Leishmania braziliensis: Protein, carbohydrate, and antigen differences between log phase and stationary phase promastigotes in vitro

EXPERIMENTAL

63, 352-359 (1987)

PARASITOLOGY

Leishmania braziliensis: Protein, Carbohydrate, and Antigen Differences between Log Phase and Stationary Phase Promastigotes in Vitro

Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, DC 20307, U.S.A.

EILEEN D. FRANKE U.S. Naval Medical Research Unit 2, Jakarta, Indonesia

PATRICK

B. MCGREEVY

Walter Reed Army Institute of Research, U.S. Army Medical Research Unit at Brasilia, Brazil AND

RAYMOND E. KUHN Wake Forest University,

Winston-Salem, North Carolina 27109, U.S.A.

(Accepted for publication 26 September 1986) GRGGL, M., FRANKE, E. D., MCGREEVY, P. B., AND KUHN, R. E. 1987. Leishmania braziliensis: Protein, carbohydrate, and antigen differences between log phase and stationary phase promastigotes in vitro. Experimental Parasitology 63, 352-359. When Leishmania species are grown in vitro, parasites from the stationary phase differ from those in log phase growth in being more infective and more resistant to complement and macrophage mediated killing. In the present study, log phase and stationary phase promastigotes of Leishmania braziliensis panamensis were compared at the molecular level. Differences in polypeptide and glycoprotein composition and antigenicity between log and stationary phase promastigotes of L. b. panamensis were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the former showed that two polypeptides were unique to log phase promastigotes and one was unique to stationary phase promastigotes. There were also differences in surface lectin binding characteristics of log and stationary phase promastigotes. Live stationary phase promastigotes bound more concanavalin ahd lentil lectin than log phase promastigotes, indicating a greater number of mannose residues on their surfaces. o 1987 Academic PXSS, h. INDEX DESCRIPTORS AND ABBREVIATIONS: Leishmania braziliensis panamensis; Protozoa, parasitic; Promastigote; Biochemistry; Antigenicity; Logarithmic (log); Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); Fluorescein-isothiocyanate (FITC); Phosphate buffered saline (PBS); Dulbecco’s phosphate buffered saline (DPBS); Horseradish peroxidase (HRP); Dolichos biflorus agglutinin (DBA); Griffonia simplicifolia I (GS-I); Griffonia simplicifolia II (GS-II); Concanavalin A (Con A); Peanut agglutinin (PNA); Ricinus communis agglutinin I (RCA-I); Wheat germ agglutinin (WGA); Maclura pomifera agglutinin (MPA); Soybean agglutinin (SBA); Ulex europaeus agglutinin I (UEA-I); Lentil lectin (LL); Bovine serum albumin (BSA). 1 To whom correspondence

should be addressed.

352 0014-4894/87 $3.00 Copyright 8 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

Leishmania

braziliensis:

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INTRODUCTION

During growth in vitro and in vivo, promastigotes of some strains of the genus Leishmania undergo a progressive increase in infectivity for rodent hosts (Giannini 1974; Keithly and Bienen 1981; Sacks and Perkins 1984, 1985). This enhanced infectivity of stationary phase promastigotes may be the result of their relative abilities to survive host defense mechanisms. Leishmania braziliensis panamensis, for instance, develops during growth in vitro from complement susceptible log phase promastigotes to complement resistant stationary phase promastigotes (Franke et al. 1985). The increased infectivity of stationary phase promastigotes of L. 6. panamensis (Franke et al. 1985) may also relate to their ability to survive the lethal effect of normal serum before uptake by host macrophages. In addition, differences have been found in the ability of stationary phase promastigotes to survive the leishmanicidal mechanisms of normal resident macrophages (Sacks et al. 1985). The purpose of the present investigation was to compare the protein composition and antigenicity of promastigotes of L. b. panamensis from log and stationary phase cultures using SDS-PAGE and immunoblotting techniques. Further characterization of log and stationary phase promastigotes involved an analysis of glycoconjugates using plant lectins on Western blots. Finally, FITC conjugated lectins were used to examine surface saccharides of promastigotes from log and stationary phase cultures. The relationship between antigenicity, protein and saccharide composition, and infectivity of promastigotes is discussed. MATERIALSANDMETHODS Leishmania

braziliensis

panamensis,

strains

MHOMIPA/82/WR470 and MHOMIPAl83lWR539, were isolated from cutaneous lesions on two patients who acquired their infections in Panama. Cultures were initiated with stabilates of the primary isolates

353

and were maintained in Schneider’s Drosophila Medium (Franke et al. 1985). Homologous sera were obtained from the two patients prior to and following one course of treatment with Pentostam. Control sera were obtained from three donors, one of whom had worked with Leishmania spp. in the laboratory but had no history of infection. The blood was allowed to clot for 1 hr at room temperature and for 3 hr at 4 C before it was centrifuged. Serum was stored in 1 ml aliquots at -70 C. Soluble L. b. panamensis antigens were prepared from log and stationary phase cultures. Promastigotes were harvested and washed five times by centrifugation in PBS, pH 7.2, before frozen at -70 C, until used. Whole cell lysates were prepared by solubilization of the log and stationary promastigote pellets. Approximately lOi cells were resuspended in 10 ml of cold 0.05 M Tris-HCl, pH 7.8, containing 10 mM Chapso (Calbiochem-Behring, La Jolla, CA, U.S.A.) in the presence of enzyme inhibitors (100 U/ml aprotinin, 2 mM pepstatin A, and 2 mM phenylmethylsulfonyl fluoride; Sigma, St. Louis, MO, U.S.A.). The cells were rotated for 12 hr at 4 C and then disrupted by sonication on ice using a Biosonik III sonicator (Rochester, NY, U.S.A.) three times for a total of 3 min, at 20 kHz with 30 set cooling intervals. The sonicates were then centrifuged at 105,OOOg for 1 hr at 4 C. The resulting pellets were extracted again in the detergent:enzyme inhibitor mixture (1: 1, v/v) and sonicated twice. The sonicates were centrifuged at 105,OOOg for 1 hr at 4 C to remove final cellular debris. The supematant fluid (antigen) was dialyzed against 0.05 M Tris-HCl, pH 7.8, for 48 hr at 4 C and concentrated to 10 mg of protein/ml (Bradford 1976) using Aquacide II-A (Calbiochem-Behring). The antigen preparations were stored at -70 C in aliquots until used. Antigen preparations were analyzed by SDSPAGE, using 10% polyacrylamide resolving gels with 5% polyacrylamide stacking gels in the Laemmli (1970) buffer system. Each sample was mixed with an equal volume of sample buffer (0.08 M Tris-HCI, pH 6.8,O. 1 M dithiothreitol, 2% SDS, 10% glycerol, 0.2% bromphenol blue) and heated at 100 C for 2 min. The wells received 20 ~1 of the antigen sample buffer mixture. Electrophoresis was performed at 14 C at 8 W (constant wattage) until the tracking dye reached the bottom of the gel. Resolved proteins were then either transferred to nitrocellulose membranes or stained with Coomassie blue or silver stain (Morrissey 1981). Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose membrane (Western blotting) was performed as described by Grog1 and Kuhn (1985). Briefly, electrophoretically separated crude antigen extracts were transferred to nitrocellulose membranes (Bio-Rad, Richmond, CA, U.S.A.) using a Bio-Rad Trans-blot cell. The gels were washed with

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300 ml of 1.0, 0.5, and 0.1% Triton X-100 in transfer buffer (25 mM Tris, pH 8.3, 192 mM glycineRO% methanol) for 30 min each. Nitrocellulose membranes were incubated for 15 min in the transfer buffer at 4 C before use. Transfers were done at 60 V (0.21 A) for 12 hr at 4 C in degassed transfer buffer with continuous stirring. Control blots were stained with amido black (Bumette 1981). After antigens were transferred to nitrocellulose, the remaining protein binding sites were blocked with 3% (v/v) BSA (Fraction V; Sigma), for 2 hr at 37 C on a rocking platform. The blots were washed for 30 min in two changes of 0.1% Tween 20 in DPBS at room temperature. To detect antigens, blots were incubated for 1 hr at 37 C with gentle shaking in control serum or patient’s serum at a dilution of 1:30 in 10% fetal bovine serum (in DPBS; v/v). The membranes were washed three times for 30 min as before, then incubated for 1 hr at 37 C in HRP conjugated, affinity purilied antihuman IgG (heavy and light chains specific; Cappel Laboratories, Cochranville, PA, U.S.A.) at a 1:lOOOdilution in DPBS. After washing as before, the blots were incubated with the substrate solution of 0.05% (w/v) 4-chloro-I-naphthol in methanol with 0.15% hydrogen peroxide until the color developed (lo-20 min). The binding of lectins to glycopeptides of strain WR470 was detected according to described methods (Grog1 et al. 1985). Blots were incubated in 3% bovine serum albumin in 0.15% Tween 20 (in DPBS, pH 7.2) for 2 hr at room temperature to block free protein binding sites. Periodic acid treatment was carried out to prevent possible binding of lectins to carbohydrates on contaminating serum proteins in the BSA. This was followed by a 2 hr incubation with one of the following peroxidase labeled lectins at a final concentration of 100 g/ml: DBA, GS-I, GS-II, Con A, PNA, WGA, MPA, RCA-I, SBA or UEA-I. All lectins were supplied by E-Y Laboratories, Inc. (San Mateo, CA, U.S.A.). Specific competing sugars incubated with lectin blot mixtures at a final concentration of 0.2 M served as controls. After reaction with lectins, blots were washed in DPBS containing 0.1% Tween 20 three times for 10 min each time. Lectin binding was detected by incubating the blots for lo-20 min in a fresh preparation of 0.05% 4-chloro-1-naphthol, 0.15% hydrogen peroxide in 20 mM Tris, 500 mM NaCl, pH 7.5. The binding of a panel of FITC conjugated plant lectins to the surface of intact promastigotes of strain WR470 was determined using a modification of the technique described by Choromanski et al. (1984). The FITC conjugated lectins used in this study were obtained from E-Y Laboratories and Sigma. The lectins tested were PNA, WGA, SBA, UEA-I, DBA, MPA, Con A, and LL. All lectin-promastigote incubations had controls of specific competing sugars. The competing sugar was D-galactose for PNA and SBA,

N-acetyl-rr-glucosamine for DBA and mannose, and u-methyl-D-mannoside for Con A and LL. Promastigotes from log or stationary phase cultures were harvested by centrifugation and washed twice in DPBS and once in DPBS containing 0.1% sodium azide. The assay was performed in glass tubes by incubating 0.5 ml promastigote suspension (2 x lo6 cells/ml) with 0.5 ml lectin (200 &ml) in DPBS and either 0.5 ml DPBS or 0.5 ml of the competing sugar (0.2 M). After incubation in the dark at 4 C for one hr, cells were washed twice in DPBS and fixed (1: 1; v/v) with 10% Formalin (in DPBS) for 10 min at 4 C. Fixed cells were washed three times in DPBS and resuspended in 0.9 ml DPBS. Promastigote suspensions (0.3 ml) were centrifuged onto slides using a Cytospin 2 centrifuge (Shandon Instrument, Sewickley, PA, U.S.A.) at 800 rpm for 4 min. Slides were mounted in 70% glycerol in DPBS and were observed with a Leitz fluorescence microscope equipped with an HBO 100 W mercury lamp and filters selective for FITC. RESULTS

Polypeptide profiles of log and stationary phase promastigotes of two strains of Leishmania braziliensis panamensis, when analyzed by SDS-PAGE and silver staining, are shown in Fig. 1. Polypeptides unique to log phase promastigotes had molecular weights of 31, 66, and 102 kDa, while a polypeptide of 79 kDa was detected only on stationary phase promastigotes. Results from the antigenic analysis (immunoblots) of soluble extracts of log and stationary phase promastigotes of two strains of L. 6. panamensis identified by a patient’s (WR470) serum is shown in Fig. 2. A greater number of antigens from log phase promastigotes (24 of WR470 and 28 of WR539) were detected by immunoblotting than from stationary phase promastigotes (16 of WR470 and 13 of WR539). Serum obtained from the patient from which WR539 was isolated identified fewer antigens and staining was less intense (data not shown). Again, more components from log phase promastigotes (7 of WR470 and 9 of WR539) were recognized as antigens compared to stationary phase promastigotes (4 each of WR470 and WR539). One to four antigens of log phase promastigotes were recognized by control sera; however,

Leishmania braziliensis: MOLECULARGROWTHCHANGESIN

116 92.5

“0 -

X

66.2

F

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w 3

45.0 5 i

%

-I

31.0

VITRO

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116 92.5

66.2

45.0

z 21.5

ii

31.0

14.4 A

B

C

D

FIG. 1. Polypeptide

composition of Leishmanin strain WR470, from (A) log phase cultures and (B) stationary phase cultures, and WR539 from (C) log phase cultures and (D) stationary phase cultures.

braziliensis

panamensis,

bands indicating reactions were very faint. Antigens of stationary phase promastigotes were not detected by control sera. Of all the lectins tested, only PNA, WGA, DBA, and MPA labeled glycoconjugates of promastigotes on Western blots (Fig. 3). PNA bound to two glycopeptides of molecular weights 59 and 61 kDa on blots of stationary phase promastigotes (data not shown), but not to blots of log phase promastigotes. WGA bound to five glycopeptides of stationary phase promastigotes ranging in molecular weight from 48 to 74 kDa. Intensely stained bands were present at molecular weights of 59, 61, and 74 kDa. The glycoconjugate of 61 kDa was unique to stationary phase promastigotes. On blots of log phase promastigotes, WGA bound to six glycopeptides ranging in molecular weight from 48 to 170 kDa. Three

A

El C D FIG. 2. Serum from patient infected with Leishmania braziliensis panamensis, strain WR470, was used to detect antigens of promastigotes following electrophoretic separation and blotting on nitrocellulose. Parasite strain WR470 from (A) log phase cultures and (B) stationary phase cultures, and WR539 from (C) log phase cultures and (D) stationary phase cultures.

bands unique to log phase promastigotes were at 152, 165, and 170 kDa. Bands at 48 and 74 kDa were much less intense in log phase than in stationary phase promastigotes. Blots of log phase promastigotes had 14 glycopeptides between 33 and 90 kDa that bound DBA. Three glycopeptides of stationary phase promastigotes, at molecular weights of 52, 58, and 60 kDa, reacted with DBA. The glycoconjugate at 58 kDa was unique to stationary phase promastigotes. MPA bound to glycopeptides of molecular weights 56, 61, and 62 kDa from stationary phase promastigotes. There were six MPA-binding glycoconjugates between 53 and 90 kDa on blots of log phase promastigotes. The FITC-lectins that bound to the sur-

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I I 692.566.2-

FIG. 3. Detection of lectin binding proteins in whole cell extracts of log (L) and stationary (S) phase promastigotes of Leishmania braziliensis panamensis following electrophoretic separation and transfer to nitrocellulose membranes. The lectins used were (A) WGA, (B) DBA, and (C) MPA.

faces of promastigotes were LL and Con A. All other lectins tested did not bind to the surfaces of promastigotes. In the presence of FITC-Con A, stationary phase promastigotes were more intensely fluorescent than log phase promastigotes (Fig 4). FITC-LL did not bind to promastigotes from log phase cultures, but did bind to stationary phase promastigotes (data not shown). Intensity of fluorescence on promastigotes from stationary phase cultures incubated in FITC-LL was less than that of stationary promastigotes incubated in FITC-Con A. Binding of FITC-LL and FITC-Con A to promastigotes was inhibited by mannose and a-methyl-D-mannoside. DISCUSSION

Stationary phase promastigotes of Leishbraziliensis panamensis are more resistant to the lethal effects of complement and more infective for the vertebrate mania

host than promastigotes from log phase cultures (Franke et al. 1985). In the present investigation, a comparison of log and stationary phase promastigotes showed differences in polypeptide composition, antigenicity and lectin binding characteristics between organisms in the two growth phases. Electrophoretic separation of whole cell extracts in the presence of SDS showed that three polypeptides were unique to log phase promastigotes and one was unique to stationary phase promastigotes. Patients’ sera detected fewer antigens of stationary phase promastigotes on Western blots than of log phase promastigotes of the same strain. Qualitative and semiquantitative differences in glycoprotein composition of log and stationary phase promastigotes were detected by lectin binding to Western blots of electrophoretically separated polypeptides. The lectins WGA, DBA, and MPA bound to blots of both log and stationary phase promastigotes, while PNA bound

Leishmania

braziliensis:

MOLECULARGROWTHCHANGES

ZNVZTRO

357

FIG. 4. Promastigotes from log phase cultures (A) and stationary phase cultures (B) of Leishmania panamensis after incubation with FITC-Con A. Only the fiagellar pocket of the log phase promastigotes stained with FITC-Con A, while the entire body and flagellum of stationary phase promastigotes bound Con A.

braziliensis

only to blots of stationary phase promastigotes. Binding of FITC-labeled lectins to intact promastigotes also showed differences in cell surface lectin receptors between the two growth phases. Although Con A bound to the surfaces of whole promastigotes from log and stationary phase cultures, for undetermined reasons it did not bind to sugar moieties of L.b. panamensis promastigote glycoconjugates on nitrocellulose blots. Perhaps the molecule which bound Con A is not solubilized effectively in Tris-Chapso buffer, or is a high molecular weight glycoprotein which did not transfer efficiently and bind to the nitrocellular membrane, or is a low molecular weight component that penetrated the nitrocellulose membrane. Alternatively, treatment with SDS and boiling may have destroyed or changed the configuration of the glycoprotein although temperature is generally known not to change the oligosaccharide, which is the portion that the lectin recognizes. Our results indicated that L.b. panamensis from infective stationary phase cultures possessed more Con A and lentil lectin binding molecules on their surfaces than promastigotes from log phase cultures

indicating a greater number of exposed mannose moieties. Using both fluorescent and agglutination assays, Ayesta et al. (1985) showed that promastigotes of a pathogenic strain (NR) of L. braziliensis bound Con A, whereas a nonpathogenic strain (LBY) of L. braziliensis did not. The association of increased mannose moieties with infective stage promastigotes is not restricted to L. braziliensis as it has also been observed in L. trupica (Ebrahimzadeh and Jones 1983). In contrast, Sacks et al. (1985) found that log and stationary phase promastigotes of L. major have equivalent densities of mannose on their surfaces. However, the expression of other carbohydrates on the surface of L. major did change with development as log phase noninfective promastigotes agglutinated in low concentrations of PNA and RCA, while infective stationary stage promastigotes did not agglutinate. The carbohydrate composition of the surface membrane of promastigotes changes during the transition from the dividing log phase to the nondividing infective phase, and the type of carbohydrates involved in this change, therefore, may not be the same for all species of Leishmania.

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Glycoconjugates of Leishmania spp. macrophages may be enhanced by interacpromastigotes have been shown to be di- tion of mannose residues on the parasite rectly involved in the initiation or early surface with lectinlike receptors on the stages of macrophage infection (Chang macrophage surface, or the increased man1981; Mitchell and Handman 1985; Nolan nose residues on the surface in combinaand Farrell 1985). The expression of man- tion with C3b may be prerequisites for the nose on the surface of L. b. panamensis uptake and survival of stationary promastistationary phase and the ability to bind C3b gotes in macrophages, is still a matter of without lysis (Franke et al. 1985) may be speculation. However, it is conceivable preadaptations required to infect vertebrates. that the biochemical changes found to While resistance to complement lysis en- occur during the switch between log phase hances survival in the extracellular com- promastigotes to stationary phase promaspartment, surface mannose and C3b may tigotes may be preadaptations that increase facilitate recognition and ingestion by mac- the infectivity of Leishmania braziliensis rophages. Mannose and C3b on the surface panamensis for its vertebrate host. of L. donovani act as ligands which bind to ACKNOWLEDGMENT the mannosyl/fucosyl (MFIUCR3) receptor We thank J. Andujar and A. Wolf for their excellent and to the type three complement receptor, technical assistance. respectively (Blackwell 1985; Blackwell et al. 1986). Likewise, the mechanism of atREFERENCES tachment of an infective strain of L. tropica AYESTA, C., ARGUELLO, C., AND HERNANDEZ, A. G. promastigotes to macrophages was pri1985. Leishmania braziliensis: Cell surface differences in promastigotes of pathogenic and nonpathomarily via a mannose receptor interaction genie strains. Experimental Parasitology 59, (Ebrahimazadeh and Jones 1983). It is not 185-191. known if the MFRCR3 recognition system BLACKWELL, J. M. 1985. Receptors and recognition operates for all Leishmania species, and it mechanisms of Leishmania species. Transactions of is possible that there are other recognition the Royal Society of Tropical Medicine and Hygiene 19, 606-612. systems. For example, the binding of L. major to macrophages is inhibited by the BLACKWELL, J. M., MCMAHON-PRATT, D., AND SHAW, J. 1986. Molecular biology of Leishmania. Fab fragment of a monoclonal antibody Parasitology Today 2, 45-54. (WIC-79.3) that reacts to a surface glyco- BRADFORD, M. M. 1976. A rapid and sensitive conjugate which is rich in galactose and method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. lactose (Handman and Goding 1985). The Analytical Biochemistry 12, 248-254. mere presence of mannose and C3b on the surface of stationary phase promastigotes BURNETTE, W. N. 1981. “Western Blotting”: Electrophoretic transfer of proteins from sodium dodecyl of L. b. panamensis is not proof that they sulfate-polyacrylamide gels to unmodified nitrocelare ligands, necessitating the need for furlulose and radiographic detection with antibody and radioiodinated protein A. Annals of Biochemistry ther studies. 112, 195-203. Based on the observation that patients’ K.-P. 1981. Leishmania donovani-macrosera identified fewer polypeptides on CHANG, phage binding mediated by surface glycoproteinsl Western blots of stationary phase than of antigens: Characterization in vitro by a radioisolog phase promastigotes, promastigotes topic assay. Molecular and Biochemical Parasitology 4, 67-76. from stationary phase cultures may be less immunogenic to the vertebrate host. Sta- CHOROMANSKI, L., HONIGBERG, B. M., AND HONHON, P. M. 1984. Trypanosoma (Nannomonas) tionary phase promastigotes of L. b. panacongolense: Analysis by fluorescein-conjugated mensis bound more Con A and lentil lectin plant lectins of surface saccharides of cloned variant than log phase promastigotes. Whether the antigen types differing in infectivity for mice. Journal of Parasitology 70, 634-643. uptake and survival of promastigotes in

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