Roles of free GPIs in amastigotes of Leishmania

Molecular and Biochemical Parasitology 99 (1999) 103 – 116

Roles of free GPIs in amastigotes of Leishmania  Kojo Mensa-Wilmot a,*, Nisha Garg a, Bradford S. McGwire b, Hong-Gang Lu b, Li Zhong b, Dora Abena Armah a, Jonathan H. LeBowitz c, Kwang-Poo Chang b b

a Department of Cellular Biology, Biological Sciences Building, Uni6ersity of Georgia, Athens, GA 30602, USA Department of Microbiology and Immunology, Uni6ersity of Health Sciences, Chicago Medical School, 3333 Green Bay Road, N. Chicago, IL 60064, USA c Department of Biochemistry, Purdue Uni6ersity, Lafayette, IN 47907, USA

Received 18 May 1998; accepted 20 December 1998

Abstract Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Glycosylated phosphatidylinositols; Leishmania amazonensis; Replication; Viability

Abbre6iations: AHM, 2,5-anhydromannitol; EtN, ethanolamine; gp63, 63 kDa GPI-anchored glycoprotein of Leishmania; GIPL, glycoinositolphospholipid; GPI, glycosylphosphatidylinositol; GPI-PLC, glycosylphosphatidylinositol-specific phospholipase C; GlcN, glucosamine; HPTLC, high performance thin layer chromatography; Ins, inositol; Man, mannose; Man3-GlcN-PI, Man(1a2)Man(1a6)Man(1a4)GlcN(1a6)inositol-1-phospho-glycerolipid; NP40, nonidet p-40; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; TLC, thin layer chromatography; VSG, variant surface glycoprotein of Trypanosoma brucei. * Corresponding author. Tel.: +1-706-5423355; fax: + 1-706-5424271; e-mail: [email protected].  Note: Dedicated to Professor Moises Agosin (University of Georgia). 0166-6851/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 9 9 ) 0 0 0 0 3 - 1

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1. Introduction Human leishmaniasis is transmitted by a sand fly that injects promastigotes, the insect vector stage of Leishmania, into a vertebrate during a blood meal. Promastigotes infect mononuclear phagocytes of the reticuloendothelial system and differentiate into amastigotes. This intracellular stage of Leishmania resides in a membrane-bound parasitophorous vacuole. Following many rounds of division amastigotes lyse the macrophages and the released parasites infect new host cells. Little is known about the requirements of amastigotes for survival and replication. Free glycosylphosphatidylinositols (GPIs) are abundant on the plasma membranes of mammalian cells and the Leishmania [1 – 3]. L. major promastigotes contain 1 – 5×106 molecules cell − 1 of lipophosphoglycan (LPG), a GPI-anchored polysaccharide, and 5×105 molecules of gp63, a GPI-anchored protease [4,5]. Two groups of ethanolamine (EtN)-containing GPIs (EtN-GPIs) are found in L. mexicana: protein-GPIs containing a ‘conserved core’ of EtN-phospho-Mana12Mana1-6Mana1-4GlcN-PI, and glycoinositolphospholipids (GIPLs) (107 cell − 1), e.g. EPiM3, EtN-phospho-Mana1-6(Mana1-3)Mana14GlcN-PI [6]. The functions of these GPIs in the Leishmania have remained elusive because null mutants in the glycan (i.e. Mana1-4GlcN-Ins) that is common to all three GPIs have not been reported (reviewed in [1,7–10]). With the aim of investigating the effects of a GPI deficiency on intracellular parasitism, GPI-deficient L. amazonensis were generated by stable transfection with a gene from Trypanosoma brucei encoding a GPI-specific phospholipase C (GPIPLC) [11–15]. GPI-PLC is an integral membrane protein which appears to co-localize with free GPIs on the cytoplasmic side of the endoplasmic reticulum (ER) membranes [16 – 19]. There, it may cleave GPI intermediates (reviewed in [1,9]), especially those occurring after Man1GlcN-PI [18 – 20]. In this work, the engineered strain was shown to be deficient in EtN-GPIs. Amastigotes of this strain were more sensitive than corresponding promastigotes to the loss of EtN-GPIs; their growth was inhibited, and virulence of the parasites was re-

duced. GPIs may, therefore, be essential for amastigote replication. Based on these observations a hypothetical explanation for the apparent absence of a GPI-PLC in Leishmania is advanced.

2. Materials and methods

2.1. Cell culture and transfection Parasites were derived from the virulent stock 12 of L. amazonensis LV78 [21]. Promastigotes were transfected with pX63NEO.GPI-PLC [18] or with pX63NEO [22]. Transfectants were selected with 50 mg ml − 1 G418 and adapted to grow in medium with 200–800 mg ml − 1 G418. Amastigote lines were obtained by culturing transfected promastigotes [23] at pH 5.3, 33°C [24]. These axenic amastigotes were maintained in medium containing G418.

2.2. Cell lysis, partial fractionation and GPI-PLC assay Parasite cells were lysed hypotonically, and a detergent fraction prepared as described [18]. GPIPLC activity was determined using [3H]myristatelabeled variant surface glycoprotein (VSG) as substrate [25].

2.3. Metabolic labeling [3H]Ethanolamine (EtN) labeling of promastigotes was performed as described [18]. Amastigotes were labeled at 33°C either with 100 mCi ml − 1 of [1-3H]EtN in Grace’s medium containing 20% heat-inactivated fetal bovine serum (FBS) and 25 mM HEPES, pH 5.3, or [6-3H]galactose (40 Ci mmol − 1, Amersham, Arlington Heights, IL) at 50 mCi ml − 1 in glucose-free RPMI containing 25 mM HEPES, pH 5.3 supplemented with FBS (Hyclone, Logan, VT).

2.4. Glycolipid extraction and analysis Pellets of [3H]EtN-labeled cells (2× 108 promastigotes, or 2× 109 amastigotes) were each extracted with chloroform/methanol (2:1, v/v)

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(CM), and subsequently with ice-cold chloroform/ methanol/water (10:10:3) (CMW) as described [18]. Dried organic extracts were resuspended in 20 ml of butanol. Glycolipids therein were resolved by thin layer chromatography (TLC) on Silica gel 60 in CMW (10:10:2.7), and detected by fluorography with pre-flashed Hyperfilm™ (Amersham). Quantitative densitometry of TLC fluorographs was performed on an IS-1000 Digital Imaging System (Alpha Innotech Corporation).

2.5. GPI-PLC digestion of glycolipid extracts CMW extracts from [3H]EtN-labeled pX63NEO/L. amazonensis (5 ×107 promastigotes or 5 ×108 amastigotes per sample) were each resuspended in 100 ml of 1X AB. These samples were incubated with or without recombinant GPIPLC (200 ng) [26] at 37°C for 3 h. Reaction was terminated and prepared [18] for TLC in CMW (10:10:2.7).

2.6. Partial structural analysis of AmGPI-Y and AmGPI-Z 2.6.1. Phospholipase C digestion [3H]EtN-labeled glycolipids (2× 107 cell equivalents), TLC-purified AmGPI-Y and AmGPI-Z (10 000 cpm), or standard Man1 – 3 GlcN-PI (10 000 cpm) were each digested with 100 U of GPI-PLC [25] for 5 h as described [18]. 2.6.2. Exoglycosidase treatment Glycolipids were treated with 2 U jack bean a-mannosidase (Oxford Glycosystems, Rosedale, NY) according to manufacturer’s instructions except that 0.3% sodium taurodeoxycholate was added to the buffer. Cleavage products were extracted with 300 ml water-saturated n-butanol, followed by two back extractions with 300 ml water. Butanol-soluble products were then dried under nitrogen, and resuspended in 10 ml n-butanol for TLC. Products from various treatments were resolved by TLC on silica gel 60 in CMW (10:10:3) or CHCl3:MeOH:0.25% KCl (55:45:10) and detected by fluorography.

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2.7. Characterization of the neutral glycans from AmGPI-Y and AmGPI-Z 2.7.1. Purification of neutral glycans GPIs (from 1–3×109 parasites) were labeled metabolically with [3H]EtN and scraped, after HPTLC in CMW, from HPTLC plates. Following dephosphorylation and deamination, the resulting neutral glycans from amastigote GPIs were radiolabeled with sodium [3H]borohydride (Dupont NEN, Wilmington, DE) [27]. The [3H]glycans were purified by HPTLC in propanol:acetone:water (PAW, 9:6:5), scraping, and elution with PAW. The eluates were dried, resuspended in 100 ml of 40% propanol, and stored at − 20°C until use. For standards, [3H]Man3-GlcN-PI synthesized in a T. brucei cellfree system [15,28] in the presence of PMSF [29] was purified from an HPTLC plate and labeled with [3H]borohydride as described above to generate [3H]Man-[3H]AHM. [3H]Man4-GlcN-PI was obtained by metabolic labeling of L. mexicana [1] and converted to [3H]Man4-[3H]AHM. 2.7.2. Exoglycosidase digestion Neutral [3H]glycans were digested with Jack bean a-mannosidase (JBAM; 2.5 U ml − 1, Boehringer Mannheim, Indianapolis, IN) and prepared for HPTLC/fluorography as described [27]. 2.7.3. HPTLC of neutral glycan fractions Glycans were resolved on aluminum-backed silica gel 60 HPTLC plates which were developed sequentially in (1) 1-propanol/acetone/water (9:6:5, v/v/v), (2) 1-propanol/acetone/water (5:4:1, v/v/v) and (3) 1-propanol/acetone/water (9:6:5, v/v/v) [27]. 2.8. Phospholipid extraction and analysis A pellet of 2 × 108 Leishmania was washed in phosphate-buffered saline and extracted with 600 ml of chloroform, and subsequently with 300 ml of methanol. Extraction was facilitated by sonication (6 min) and repeated inversion. The delipidated debris was pelleted at 14 000× g (10 min) and the supernatant recovered. The pellet was re-extracted with 900 ml of chloroform/methanol (2:1), the

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eluate was pooled with the previous extract and dried. A chloroform:methanol (1:1) (400 ml) extract of the resulting pellet was added to the tube in which the previous eluate had been dried. The organic extract was extracted with 0.88% KCl, and the aqueous layer washed with methanol:water (1:1 v/v) [30]. Lipids were recovered as described [30], dried, and stored at − 20°C. Just before use, lipids were dissolved in 10 ml of chloroform and applied to HPTLC plates. Lipids were developed for 4 cm in methyl acetate/propan-2-ol/chloroform/methanol/0.25% aqueous KCl (10:10:10:4:3.6, vol./vol.) and 8 cm in hexane/diethylether/glacial acetic acid (20:5:0.5, vol./vol.) [31]. Phospholipids were visualized by spraying with molybdenum blue (Sigma, St. Louis, MO).

are depicted in Figs. 1 and 2. Two glycolipids AmGPI-Z and AmGPI-Y were found predominantly in amastigotes. Both were cleaved by T. brucei GPI-PLC in vitro (Fig. 1), indicating that they are GPIs whose inositol moieties are not

2.9. Infections of macrophage and hamster In all cases, transfected promastigotes cultured in the presence of G418 were inoculated into drug-free medium and grown to stationary phase before use. During in vitro infection, 4× 107 promastigotes were added to 4×106 J774G8 mouse macrophages [21]. The number of intracellular parasites per macrophage was obtained by microscopic examination of about 200 macrophages. The total number of amastigotes per flask was estimated as described [21]. Cultures were counted every 3–5 up to 35 days. Male Syrian Golder hamsters (60 – 80 g) were divided into groups of four for in vivo infection. Each hamster was inoculated subcutaneously with 2×107 pX63NEO/L. amazonensis or pGPI-PLC/ L. amazonensis at the nose tip. Infections with axenic amastigotes or promastigotes were performed similarly. Lesions developed were measured with a caliper once or twice a week for up to 3 months.

3. Results

3.1. Partial structures of amastigote GPIs TLC profiles of polar glycolipids obtained from [3H]EtN-labeled promastigotes and amastigotes

Fig. 1. AmGPI-Z and AmGPI-Y are expressed in amastigotes, and are cleaved by GPI-PLC in vitro. A chloroform/methanol/ water extract of [3H]EtN-labeled pX63NEO/L. amazonensis was incubated in IX AB with or without purified GPI-PLC (see Experimental Procedures). Butanol extractable cleavage products were analyzed by TLC (chloroform:methanol:water (10:10:3))/fluorography. Lane 1, pX63NEO/L. amazonensis amastigotes; lane 2, pX63NEO/L. amazonensis amastigotes with GPI-PLC digestion; lane 3, pX63NEO/L. amazonensis promastigotes; lane 4, pX63NEO/L. amazonensis promastigotes with GPI-PLC digestion.

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Fig. 2. AmGPI-Y is resistant to digestion by a-mannosidases HPTLC-purified [H3]EtN-labeled AmGPI-Z and AmGPI-Y (10,000 dpm) were treated with jack bean a-mannosidase (JBAM), Aspergillus saitoi a-mannosidase (ASAM), and partial alkali hydrolysis. Butanol extractable cleavage products were resolved by HPTLC and detected by fluorography. [H3]Man3-GlcN-PI from T. brucei served as control. Lane 1, [3H]Man1 – 3GlcN-PI from T. brucei; lane 2, [3H]Man1 – 3GlcNPI/ASAM; lane 3, [3H]Man1 – 3GlcN-PI/JBAM; lane 4, [3H]Man1 – 3GlcN-PI/mild base; lane 5, AmGPI-Y; lane 6, AmGPI-Y/ASAM; lane 7, AmGPI-Y/JBAM; lane 8, AmGPIY/mild base.

acylated [32,33]. [3H]EtN-labeled AmGPI-Y and AmGPI-Z were resistant to digestion by a-mannosidase (JBAM) (Fig. 2), suggesting the absence of unsubstituted mannosyl residues (see evidence for the presence of mannose below). Control [3H]Man3-GlcN-PI from T. brucei was cleaved by JBAM (Fig. 2), as expected. Cleavage by partial base treatment (Fig. 2, lane 8) indicates the AmGPI-Y contains an oxy-esterified lipid. Neutral glycans generated from both AmGPI-Y and AmGPI-Z (AmGPI-Y.NG and AmGPIZ.NG, respectively) were analyzed (Fig. 3A, lanes 3 and 4). The predominant component had similar HPTLC mobility as Man3-AHM from glycolipid A

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of T. brucei (Fig. 3A, lane 1), indicating that both glycolipids had Man3-GlcN headgroups. Both neutral glycans were susceptible to JBAM (Fig. 3B, lanes 5 and 8, respectively). Cleavage by partial acetolysis (Fig. 3B, lanes 6 and 9) suggests the existence of Mana1,6Man linkages in both GPIs. We conclude from these observations that the headgroups of both AmGPI-Y and AmGPI-Z are most likely Man3GlcN. Since both GPIs are metabolically labelled with EtN, we infer that AmGPI-Y (and also AmGPI-Z) is composed of EtN-phospho-Man3-GlcN-Ins-phospho-lipid, assuming the presence of only one phospho-EtN group per molecule of AmGPI-Y (or AmGPI-Z). The broadness of the AmGPI-Y spot (Figs. 1 and 2) suggests that the constituent alkyl or acyl chains might be heterogeneous. Finally, since the polar headgroups of AmGPI-Z and AmGPI-Y appear to have identical sizes, the difference in TLC mobility between the two GPIs might be due to the presence of longer acyl chains in the former. Extensive structural analysis of these glycolipids is beyond the objectives of this study, since our purpose was limited to the demonstration of the glycolipids as GPIs (Fig. 1).

3.2. GPI-PLC-expressing amastigotes are deficient in GPIs GPI-anchored proteins are expressed in promastigotes of Leishmania. On the contrary, GPIanchored proteins were not detectable in amastigotes of L. amazonensis by metabolic labeling with [3H]EtN combined with SDS-PAGE fluorography (not shown). This observation agrees with previous work by Overath’s group [34], and led us to consider possible physiological roles of amastigote GPIs, now that these glycolipids were unlikely to serve as anchors for proteins. AmGPI-Z and AmGPI-Y were 10-fold less abundant in GPI-PLC-expressing L. amazonensis (pGPI-PLC/L. amazonensis) (Fig. 4A, lane 2) than in control parasites containing the expression vector only, i.e. pX63NEO/L. amazonensis (Fig. 4A, lane 1). Hence, pGPI-PLC/L. amazonensis is deficient in the two [3H]EtN-containing GPIs. This deficiency may start out by GPI-PLC affecting the turnover of AmGPI-Z and AmGPI-Y,

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Fig. 3. Neutral glycans from AmGPI-Z and AmGPI-Y co-migrate with Man3-GlcN. (A) Neutral [3H]glycans were subjected to HPTLC and detected by fluorography. Lane 1, Man3-AHM from T. brucei; lane 2, Man4-AHM from L. mexicana; and lane 3, AmGPI-Y neutral glycan; lane 4, AmGPI-Z neutral glycan. (B) Chemical and enzymatic cleavages of neutral glycans [3H]glycans were either undigested or subjected to the indicated treatments and analyzed by HPTLC/fluorography. Lane 1, Man3-AHM/untreated; lane 2, Man3-AHM/JBAM; lane 3, Man3-AHM/acetolysis; lane 4, AmGPI-Y.NG/untreated; lane 5, AmGPI-Y.NG/JBAM; lane 6, AmGPI-Y.NG/acetolysis; lane 7, AmGPI-Z.NG, untreated; lane 8, AmGPI-Z.NG/JBAM; lane 9, AmGPI-Z.NG/acetolysis.

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Fig. 4. pGPI-PLC/L. amazonensis is deficient in EtN-GPIs. (A) GPIs from chloroform:methanol:water extracts. Glycolipids from [3H]EtN-labeled amastigotes (2.5 ×108 cell equivalents) (lanes 1 and 2) or [3H]Gal-labeled amastigotes (5 × 107 cells) (lanes 3 and 4) were analyzed by TLC/fluorography. Lane 1, pX63NEO/L. amazonensis; lane 1; pX63NEO/L. amazonensis axenic amastigotes; lane 2, pGPI-PLC/L. amazonensis axenic amastigotes; lane 3, [3H]gal-labeled glycolipids from pX63NEO/L. amazonensis; lane 4, [3H]gal-glycolipids from pGPI-PLC/L. amazonensis. Glycolipids present in equal quantities in extracts of the same stage or expressed in greater quantities in pGPI-PLC/L. amazonensis are marked with asterisks. (B) Neutral lipids from [3H]EtN-labeled cells. Extracts were incubated in 1X AB with or without GPI-PLC, and analyzed as described in panel A. Lane 1, pX63NEO/L. amazonensis, no GPI-PLC digestion; lane 2, GPI-PLCL. amazonensis, no GPI-PLC digestion; lane 3, pX63NEO/L. amazonensis treated with GPI-PLC; lane 4, pGPI-PLC/L. amazonensis, with GPI-PLC.

cleaving them (or a biosynthetic intermediate preceding their production). The Leishmania GPI biosynthetic machinery appears to be unable to replenish EtN-GPIs as fast as GPI-PLC can

cleave them. Evidence for this comes from studies of promastigotes in which an EtN-GPI deficiency led to secretion of gp63 into the culture medium because of a shortage of cellular GPI anchors [18].

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There is no evidence of a generalized reduction in amastigote metabolism because of GPI-PLC expression. Three observations support this assertion: (1) the TLC profile of glycolipids extracted from [3H]Gal-labeled amastigotes is comparable for both pX63NEO/L. amazonensis and pGPIPLC/L. amazonensis (Fig. 4A, lanes 3 and 4); (2) a fortuitous internal ([3H]EtN-labeled lipid) control marked with asterisk in lanes 1 and 2 of Fig. 4A, is present in equivalent amounts in both pX63NEO/L. amazonensis and pGPI-PLC/L. amazonensis, and (3) no significant differences were observed in the [3H]EtN-labeled neutral lipids present in the chloroform/methanol extracts of pX63NEO/L. amazonensis and pGPI-PLC/L. amazonensis (compare Fig. 4B, lanes 1 and 2). Thus, in vivo GPI-PLC appears to deplete GPIs selectively.

conclude that the GPI-PLC does not cause a PI deficiency in Leishmania.

3.4. Effect of a GPI deficiency on amastigote growth The GPI deficiency appears to affect amastigote replication. Promastigotes of pGPI-PLC/L. amazonensis had a growth rate similar to pX63NEO/ L. amazonensis promastigotes in Medium 199 containing 400 mg ml − 1 or less of G418 (Fig. 6). In 800 mg ml − 1 of G418, pX63NEO/L. amazonensis grew normally, as compared to wild-type untransfected cells growing in the absence of drug, but the generation time of pGPI-PLC/L. amazonensis doubled (data not presented). In addition, the latter cells appeared to enter stationary phase when their density was two to three-fold less than that of pX63NEO/L. amazonensis (data

3.3. GPI-PLC does not cause a phosphatidylinositol deficiency in Leishmania Phosphatidylinositol is a very poor substrate for GPI-PLC [12,14,35]. However, PI can be cleaved if an excess amount of GPI-PLC is used in combination with prolonged incubation of enzyme and phospholipid [14,36]. We demonstrate here that no deficiency of PI accompanied GPIPLC expression in Leishmania. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and sphingomyelin (SM) are not substrates for GPIPLC. Hence, by comparing the relative proportion of PI to these phospholipids one could determine whether a PI deficiency arose when GPI-PLC was expressed in Leishmania, which have no endogenous GPI-PLC. First, a comparison to known amount of standard phospholipids (Fig. 5, lanes 1–4) enabled us to obtain an estimate of the quantity of phospholipids in a given number of cells. The major phospholipids in amastigotes were PI (approximately 2 mg per 108 cells), PC (2–4 mg per 108 cells), PE (approximately 2 mg per 108 cells), and PS (1 mg per 108 cells) (Fig. 5). More importantly, the ratio of PI to any of the other phospholipids was comparable between cells with and without GPI-PLC (Fig. 5, compare lane 5 to lane 6). From these data we

Fig. 5. Effect of T. brucei GPI-PLC on phospholipids in Leishmania. Cellular lipids (from 108 amastigotes) or phospholipid standards (PE, PI, PS, PC and SM) were resolved by HPTLC and detected with molybdenum blue. Lane 1, 4 mg each PE, PI, PS, PC and SM; lane 2, 2 mg each of phospholipid; lane 3, 1 mg each of phospholipids; lane 4, 500 ng each of phospholipids; lane 5, amastigote phospholipids from pX63NEO/L. amazonensis; lane 6, amastigote phospholipids from pGPI-PLC/L. amazonensis.

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50 amastigotes per infected macrophage (Fig. 7A). In contrast, GPI-deficient L. amazonensis gradually decreased in number after day 15, reaching at day 27 a level of approximately five amastigotes per macrophage, 4-fold less than the number ob-

Fig. 6. Growth of promastigotes in medium containing 400 mg ml − 1 G418. Promastigotes of pX63NEO/L. amazonensis (“) and pGPI-PLC/L. amazonensis () were seeded in complete medium containing 400 mg ml − 1 G418. Parasites were counted daily with a haemocytometer.

not shown). (These observations are in agreement with previous findings with GPI-PLC transfectants of L. major promastigotes [18].) GPI-PLC expression in L. amazonensis can be modulated by G418 concentration to a level that has little effect on promastigote growth (Fig. 6) but surprisingly arrests the replication of amastigotes (Fig. 7). We examined the ability of promastigotes to establish and sustain intracellular parasitism in a mouse J774G8 macrophage cell line. These cells were infected with parasites that had been cultured continuously in either 400 or 800 mg ml − 1 of G418 but were grown to stationary phase in G418-free medium just before inoculation. Both pX63NEO/L. amazonensis and pGPI-PLC/L. amazonensis infected the macrophages to a similar extent, as assessed 5 days after infection: 85 – 100% of the macrophages were infected, and each harbored an average of 18 parasites (Fig. 7A). Significant differences were observed between the two transfectants in their intracellular proliferation as amastigotes (Fig. 7A): Control L. amazonensis increased in number steadily (i.e. beyond day 15), reaching at day 27 an average of about

Fig. 7. Proliferation of amastigotes inside macrophages. (A) Mouse macrophages were infected with promastigotes. Average number of intracellular parasites per infected macrophage was determined microscopically every 3 – 5 days. (“) pX63NEO/L. amazonensis amastigotes; () pGPI-PLC/L. amazonensis amastigotes. (B) Total number of parasites per culture in (A) (“) pX63NEO/L. amazonensis; () pGPI-PLC/ L. amazonensis.

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Fig. 8. Diminished virulence of pGPI-PLC/L. amazonensis. Hamsters were infected with promastigotes. Lesions produced were measured at stated intervals post-infection. (“) pX63NEO/L. amazonensis; () pGPI-PLC/L. amazonensis.

served initially at day 5. GPI-deficient Leishmania harbored 10-fold less amastigotes per macrophage at day 27 than control parasites (Fig. 7A). Consequently, a 10-fold difference in the total number of parasites per culture was observed between the two groups of parasites: at day 27 there were pGPI-PLC/L. amazonensis 2.1 9 0.7 × 107 amastigotes as compared to 2.290.35 ×108 amastigotes of pX63NEO/L. amazonensis (Fig. 7B).

3.5. GPI-deficient Leishmania are less 6irulent in an animal model of cutaneous leishmaniasis Virulence of GPI-deficient L. amazonensis and pX63NEO/L. amazonensis was compared by their ability to produce lesions in hamster. Although both parasites produced lesions, those caused by GPI-deficient L. amazonensis developed slower with a delay of several weeks and were smaller. The lesions developed exponentially in size between weeks 4 and 6 with pX63NEO/L. amazonensis, and between weeks 4 and 9 with pGPI-PLC/L. amazonensis infection (Fig. 8). At week 6, lesions produced by pX63NEO/L. amazonensis were four times larger than those produced by the GPI-deficient parasites (Fig. 8). Although all lesions became fully developed be-

tween weeks 9 and 10, they were twice larger in pX63NEO/L. amazonensis infected hamsters than in the pGPI-PLC/L. amazonensis infections. Parasites were 20-fold fewer in lesions caused by GPI-deficient L. amazonensis (1× 108 at week 10) than those found in pX63NEO/L. amazonensis-infected hamsters (2×109 parasites at week 10) (Fig. 8). These data are consistent with the magnitude of the difference observed in replication of pGPI-PLC/L. amazonensis and pX63NEO/L. amazonensis in macrophage infections (Fig. 7B). We surmise that a GPI-deficient strain of L. amazonensis is less virulent than the control pX63NEO/L. amazonensis, since the former produced smaller lesions with fewer amastigotes.

3.6. GPI-deficient amastigotes replicate poorly during axenic culture Presumably, the reduced virulence of pGPIPLC/L. amazonensis in hamster (Fig. 8) was due to the inability of amastigotes to replicate. To test this hypothesis, axenic amastigotes of both lines were cultured without macrophages in media containing 800 mg ml − 1 G418. At this drug concentration, amastigotes from pX63NEO/L. amazonensis grew, whereas those from GPI-deficient pGPI-PLC/L. amazonensis did not (data not shown). In 400 mg ml − 1 of G418 (Fig. 9), a drug concentration with no discernible effect on promastigote proliferation (Fig. 6), GPI-deficient amastigotes grew poorly, although repeated subculturing was possible. In contrast, the control cells grew exponentially (Fig. 9). Coincident with the entry of control pX63NEO/L. amazonensis into the stationary phase beyond day 5 (Fig. 9), GPI-deficient amastigotes began dying off, leading to a reduction in number (Fig. 9). At day 8, there were 20-fold more control cells (pX63NEO/ L. amazonensis, 3 × 107 ml − 1) than GPI-deficient amastigotes (1.5× 106 ml − 1) (Fig. 9). We quantitated GPI-PLC levels to determine whether the observed cytostasis of pGPI-PLC/L. amazonensis amastigotes was due to a large increase of GPI-PLC activity in this stage of the life cycle. Results obtained from cells grown in medium with 800 mg G418 ml − 1 indicate other-

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wise: promastigotes expressed 2.5-fold more GPIPLC per cell than amastigotes (93.2 U per 108 cells vs 36.2 U per 108 cells). Thus, both stages of transfectants were comparable in the specific activity of GPI-PLC, since promastigotes contain 2 – 4-fold more total protein than amastigotes. Hence, cytostasis of pGPI-PLC/L. amazonensis amastigotes is not due to a dramatic stage-specific increase in the GPI-PLC activity. Together, these data suggest that: (1) a GPI deficiency of L. amazonensis causes cytostasis of amastigotes; and (2) amastigotes appear to be more susceptible to a GPI deficiency than promastigotes.

4. Discussion

4.1. Deficiency of two amastigote GPIs is associated with cytostasis Genes which may be used for targeted mutagenesis of the ‘EtN-GPI core’ (EtN-Man3-GlcN-Ins1-phospho-lipid) biosynthetic pathway have not

Fig. 9. Growth of axenic amastigotes in medium containing 400 mg ml − 1 G418. Growth rate of axenic amastigotes seeded at 5 × 106 ml − 1 was determined. (“) pX63NEO/L. amazonensis; () pGPI-PLC/L. amazonensis.

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been isolated from any protozoan parasite as yet. Consequently, mutants in the ‘EtN-GPI core’ have not been described in the Leishmania. Given our interest in studying the effects of a GPI deficiency on intracellular parasitism, we generated phenotypic GPI mutants by transfecting Leishmania with a GPI-PLC gene from T. brucei. GPI-PLC cleaves GPI intermediates in vivo conferring on the transgenic strain a phenotype equivalent to a dominant negative loss-of-function mutation in the GPI pathway [18]. Our studies were greatly facilitated by using an episomal vector for GPI-PLC expression [22]. GPI-PLC content of L. amazonensis transfectants was regulated indirectly by adjusting the level of G418: raising the drug concentration from 125 to 500 mg ml − 1 increased the amount of GPI-PLC expressed from 499 to 1241 U per 109 cells. Consequently, GPI-PLC effects could be rendered ‘conditional’ to G418 concentrations, enabling us to explore the biological consequences of this potentially lethal biochemical deficiency. Because there have been no reports of Leishmania that are completely devoid of GPIs, to our knowledge, it is possible that GPIs are so essential to the parasites that it will be extremely difficult to obtain null mutants without employing special selection schemes after mutagenesis. The difficulty of pGPI-PLC/L. amazonensis to grow in medium containing 800 mg ml − 1 G418 and the normal growth of control pX63NEO/L. amazonensis provides indirect support for this suggestion. Unlike promastigotes, amastigotes of Leishmania amazonensis do not appear to have a large quantity of GPI-anchored proteins. Therefore, it seems unlikely that AmGPI-Z and AmGPI-Y are used for anchoring such proteins. Instead, AmGPI-Z and AmGPI-Y may be analogous to GIPLs, which are found unattached to macromolecules on the plasma membrane of eukaryotes, including Leishmania [3,9,37]. Since their depletion is associated with amastigote cytostasis, AmGPI-Y and AmGPI-Z appear to be required for viability and/or replication of the parasite. The most likely explanation for cytostasis of pGPI-PLC/L. amazonensis (Figs. 6–8), in the apparent absence of GPI-anchored proteins, rests on the depletion of AmGPI-Z and

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AmGPI-Y per se. The possibility remains, however, that AmGPI-Y serves as the anchor for essential GPI-anchored proteins that are present at undetectable levels. Regardless, our observations raise the possibility that Leishmania infections may be intervened by drugs interfering with GPI biosynthesis (reviewed in [7]). Evidence indicates that a PI deficiency is not responsible for the phenotype observed for GPIPLC-expressing amastigotes. PI deficiency could not be documented in Leishmania which express the T. brucei GPI-PLC (Fig. 5). This result is not surprising for three reasons. First, PI is a major phospholipid in Leishmania; there are about 1.2 ×107 molecules cell − 1. Second, PI is a very poor substrate for GPI-PLC, to the extent that when a GPI and PI are presented simultaneously to the enzyme as substrates, the GPI is preferentially cleaved (Morris, J.C. and K. MensaWilmot, unpublished). In effect, the relatively large pool of PI coupled with it being a poor substrate make it unlikely that a deficiency of that phospholipid occurs when GPI-PLC is expressed in Leishmania. Finally, PI inhibits GPI-PLC cleavage of GPI [33]. It is reasonable, given these observations, to ascribe the phenotype of GPIPLC-expressing Leishmania to a GPI deficiency.

4.2. A GPI deficiency decreases 6irulence of Leishmania Lipophosphoglycan (LPG) (reviewed in [10]) and gp63 [38,39] are virulence factors in promastigotes of Leishmania. Expression of both molecules is down regulated in amastigotes, indicating that those macromolecules may play roles less significant in the parasite after establishment of an infection. The studies reported here, (Figs. 7 – 9), suggest that EtN-containing GPIs may be virulence factors in amastigotes.

4.3. GPI-PLC-like acti6ity and intracellular parasitism: possible effects on transmission of Leishmania GPI-PLC which is endogenous to T. brucei has not been found in Leishmania [18,40]. This is surprising because both parasites express over

5× 105 molecules of GPI-anchored macromolecules per cell, namely LPG and gp63 in Leishmania promastigotes, and VSG and procyclin in T. brucei bloodstream form and procyclics, respectively. Since GPI-PLC of T. brucei can cleave VSG [11], GPI-PLC might be expected to exist in Leishmania, similarly, to release gp63. Did Leishmania originally have GPI-PLC that was subsequently lost? Or was the GPI-PLC gene acquired by T. brucei after its evolutionary separation from Leishmania? A complete answer to this question rests on the identification of GPI-PLC or its homologue in an ancestral kinetoplastid from which these organisms diverge. In the meantime, the following observations might be worthy of mention: First, T. brucei has no intracellular stage, and GPI-PLC is expressed in this parasite without any discernible growth-inhibitory effects. In fact, deletion of the GPI-PLC gene results in decreased virulence of the parasite [41]. Second, proliferation of T. cruzi that has been engineered to express T. brucei GPI-PLC is dramatically inhibited at the intracellular amastigote stage [20]. Third, a GPI-PLC gene is detectable in T. equiperdum (Shapiro, T. and Mensa-Wilmot, K., unpublished) which like T. brucei has no amastigote stage. We propose that a membrane-bound GPI-PLClike activity is most likely incompatible with efficient intracellular parasitism. Thus, Leishmania ‘variants’ with GPI-PLC-like activities might have been selected against at the amastigote stage of parasite evolution. References [1] McConville MJ, Ferguson MAJ. The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem J 1993;294:305 – 24. [2] Ueda E, Sevlever D, Prince GM, et al. A candidate mammalian glycoinositol phospholipid precursor containing three phosphoethanolamines. J Biol Chem 1993;268:9998– 10002. [3] Singh N, Liang L-N, Tycocinski ML, Tartakoff AM. A novel class of cell surface glycolipids of mammalian cells. J Biol Chem 1996;271:12879– 84. [4] Turco SJ, Descoteaux A. The lipophosphoglycan of Leishmania parasites. Annu Rev Microbiol 1992;46:65 – 94.

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