublished data), it was possibleto compare mRNA expression levels in axeni-cally culturedL. amazonensisamastigote-likeorganisms and promastigotes using Northernblot analysis. As illustrated in Fig. 6A, specificP-4 mRNA of 2.8, 5.6, and 7.0 kb was ex-pressed only in axenic amastigotes, not in pro-mastigotes. To verify the amount of RNAloaded onto the gel (Fig. 6B), the same mem-brane was reprobed with the Ld p23 peptide/gene probe (Campos-Netoet al.1995). Compa-

FIG. 5. Immunoprecipitation by mAb A-2. Autoradio-graphic results ofL. amazonensisamastigote-like organisms(2) and promastigotes (3) metabolically labeled with[35S]methionine, lysed, and precipitated with mAb A-2. Thecontrol (1) was as in Fig. 4.

FIG. 4. Comparison ofL. amazonensisaxenic amastigote-like organisms and promastigotes by Western blot analysis.Transfer of parasite antigens from 12% SDS gels was followed by incubation of membrane strips with mAbs as indicatedat the top of each panel. The culture supernatant from the nonsecreting parental myeloma cell line (NS-1) was used as anegative control. The second antibody was anti-mouse IgG;125I-labeled (mAb A-1, P-2, P-5, P-8, P-9, and M-2) or alkalinephosphatase conjugated (mAb F-4). The lanes for F-4 were enlarged from a mini-gel, resulting in a more diffuse band thanwas in the original. At the bottom of the figure “a” denotes axenic amastigote and “p” denotes promastigote.



rable levels of mRNA (0.7 kb) were expressedin both amastigotes and promastigotes.Infectivity.Axenic amastigote-like organisms

infected peritoneal macrophages from BALB/cmice (Fig. 7) and J774.G8 macrophages. Thelarge parasitophorous vacuoles indicated thatthe parasites were intracellular. In addition, du-plicate cultures of axenic amastigotes were un-able to survive extracellularly without macro-phages. Replication of axenic amastigoteswithin macrophages was indicated by the pres-ence of two nuclei or two kinetoplasts per cell(Fig. 7, arrows). Organisms recovered fromJ774.G8 macrophages after 11 days in culturedifferentiated into promastigotes at pH 7, 24°C.After one passage at pH 4.6, 32°C these pro-mastigotes were capable of transforming backto axenic amastigotes at pH 4.6, 32°C. Lesionsof at least 8 mm were produced in BALB/c micewithin 3 months after the injection of axenicamastigotes. Although a quantitative analysis ofinfectivity was not carried out, the lesions werecomparable in size to those produced by direct

inoculation of nonadaptedL. amazonensisle-sion amastigotes. These results confirmed theobservations fromin vitro macrophage culturesand indicated thein vivo infectivity of the ax-enic organisms.

DISCUSSION

The present report documents the morpho-logical, growth, and antigenic characteristics ofL. amazonensisaxenic amastigotes and furtheremphasizes the need to carefully monitor andevaluate axenic amastigote populations. At thelight microscope levelL. amazonensisaxenicamastigotes clearly were morphologically anamastigote-like, not a promastigote-like popu-lation. However, during the adaptation proce-dures it became clear that in the absence of theappropriate conditions for continuous cultiva-tion, organisms could be morphologically amas-tigote-like and then either become more pro-mastigote-like or fail to survive subsequent sub-passages. Physiological and morphologicalrelevance of data obtained from such popula-

FIG. 6. Northern blot ofL. amazonensispromastigotesand amastigotes. Total RNA was isolated fromL. ama-zonensisamastigote-like organisms (A) or from promasti-gotes (B). Approximately 20mg of total RNA per lane wasused for Northern blot analysis. The membrane was probedwith a 32P-labeled DNA fragment that was specific for theprotein recognized by mAb P-4 (A), stripped, and reprobedwith a labeled DNA fragment that encodes the Ld p23 pep-tide (B) which is expressed in both the promastigote andamastigote stages.

FIG. 7. Infection of BALB/c peritoneal macrophageswith L. amazonensisaxenic amastigote-like organisms. Ad-herent macrophages were infected at a ratio of 5:1 at 32°Cin 5% CO2 and free parasites were removed after 5 hr.Cultures were incubated at 37°C in 5% CO2 for 72 hr. Notethe two kinetoplasts (small arrow) and two nuclei (largearrow) in reproducing organisms. Parasites in duplicate cul-tures without macrophages did not survive. Bar, 5mm.



tions may not be applicable to the amastigotereproducing in the vertebrate host and shouldnot be presented as characteristic of “axenicamastigotes.” As an example, the absence oftrue megasomes inL. amazonensisnoted after 2days at 34°C reported by Leonet al. (1994) wasmost likely due to the fact that these organismswere merely “heat-shocked” and not physi-ologically differentiated amastigotes. Studyingthe differentiation from promastigote to amas-tigote within mouse macrophages, Galvao-Quintaoet al. (1990) found that typical mega-somes were not identified until Day 5 after in-fection. It can be inferred that the developmentof the megasome is a complex differentiationprocess that may not be induced after heat-shock in short-term culture. This is undoubtedlytrue for various aspects of amastigote differen-tiation and development. It would avoid confu-sion if organisms that cannot be maintained asamastigotes were referred to as “heat-shockedamastigote-like” forms, “pH-induced amasti-gote-like” forms, etc. and if reports of amasti-gote-like organisms that can be maintained inaxenic culture included or referenced support-ing characterization.Although an increase in temperature and a

decrease in pH have been used to obtain amas-tigote-like populations of otherLeishmaniaspe-cies (Bates 1994; Joshiet al. 1993; for reviewsee Zilberstein and Shapira 1994), this is thefirst report of continuous cultivation of a speciesat the low pH of 4.6 at 32°C. However, a sub-population of promastigotes ofL. majoradaptedto growth at pH 4.5, 26°C synthesized an amas-tigote protein (reviewed in Zilberstein and Sha-pira 1994). Although the biochemical mecha-nisms responsible for differences amongLeish-maniaspecies are unclear at this point, the lowculture pH requirement of axenic amastigotesmay indicate unique physiologic features ofL.amazonenesis.It may be significant that the pHof the parasitophorous vacuole of macrophagesinfected in vitro with L. amazonensiswas re-ported as pH 4.74 to pH 5.26 (Antoineet al.1990) and was lower forL. amazonensisthanfor L. donovani(Chang 1980a).The conclusion that the population described

in the present report was amastigote-like wasbased on the presence of amastigote character-istics and the absence of promastigote traits. Inan earlier report (Traub-Csekoet al. 1993),Northern blot analysis detected higher concen-trations of mRNA specific for a 27-kDa cyste-ine proteinase inL. amazonensislesion amasti-gotes andL. pifanoi axenic amastigotes than inpromastigotes. This proteinase was identifiedby mAb A-2 and localized to megasomes inamastigotes, but was absent in promastigotes ofboth species (Duboiseet al. 1994). The pres-ence of this proteinase inL. amazonensisaxenicamastigotes and its similarity to theL. pifanoiproteinase were substantiated by immunofluo-rescence and immunoprecipitation analyses us-ing mAb A-2 and by Western blot analysis us-ing mAb P-2.L. amazonensisaxenic amasti-gotes will be valuable in continuing studiesaimed at elucidation of the biological functionof amastigote cysteine proteinases, particularlyas the genes are upregulated during transforma-tion of the promastigote to the amastigote stage.The present results also clearly established

the preferential expression of antigens recog-nized by mAbs A-1, P-4, P-5, and P-8 in axenicL. amazonensisamastigotes as compared withpromastigotes and the differential synthesis ofmRNA specific for the molecules reactive withmAb P-4. The differential antigen expressionwas in agreement with previous immunologicalcomparisons ofL. amazonensisamastigotesfrom lesion or from the J774.G8 macrophagecell line with L. amazonensispromastigotes(Duboise 1994; Pan and McMahon-Pratt 1988).The antigens recognized by mAbs P-4 and P-8have been purified fromL. pifanoiaxenic amas-tigotes and used to induce significant protectionagainst murine infection withL. pifanoi andpartial protection against nonhomologous infec-tion with L. amanzonensis(Soonget al. 1995).The expression of these antigens by axenicL.amazonensisamastigote-like organisms pro-vides readily available homologous material forfuture immunization studies.Verification that theL. amazonensisaxenic

amastigote-like organisms characterized in thepresent study were not simply morphologically



modified promastigotes was further supportedby the absence of the paraxial rod protein rec-ognized by mAb F-4 and the dramatically re-duced expression of the promastigote moleculesreactive with mAbs M-2 and P-9. The promas-tigote gp46 (detected by mAb M-2) has beenused to elicit protective immunity in mice andthe molecules reactive with mAb P-9 wereamong the first to be synthesized during thetransformation of amastigote to promastigote(Champsi and McMahon-Pratt 1988; Duboise1994).L. amazonensisaxenic amastigotes willprovide a system in which to study the regula-tion of these as well as other promastigote mol-ecules.Numerous reports have identified ultrastruc-

tural, biochemical, metabolic and antigeniccharacteristics specific to the amastigote stageof Leishmania(Dell and Engel 1994; Ilget al.1995; Joshiet al. 1993; Medina-Acostaet al.1993; Souzaet al. 1994; Strauset al. 1993;Winter et al.1994; for review see Moody 1993and Panet al. 1993). It will now not only beeasier to study amastigote molecules ofL. ama-zonensisin more detail and in the absence ofcontaminating host components, but also to fur-ther assess the extent and function of host de-rived molecules associated with the amastigote(Schneideret al. 1993; Winteret al. 1994). Inaddition, axenic amastigotes provide an idealsystem for identifying specific amastigote nu-tritional requirements as control targets. Thecontinuous cultivation ofL. amazonensisamas-tigote-like organisms brings us one step closerto a better understanding of the biology ofL.amazonensisamastigotes.

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Received 3 November 1995; accepted with revision 22March 1996


Leishmania amazonensis:Cultivation and Characterization of Axenic Amastigote-like Organisms

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