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

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EXPERIMENTAL PARASITOLOGY ARTICLE NO. 0053

83, 94–105 (1996)

Leishmania amazonensis: Cultivation and Characterization of Axenic Amastigote-like Organisms V. H. HODGKINSON,1 LYNN SOONG, S. MONROE DUBOISE,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. Leishmania amazonensis: Cultivation and characterization of axenic amastigote-like organisms. Experimental Parasitology 83, 94–105. Extracellular amastigote-like forms of Leishmania amazonensis can be maintained in axenic culture at 32°C, pH 4.6, with a generation time of approximately 17 hr. This species of Leishmania is of particular interest since it has been associated with cutaneous, diffuse cutaneous, and mucocutaneous forms of the disease. Immunofluorescence, Western and Northern blot analyses, and immunoprecipitation have been used to estimate the expression levels of amastigote or promastigote 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 reactive with mAb P-4 have been employed. The amastigote-like organisms were infective for peritoneal and J774.G8 macrophages and BALB/c mice. While amastigote-like forms maintained at pH 4.6, 32°C transformed to promastigotes when transferred to pH 7.3, 24°C, transformation of promastigotes to amastigote-like organisms required a period of growth at pH 4.6 24°C prior to transfer to 32°C. This is the first report of the axenic cultivation of L. amazonensis amastigote-like organisms. This species grows in continual culture at a lower pH than any other species characterized to date. These organisms will prove useful in further studies of the biochemistry, immunology, developmental biology, and molecular biology of this parasite. © 1996 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Leishmania amazonensis; protozoa, parasitic; axenic amastigote; immunofluorescence; Western blot; Northern blot; morphology; FCS, fetal calf serum; PBS, phosphate-buffered saline; IgG, immunoglobulin G; bp, base pair; mRNA, messenger ribonucleic acid; kDa, kilodalton; gp, glycoprotein; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; mAb, monoclonal antibody; IMDM, Iscove’s modified Dulbecco medium.

INTRODUCTION

also been reported to be a causative agent of mucocutaneous and cutaneous leishmaniasis. It is thus critical that Leishmania control measures be effective against this species. It is the intracellular, amastigote stage of Leishmania spp. that is associated with the vertebrate pathology. Survival of this stage within phagolysosomes was first demonstrated by Alexander and Vickerman (1975) for L. mexicana and by Chang and Dwyer (1976) for L. donovani. The promastigote is the flagellated stage that exists in the insect vector and grows in axenic culture at 24°C, pH 7.3. Successfully combating the pathology that is produced by infection is dependent upon elucidating and understanding the characteristics of the amastigote stage. Until recently this has been hampered by the dependence on parasites obtained from in-

Leishmania spp. have been identified in all continents except Australia and Antartica (Peters and Killick-Kendrick 1987) and present a wide range of clinical disease manifestations. Although with any species of Leishmania, the host immune response influences the course of the disease (Sher and Coffman 1992), L. amazonensis infection is associated with the devastating diffuse cutaneous leishmaniasis and has 1 To whom correspondence should be addressed at Biology Department, Fairfield University, Fairfield, CT 06430. Fax: (203)254-4253. E-mail: [email protected]. 2 Present address: Harvard Medical School, Department of Microbiology, New England Regional Primate Research Center, 1 Pinehill Drive, P. O. Box 9102, Southborough, MA 01772.

94 0014-4894/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Leishmania amazonensis: AXENIC AMASTIGOTE-LIKE ORGANISMS fected animals. Several Leishmania species have been cultured axenically as amastigotelike organisms (Bates 1994; Castilla et al. 1995; Eperon and McMahon-Pratt 1989a; Joshi et al. 1993; Pan 1984; Pral et al. 1993; for review see Pan et al. 1993), but there has been extensive variation in the number and type of criteria that have been used to justify the designation “amastigote-like,” the culture conditions employed, and the reporting of inoculated culture medium pH. Studies have indicated that parasite culture conditions vary for each species and that stringent evaluation of each extracellularly cultured amastigote strain is critical. Morphology and reproductive rate differences between the promastigote and amastigote can be used to identify potential amastigote-like populations, but documentation of additional amastigote characteristics and the absence of promastigote characteristics is necessary to establish the relationship of axenic culture amastigotes to amastigotes reproducing in the vertebrate host. Stage-specific mAbs have provided a particularly sensitive tool for identifying multiple amastigote and promastigote specific molecules (Eperon and McMahon-Pratt 1989b; Jaffe and Rachamin 1989; Pan and McMahonPratt 1988), thus expanding the characterization of axenic amastigote-like organisms beyond morphological or single enzymatic analyses. Using a DNA probe and mAbs that were specific for amastigote or promastigote molecules, we have analyzed molecular and immunological characteristics of L. amazonensis amastigote-like organisms. The present report documents for the first time the production of amastigote molecules by L. amazonensis that can be maintained continually as amastigote-like organisms in axenic culture. Each one of these molecules represents a potential vaccine candidate and/or a target for parasite control. MATERIALS

AND

METHODS

Parasite cultures. The strain of L. amazonensis (MHOM/ BR/77/LTB0016, obtained originally from Dr. P. Marsden) was cultured as promastigotes at 24°C in Schneider’s Drosophila medium, pH 7.3 (GIBCO, Grand Island, NY), supplemented with 20% FCS (heat inactivated at 56°C for 75 min) and gentamicin (50 mg/ml). The same medium was

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used for culturing amastigotes at 32°C except that the pH was adjusted to 4.6 using HCl and gentamicin was present at either 12.5 or 25 mg/ml. Promastigotes recently transformed from lesion-derived amastigotes were used as the starting population for adaptation to axenic amastigotes. The feasibility of beginning with potentially attenuated long-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 suitability for growth and maintenance of L. amazonensis. Complete medium was stored at 4°C for no longer than 2–4 weeks. In order to maintain amastigote morphology in >95% of the organisms it was necessary to passage these cultures once they reached a concentration of 1 × 107–2 × 107/ml. Unless indicated otherwise, all results presented were from amastigotes growing at 32°C, pH 4.6. Amastigote infectivity was monitored and maintained by periodic infection of J774.G8 macrophage cell line (Chang 1980b; Eperon and McMahon-Pratt 1989a), BALB/c peritoneal macrophages, and/or subcutaneous injection of BALB/c mice in the foot (5 × 105–5 × 106 axenic amastigotes per mouse). Macrophages were collected following intraperitoneal injection of 5 ml of 3% FCS-PBS and washed. Cells were seeded in 8-well Lab-Tek Chamber Slides (Nunc, Inc., Naperville, IL) and incubated for 3 hr at 37°C in 5% CO2 in IMDM (GIBCO) supplemented with 10% FCS, 5 × 10−5 M 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO), 50 mg/ml gentamicin, 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate. After 3 hr, cultures were inoculated with axenic amastigotes at a 5:1 ratio at 32°C. Free parasites were removed by washing after 5–6 hr of incubation. Infected cultures were incubated at 37°C. Chamber slides were washed, air dried, fixed with methanol, and stained with Giemsa (Sigma Chemical Co.). Growth kinetics. Complete Schneider’s medium was inoculated 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 promastigotes and with 0.4% erythrosin B in PBS for amastigotes (Hodgkinson et al. 1980). The density of the cultures was determined from hemocytometer counts and the pH of the medium was determined after the organisms were removed by 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 McMahonPratt 1987), A-2 (Traub-Cseko et al. 1993), and F-4 (Ismach et al. 1989). Table I summarizes the characterization of the molecules recognized by these antibodies. The mAb A-1 (C3A1A8) was produced as previously described (Pan and McMahon-Pratt 1988) by the fusion of NS-1 mouse plasmacytoma cells with the spleen cells of a BALB/c mouse, immunized with a membrane preparation of L. amazonensis lesion-derived amastigotes. The supernatant from cultures of NS-1 cells (the parental myeloma cell line) was employed as the negative control. Immunofluorescence. Organisms were washed with 5%

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HODGKINSON ET AL. TABLE I Summary of Characterized Molecules Associated with Stage-Specific Leishmania Epitopes Recognized by mAbs Employed in This Study

mAb

Stage

A-1 A-2

Amastigote Promastigote Amastigote

L. L. L. L.

P-2

Amastigote

L. pifanoia,c

P-5

Amastigote

L. L. L. L. L. L. L. L. L. L. L. L.

Promastigote P-8

Amastigote Promastigote

P-9

Promastigote

M-2 F-4

Promastigote Promastigote

Species amazonensisa amazonensis amazonensisa pifanoia,c

amazonensisa pifanoia,c amazonensis pifanoi amazonensisa pifanoia,c amazonensis pifanoi amazonensis pifanoi amazonensis amazonensis

kDa 36, 40 36,b 40b 27 27 40, 45 36, 40 34, 43 19.5, 36,b 40b 34,b 43b 36, 40 34, 43 19.5, 36,b 40b 21, 34,b 43b >100 >100 46 70

Reference Duboise 1994 Duboise 1994 Duboise 1994 Pan et al. 1993 Duboise et al. 1994 Pan et al. 1993 Duboise et al. 1994 Duboise 1994 Pan and McMahon-Pratt 1988 Duboise 1994 Pan and McMahon-Pratt 1988 Duboise 1994 Pan and McMahon-Pratt 1988 Duboise 1994 Pan and McMahon-Pratt 1988 Duboise 1994 Pan and McMahon-Pratt 1988 Kahl and McMahon-Pratt 1987 Ismach et al. 1989

a

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

FCS in PBS (pH 7.0 for promastigotes, pH 4.6 for amastigotes) and checked for viability using erythrosin B. Parasites were then dispensed on 12-well slides (Cel-Line Associates, Newfield, NJ), rapidly air-dried, and evaluated within 2–3 days. Organisms that were to be assayed with mAb M-2 were fixed with absolute methanol for 5 min prior to incubation with the antibody. The reactions with all other mAbs were assessed on air-dried, unfixed organisms. In all experiments, cells were preincubated for 1 hr at 37°C with 5% FCS in PBS, pH 7.0 (also used for subsequent washings). Cells were incubated with dilutions of specific mAbs for 45 min at 24°C, washed, incubated with dilutions of rhodamine-labeled anti-mouse IgG (Jackson ImmunoResearch 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 examined with a Leitz Orthoplan microscope (Wetzler, Germany). A minimum of 200 organisms were evaluated. Negative controls were incubated with NS-1 culture supernatant and/or the second antibody alone. Western blot analysis. Proteins were electrophoretically transferred from 12% SDS–PAGE gels (Laemmli 1970) to nitrocellulose (0.45 mm) or Immobilon-P (0.45 mm, Millipore Corp., Bedford, MA) and visualized according to the method of Towbin et al. (1979) as modified by Pan and McMahon-Pratt (1988). Alternatively, antigens were visualized using a modification of the procedure described by

Ey and Ashman (1986). Strips were incubated for 2 hr at 24°C in anti-mouse IgG (1:1000) conjugated to alkaline phosphatase (Bio-Rad Laboratories, Hercules, CA), followed by a 5- to 10-min incubation in 150 mM veronal buffer, pH 9.5. The protein bands were developed using an alkaline phosphatase substrate (BCIP/NBT, Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD) and the reaction was stopped by rinsing in distilled water. Northern blot analysis. Total RNA was isolated from 2 × 107 promastigotes or axenic amastigotes using a Micro RNA isolation kit (Stratagene, La Jolla, CA). RNA samples (20 mg per lane) were loaded onto a 1.2% agarose gel containing 1.1 M formaldehyde. Electrophoresis, transfer, and hybridization were carried out as described previously (Campos-Neto et al. 1995). The membrane was probed with a 32P-labeled 540-bp DNA fragment that was specific for a gene encoding the protein recognized by mAb P-4 (Soong et al., unpublished data). To verify the concentration of mRNA loaded in each lane, the same membrane was stripped and reprobed with a 32P-labeled Ld p23 peptide/ gene, 640-bp DNA fragment that recognizes a gene encoding a surface protein that is expressed in both amastigotes and promastigotes and conserved among different Leishmania species (Campos-Neto et al. 1995). Immunoprecipitation. Promastigotes and axenic amastigotes were metabolically radiolabeled with [ 35 S]methionine (Amersham, Arlington Heights, IL) using me-

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Leishmania amazonensis: AXENIC AMASTIGOTE-LIKE ORGANISMS thionine-free Schneider’s medium supplemented with dialyzed heat-inactivated FCS. The radiolabeled cells were immunoprecipitated using ascitic fluid containing mAb A-2 and processed as previously described (Kahl and McMahon-Pratt 1987) except that protein G, 4 fast flow resin (Pharmacia LKB, Uppsala, Sweden) was employed.

RESULTS Culture. During the adaptation process, increased expression of amastigote-specific antigens recognized by mAbs A-1 and A-2 paralleled inoculation of cultures into increasingly acidic media. Table II summarizes results from populations growing at pH 5.5 to pH 7.0 that were amastigote-like as viewed by phase microscopy. In the first three populations the number of flagellated organisms increased with additional subpassages. Stable amastigote-like populations were obtained only at 32°C, pH 4.6. This was achieved by initial temperature increments of 2°C followed by decreases of 0.5 pH units from pH 7.0 to pH 5.0 and then 0.2 pH units to pH 4.6. Culture density was always maintained below 2 × 107/ml since during the adaptation process, populations of axenic amastigote-like organisms were observed with up to 29% flagellated organisms at a density of 3.6 × 107/ml or higher. This increase in flagellated forms was eliminated if the organisms were centrifuged and resuspended in fresh medium, suggesting a stringent nutrient requirement for the maintenance of amastigotes. Amastigote-like populations are presently being maintained that exhibit

TABLE II Immunofluorescence of Nonflagellated Organisms Growing at 32°C in Media pH 4.6–7.0, Detected by Reaction with Amastigote-Specific mAbs A-1 and A-2 pH (No. of passages) 7.0 6.0 5.5 4.6

(13) (3) (1) (6)

mAb A-1a

A-2a

34 32 50 85

2 17 18 92

a Percentage of fluorescent organisms based on the evaluation of at least 200 cells.

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no more than 7% flagellated forms at concentrations as high as 8 × 107/ml. Cultures proved sensitive to gentamicin at 50 mg/ml as previously reported for axenic amastigotes of L. braziliensis and L. panamensis (Eperon and McMahon-Pratt 1989a), but 12.5 mg/ ml was never inhibitory and some populations were maintained at 25 mg/ml. The ability of axenic amastigotes to transform into promastigotes was assessed by periodic transfer to 24°C, pH 7.3. Amastigote cultures were reestablished by shifting the promastigote culture back to 32°C, pH 4.6 following at least one passage at 24°C, pH 4.6. To date, it has not been possible to maintain organisms isolated directly from a lesion as axenic amastigotes. Although Bates (1994) described induction of metacyclic forms following a decrease in pH of L. mexicana cultures, morphological metacyclic forms were not identified in pH 4.6, 24°C cultures of L. amazonensis (strain MHOM/BR/ 77/LTB0016). In addition, while low pH stationary phase metacyclic cultures of L. mexicana converted to amastigote-like organisms when shifted to a higher temperature (Bates 1994), a greater percentage of morphologically heterogeneous log phase, not stationary phase L. amazonensis organisms, survived the increase to 32°C and converted to amastigotes. Recently, this laboratory obtained axenic amastigotes by shifting a previously adapted culture directly from pH7, 24°C to pH 4.6, 32°C; however these organisms were not used in the present study. The new serum lot appears to be responsible for this, emphasizing the importance of possible growth and nutritional factors in this transformation process. Since the first successful report of continuous culture of axenic amastigotes employed media containing hemin (Pan 1984) this component was included in the initial adaptation stages using L. amazonensis. However, no difference in parasite growth rate or the percentage of nonflagellated forms was detected in medium with or without hemin. In an attempt to provide a component from the host cell that might be required to attain and maintain amastigote morphology, extracts of J774.G8 macrophages were

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added to L. amazonensis cultures, but no effect of macrophage extracts was observed. Morphology and growth. As shown in Fig. 1, the morphology of L. amazonensis axenic amastigotes was clearly distinct from the promastigotes. The growth of axenic amastigotes as round doublets and short chains was observed by phase microscopy and has also been reported as 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 of promastigotes. Log phase promastigotes exhibited a generation time of 8 hr. while the generation time for log phase axenic amastigotes was 17 hr. During the late log phase of promastigote growth the pH of the culture medium decreased while in comparable amastigote cultures the pH was increasing (Fig. 2). The increase in pH during growth has not been reported for other axenic amastigotes, but has been described for the protists Naegleria (Weik and John 1977) and Tetrahymena (Seaman 1955), growing at pH 6.5–6.7.

Antigenic characterization. The amastigotespecific antigens recognized by mAbs A-1, A-2, P-2, P-4, P-5, and P-8 (Table I) were detected by indirect immunofluorescence in L. amazonensis axenic amastigotes maintained in continuous culture, but not in promastigotes. On the other hand, mAbs M-2, F-4, and P-9 that recognized promastigote antigens usually exhibited no reaction with axenic amastigotes; however, occasionally a small subpopulation was reactive with mAb P-9. Representative reactions are seen in Fig. 3. To confirm the molecular specificity of the indirect immunofluorescence, Western blot analysis and immunoprecipitation experiments were performed (Figs. 4 and 5). All molecules recognized by the stage-specific mAbs were in the molecular weight range of those previously identified in L. amazonensis lesion and macrophage-derived amastigotes and L. pifanoi macrophage-derived or axenic amastigotes (Table I). In Fig. 4 a broad band at 36 kDa was recognized in the L. amazonensis axenic amastigote population by mAbs A-1, P-5, and P-8. This

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

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

band was resolved in one experiment using mAb A-1 into bands at 35 and 37 kDa (results not shown). The same band was absent or barely visible in promastigotes. Using mAb P-8, two bands in the axenic amastigotes, at 20 and 18 kDa were distinct from the 19.5-kDa band found in the promastigotes (Fig. 4; Duboise

1994). The previous observation of an additional band at 40–43 kDa with mAbs A-1, P-5, and P-8 in lesion and macrophage-derived amastigotes (Table I) was interpreted as the result of loading differences. However, differences due to degradation and/or processing by axenic amastigotes cannot be ruled out.

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FIG. 3. Indirect immunofluorescence of L. amazonensis axenic amastigote-like organisms and promastigotes. Axenic amastigotes (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 rhodamine conjugated anti-mouse IgG. Bar, 15 mm.

Intermediate and precursor molecules (41 and 45 kDa) of the Lpcys2 cysteine proteinase (Duboise et al. 1994) recognized by mAb P-2 were present in L. amazonensis axenic amastigotes, but not promastigotes (Fig. 4). This an-

tibody was previously shown to recognize an epitope associated with the COOH-terminal extension of the cysteine proteinase, which was removed during intracellular processing (Duboise et al. 1994). Further, the 27-kDa mature

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FIG. 4. Comparison of L. amazonensis axenic 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 indicated at the top of each panel. The culture supernatant from the nonsecreting parental myeloma cell line (NS-1) was used as a negative 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 alkaline phosphatase conjugated (mAb F-4). The lanes for F-4 were enlarged from a mini-gel, resulting in a more diffuse band than was in the original. At the bottom of the figure “a” denotes axenic amastigote and “p” denotes promastigote.

proteinase was selectively identified in L. amazonensis axenic amastigotes in immunoprecipitation experiments using mAb A-2 (Fig. 5). The molecules recognized by mAbs F-4, M-2, and P-9 were detected by immunofluorescence in all of the promastigotes examined. In the population of axenic amastigotes evaluated

FIG. 5. Immunoprecipitation by mAb A-2. Autoradiographic results of L. amazonensis amastigote-like organisms (2) and promastigotes (3) metabolically labeled with [35S]methionine, lysed, and precipitated with mAb A-2. The control (1) was as in Fig. 4.

by Western blot analysis in Fig. 4, none of the organisms appeared to react with mAbs F-4 or M-2 by immunofluorescence, but a small subpopulation (<1%) was reactive with mAb P-9. Consequently, it is likely that the very low level of expression of both the M-2 and P-9 proteins detected in the Western blot analysis was due to this subpopulation. Northern blot analysis of P-4 mRNA. The protein (35 kDa) recognized by mAb P-4 was previously demonstrated in L. amazonensis and L. pifanoi macrophage-derived, lesion amastigotes and in L. pifanoi axenic amastigotes (Duboise 1994; Pan et al. 1993). Since the gene encoding the P-4 protein has been cloned (Soong et al., unpublished data), it was possible to compare mRNA expression levels in axenically cultured L. amazonensis amastigote-like organisms and promastigotes using Northern blot analysis. As illustrated in Fig. 6A, specific P-4 mRNA of 2.8, 5.6, and 7.0 kb was expressed only in axenic amastigotes, not in promastigotes. To verify the amount of RNA loaded onto the gel (Fig. 6B), the same membrane was reprobed with the Ld p23 peptide/ gene probe (Campos-Neto et al. 1995). Compa-

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FIG. 6. Northern blot of L. amazonensis promastigotes and amastigotes. Total RNA was isolated from L. amazonensis amastigote-like organisms (A) or from promastigotes (B). Approximately 20 mg of total RNA per lane was used for Northern blot analysis. The membrane was probed with a 32P-labeled DNA fragment that was specific for the protein recognized by mAb P-4 (A), stripped, and reprobed with a labeled DNA fragment that encodes the Ld p23 peptide (B) which is expressed in both the promastigote and amastigote stages.

rable levels of mRNA (0.7 kb) were expressed in both amastigotes and promastigotes. Infectivity. Axenic amastigote-like organisms infected peritoneal macrophages from BALB/c mice (Fig. 7) and J774.G8 macrophages. The large parasitophorous vacuoles indicated that the parasites were intracellular. In addition, duplicate cultures of axenic amastigotes were unable to survive extracellularly without macrophages. Replication of axenic amastigotes within macrophages was indicated by the presence of two nuclei or two kinetoplasts per cell (Fig. 7, arrows). Organisms recovered from J774.G8 macrophages after 11 days in culture differentiated into promastigotes at pH 7, 24°C. After one passage at pH 4.6, 32°C these promastigotes were capable of transforming back to axenic amastigotes at pH 4.6, 32°C. Lesions of at least 8 mm were produced in BALB/c mice within 3 months after the injection of axenic amastigotes. Although a quantitative analysis of infectivity was not carried out, the lesions were comparable in size to those produced by direct

FIG. 7. Infection of BALB/c peritoneal macrophages with L. amazonensis axenic amastigote-like organisms. Adherent macrophages were infected at a ratio of 5:1 at 32°C in 5% CO2 and free parasites were removed after 5 hr. Cultures were incubated at 37°C in 5% CO2 for 72 hr. Note the two kinetoplasts (small arrow) and two nuclei (large arrow) in reproducing organisms. Parasites in duplicate cultures without macrophages did not survive. Bar, 5 mm.

inoculation of nonadapted L. amazonensis lesion amastigotes. These results confirmed the observations from in vitro macrophage cultures and indicated the in vivo infectivity of the axenic organisms. DISCUSSION The present report documents the morphological, growth, and antigenic characteristics of L. amazonensis axenic amastigotes and further emphasizes the need to carefully monitor and evaluate axenic amastigote populations. At the light microscope level L. amazonensis axenic amastigotes clearly were morphologically an amastigote-like, not a promastigote-like population. However, during the adaptation procedures it became clear that in the absence of the appropriate conditions for continuous cultivation, organisms could be morphologically amastigote-like and then either become more promastigote-like or fail to survive subsequent subpassages. Physiological and morphological relevance of data obtained from such popula-

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Leishmania amazonensis: AXENIC AMASTIGOTE-LIKE ORGANISMS tions may not be applicable to the amastigote reproducing in the vertebrate host and should not be presented as characteristic of “axenic amastigotes.” As an example, the absence of true megasomes in L. amazonensis noted after 2 days at 34°C reported by Leon et al. (1994) was most likely due to the fact that these organisms were merely “heat-shocked” and not physiologically differentiated amastigotes. Studying the differentiation from promastigote to amastigote within mouse macrophages, GalvaoQuintao et al. (1990) found that typical megasomes were not identified until Day 5 after infection. It can be inferred that the development of the megasome is a complex differentiation process that may not be induced after heatshock in short-term culture. This is undoubtedly true for various aspects of amastigote differentiation and development. It would avoid confusion if organisms that cannot be maintained as amastigotes were referred to as “heat-shocked amastigote-like” forms, “pH-induced amastigote-like” forms, etc. and if reports of amastigote-like organisms that can be maintained in axenic culture included or referenced supporting characterization. Although an increase in temperature and a decrease in pH have been used to obtain amastigote-like populations of other Leishmania species (Bates 1994; Joshi et al. 1993; for review see Zilberstein and Shapira 1994), this is the first report of continuous cultivation of a species at the low pH of 4.6 at 32°C. However, a subpopulation of promastigotes of L. major adapted to growth at pH 4.5, 26°C synthesized an amastigote protein (reviewed in Zilberstein and Shapira 1994). Although the biochemical mechanisms responsible for differences among Leishmania species are unclear at this point, the low culture pH requirement of axenic amastigotes may indicate unique physiologic features of L. amazonenesis. It may be significant that the pH of the parasitophorous vacuole of macrophages infected in vitro with L. amazonensis was reported as pH 4.74 to pH 5.26 (Antoine et al. 1990) and was lower for L. amazonensis than for L. donovani (Chang 1980a). The conclusion that the population described

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in the present report was amastigote-like was based on the presence of amastigote characteristics and the absence of promastigote traits. In an earlier report (Traub-Cseko et al. 1993), Northern blot analysis detected higher concentrations of mRNA specific for a 27-kDa cysteine proteinase in L. amazonensis lesion amastigotes and L. pifanoi axenic amastigotes than in promastigotes. This proteinase was identified by mAb A-2 and localized to megasomes in amastigotes, but was absent in promastigotes of both species (Duboise et al. 1994). The presence of this proteinase in L. amazonensis axenic amastigotes and its similarity to the L. pifanoi proteinase were substantiated by immunofluorescence and immunoprecipitation analyses using mAb A-2 and by Western blot analysis using mAb P-2. L. amazonensis axenic amastigotes will be valuable in continuing studies aimed at elucidation of the biological function of amastigote cysteine proteinases, particularly as the genes are upregulated during transformation of the promastigote to the amastigote stage. The present results also clearly established the preferential expression of antigens recognized by mAbs A-1, P-4, P-5, and P-8 in axenic L. amazonensis amastigotes as compared with promastigotes and the differential synthesis of mRNA specific for the molecules reactive with mAb P-4. The differential antigen expression was in agreement with previous immunological comparisons of L. amazonensis amastigotes from lesion or from the J774.G8 macrophage cell line with L. amazonensis promastigotes (Duboise 1994; Pan and McMahon-Pratt 1988). The antigens recognized by mAbs P-4 and P-8 have been purified from L. pifanoi axenic amastigotes and used to induce significant protection against murine infection with L. pifanoi and partial protection against nonhomologous infection with L. amanzonensis (Soong et al. 1995). The expression of these antigens by axenic L. amazonensis amastigote-like organisms provides readily available homologous material for future immunization studies. Verification that the L. amazonensis axenic amastigote-like organisms characterized in the present study were not simply morphologically

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modified promastigotes was further supported by the absence of the paraxial rod protein recognized by mAb F-4 and the dramatically reduced expression of the promastigote molecules reactive with mAbs M-2 and P-9. The promastigote gp46 (detected by mAb M-2) has been used to elicit protective immunity in mice and the molecules reactive with mAb P-9 were among the first to be synthesized during the transformation of amastigote to promastigote (Champsi and McMahon-Pratt 1988; Duboise 1994). L. amazonensis axenic amastigotes will provide a system in which to study the regulation of these as well as other promastigote molecules. Numerous reports have identified ultrastructural, biochemical, metabolic and antigenic characteristics specific to the amastigote stage of Leishmania (Dell and Engel 1994; Ilg et al. 1995; Joshi et al. 1993; Medina-Acosta et al. 1993; Souza et al. 1994; Straus et al. 1993; Winter et al. 1994; for review see Moody 1993 and Pan et al. 1993). It will now not only be easier to study amastigote molecules of L. amazonensis in more detail and in the absence of contaminating host components, but also to further assess the extent and function of host derived molecules associated with the amastigote (Schneider et al. 1993; Winter et al. 1994). In addition, axenic amastigotes provide an ideal system for identifying specific amastigote nutritional requirements as control targets. The continuous cultivation of L. amazonensis amastigote-like organisms brings us one step closer to a better understanding of the biology of L. amazonensis amastigotes. REFERENCES ALEXANDER, J., AND VICKERMAN, K. 1975. Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana-infected macrophages. Journal of Protozoology 22, 502–508. ANTOINE, J-C., PRINA, E., JOUANNE, C., AND BONGRAND, P. 1990. Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH. Infection and Immunity 58, 779–787. BATES, P. A. 1994. Complete developmental cycle of Leishmania mexicana in axenic culture. Parasitology 108, 1–9. C A M P O S -N E T O , A., S O O N G , L., C O R D O V A , J. L.,

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