Structural comparison of lipophosphoglycan from Leishmania turanica and L. major, two species transmitted by Phlebotomus papatasi

PARINT-01243; No of Pages 4 Parasitology International xxx (2014) xxx–xxx

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Short communication

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Petr Volf a, Paula M. Nogueira b, Jitka Myskova a, Salvatore J. Turco c, Rodrigo P. Soares b,⁎

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Article history: Received 19 December 2013 Received in revised form 13 May 2014 Accepted 15 May 2014 Available online xxxx

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Keywords: Leishmania turanica LPG Sand fly–Leishmania interaction Phlebotomus papatasi

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The lipophosphoglycan (LPG) of Leishmania major has a major role in the attachment to Phlebotomus papatasi midgut. Here, we investigated the comparative structural features of LPG of L. turanica, another species transmitted by P. papatasi. The mAb WIC 79.3, specific for terminal Gal(β1,3) side-chains, strongly reacted with L. turanica LPG. In contrast, L. turanica LPG was not recognized by arabinose-specific mAb 3F12. In conclusion, LPGs from L. major and L. turanica are similar, with the latter being less arabinosylated than L. major's. The high galactose content in L. turanica LPG is consistent with its predicted recognition by P. papatasi lectin PpGalec. © 2014 Elsevier Ireland Ltd. All rights reserved.

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1. Introduction

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Protozoan parasites of the genus Leishmania (Trypanosomatidae: Kinetoplastida) cause human diseases ranging from self-healing cutaneous lesions to fatal visceral forms. The parasite has a digenetic life-cycle alternating between a mammalian host and insect vectors, phlebotomine sand flies (Diptera: Phlebotominae). However, sand fly vectors differ in their ability to support infections with different Leishmania species; based upon experimental tests of their ability to support the development of a wide or limited range of Leishmania species, they have been classified as specific (also called restrictive) or permissive vectors. The majority of sand fly species tested to date support development of multiple Leishmania species and thus belongs to the permissive group. In contrast, there appears to exist a close evolutionary fit between Phlebotomus papatasi and Phlebotomus duboscqi with Leishmania major and Phlebotomus sergenti with Leishmania tropica, as other Leishmania species survive poorly in these sand flies [1,2]. Natural barriers to Leishmania development within the sand fly midgut during bloodmeal digestion include secreted proteolytic enzymes, the peritrophic matrix surrounding the ingested blood and likely sand fly immune reactions. As the blood digestion proceeds, parasites need to bind to the midgut epithelium to avoid being excreted

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Department of Parasitology, Faculty of Science, Charles University, Prague 2 128 44, Czech Republic Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, 30190-002 Belo Horizonte, MG, Brazil Department of Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536, USA

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Structural comparison of lipophosphoglycan from Leishmania turanica and L. major, two species transmitted by Phlebotomus papatasi

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⁎ Corresponding author at: Laboratório de Biomarcadores de Diagnóstico e Monitoração, Centro de Pesquisas René Rachou/FIOCRUZ, Av. Augusto de Lima, 1715, 30190-002 Belo Horizonte, MG, Brazil. Tel.: +55 31 3349 7760; fax: +55 31 3295 3115. E-mail address: rsoares@cpqrr.fiocruz.br (R.P. Soares).

with the blood remnant and this midgut binding is the key determinant of parasite–vector specificity [1,3]. The midgut binding is strictly stagedependent as it is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms), but is absent in the early blood meal and final stages (procyclic and metacyclic forms, respectively) [4]. The mechanism of midgut attachment has been most intensively studied in the specific vector P. papatasi infected with L. major and the role of parasite surface lipophosphoglycan (LPG) has been repeatedly demonstrated [5–10]. LPG is an abundant glycolipid that covers the entire parasite surface, including the flagellum. Its basic structure consists of a glycosyl–phosphatidyl–inositol lipid anchor attached through a hexasacharide core to a polymer of phosphoglycan repeating units terminated by a small neutral oligosaccharide cap. The PG repeating units are often modified by strain-, species-, and stage-specific side-chain sugar residues [11]. Studies using LPG-deficient parasites confirmed the crucial role of LPG in the attachment of L. major in the midgut. The ability to persist in the midgut of P. papatasi or P. duboscqi following bloodmeal excretion was completely lost in these parasites [2,7,12] and this defect was correlated with their inability to bind to midgut epithelial cells in vitro [7,12]. Leishmania turanica is widely distributed in Central Asia, Iran, Mongolia and China, often sympatrically with more pathogenic L. major. Similarly to L. major the main reservoir host of L. turanica is the great gerbil Rhombomys opimus while P. papatasi is generally considered as its main vector [13–15]. Recently, the development of L. turanica in different sand fly species was tested and mature infections with the colonization of the stomodeal valve in P. papatasi but not in P. Sergenti

http://dx.doi.org/10.1016/j.parint.2014.05.004 1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: Volf P, et al, Structural comparison of lipophosphoglycan from Leishmania turanica and L. major, two species transmitted by Phlebotomus papatasi, Parasitology International (2014), http://dx.doi.org/10.1016/j.parint.2014.05.004

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only the LPG from L. major reacted with the 3F12 antibody indicating the presence of the Ara(β1,2)Gal(β1,3) epitope (Fig. 1, 3F12, lane 1). As expected, LPG from late log stage promastigotes of L. major possessing a certain amount of Ara capping of side chains was reactive (Fig. 1, 3F12, lane 1) while L. donovani LPG whose repeat units are not terminated with Ara(β1,2) residues, was negative (lane 3). Interestingly, the LPG from L. turanica was not recognized by 3F12 antibodies (Fig. 1C, lane 2). Due to similarities between L. turanica and L. major LPG found by western blots we decided to analyze LPG repeat units of these two species with a more sensitive technique, fluorophore-assisted carbohydrate electrophoresis (FACE). LPGs were depolymerized by mild acid hydrolysis (0.02 N HCl, 5 min, 100 °C) that produced two pools: i) a glycancore-PI fraction and ii) a mixture of phosphorylated repeat units and neutral oligosaccharide caps. The mixture of repeat units and caps were separated from the glycan core-PI by n-butanol: water (2:1) partitioning. The neutral caps were then resolved from the charged repeat units by anion exchange chromatography on a column of AG1-X8 (acetate) in water; repeat units were collected with 0.3 M NaCl and desalted using a P2 column eluted with water [21]. Phosphorylated repeat units were dephosphorylated with alkaline phosphatase (Sigma) in 15 mM Tris–HCl, pH 9.0 (1 U, 16 h, 37 °C), and desalted by passage through a two-layered column consisting of AG50W-X12 cation-exchange resin over AG1-X8 anion-exchange resin (Bio-Rad) [19]. The repeat units were fluorescently labeled with 0.05 N ANTS (8-aminonaphthalene1,3,6-trisulfate) and 1 M cyanoborohydride (37 °C, 16 h) and analyzed using FACE as described [21]. The FACE profiles of dephosphorylated repeat units of L. major and L. turanica are given on Fig. 2, the assignments of the L. major FV1 structures in the figure was done as previously described [17,21]. The profile of L. turanica was very similar to L. major, indicating the presence of side-chains up to G4–G5 positions (Fig. 2). In order to provide a better biochemical characterization of the side-chains, neutral oligosaccharides of L. major and L. turanica were treated with E. coli β-galactosidase (Sigma) in 80 mM Na3PO4, pH 7.3 (4 U, 16 h, 37 °C), desalted and analyzed by FACE, see Fig. 3. The intensities of the bands co-migrating with standard maltose G2 disappeared and thus is consistent with the expected disaccharide Gal(β1,4)Man. In addition, a strong increase in the monosaccharide content was observed in both strains (Fig. 3, FV1 and BZ18, lower arrows). Similarly, the bands co-migrating with standard G3, consistent with the structure of GalβGalβMan for the trisaccharide, also disappeared in both L. major and L. turanica. As expected, the tetrasaccharide Ara-Gal-Gal-Man, terminated by arabinose, was not cleaved by the enzyme (Fig. 3, FV1, upper arrow). Interestingly, this band was also observed in the L. turanica BZ18 (Fig. 3, BZ18, upper arrow), but was much fainter compared to L. major FV1. This profile indicates the

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were observed [16]. It was proved that L. turanica is able to establish the late-stage infections in this specific vector of L. major where the unique structure of L. major LPG side chains is thought to be responsible for the specificity of sand fly–Leishmania interaction [6–8]. In this study, we therefore focused on the characterization of L. turanica LPG. The ability of L. turanica to develop in P. papatasi suggests that the LPG of this species should be either identical or similar to that of L. major to mediate the binding to P. papatasi galectin. An alternative hypothesis expected that L. turanica LPG may differ from L. major and this Leishmania species binds to the P. papatasi midgut using another mechanism. L. turanica strain BZ18 (MRHO/MN/08/BZ18) was compared with control strains for which LPG structures have been fully characterized [17,18]: L. major Friedlin V1 strain (MHOM/IL/80/Friedlin) and L. donovani LD4 strain (MHOM/SD/00/1S-2D). Promastigotes were grown in Medium 199 (Gibco Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals) (FBS), penicillin (100 U/ml), streptomycin (50 μg/ml), 12.5 mM glutamine, 40 mM HEPES, pH 7.4, 0.1 M adenine, and 0.0005% hemin, at 25 °C [19]. LPGs from late log phase parasites were extracted in solvent E (H2O/ethanol/diethylether/pyridine/NH4OH; 15:15:5:1:0.017) and the extract was dried by N2 evaporation, resuspended in 0.1 N acetic acid/ 0.1 M NaCl, and applied to a column of Phenyl-Sepharose (Sigma, 2 ml), equilibrated in the same buffer. LPG was eluted using solvent E [20]. Purified LPGs (10 μg) were resolved by SDS-PAGE (12%), transferred to a nitrocellulose membrane and blocked (5% milk in PBS–0.1% Tween 20) for 1 h. Primary antibodies were incubated for 16 h at 4 °C. Those included monoclonal antibodies of known carbohydrate specificities: WIC 79.3 (1:1000), which recognizes terminal Gal(β1,3) sequences that branch off the Gal(β1,4)Man(α1)-PO4 repeat units; CA7AE (1:1000), which recognizes the Gal(β1,4)Man(α1)-PO4 repeat units; and 3F12 (1:500), which recognizes terminal Ara(β1,2)Gal(β1,3) sequences in L. major LPG [21]. Membranes were washed (3 × 10 min) three times in PBS (2.68 mM KCl, 1.47 mM KH2PO4, 136.89 mM NaCl, 8.10 mM Na2HPO4, pH 7.4) for 10 min, then the membrane was incubated with anti-mouse IgG conjugated with horseradish peroxidase (1:10,000, 1 h) and the reaction was visualized using luminol [22]. The antibodies WIC 79.3, specific for terminal Gal(β1,3) side-chains, strongly reacted with the LPGs purified from L. major and L. turanica (Fig. 1, WIC 79.3, lanes 1 and 2); as expected, it gave a negative reaction with L. donovani LPG (lane 3). On the other hand, CA7AE antibodies, specific for multiple Gal(β1,4)Man(α1)-PO4 repeat units, recognized L. donovani LPG (Fig. 1, CA7AE, lane 3) but did not react with L. major and L. turanica (lanes 1 and 2). This is an indication that the LPG from L. turanica possesses side-chains being similar to L. major FV1, thus altering the disaccharide phosphate epitope. Finally, as shown in Fig. 1,

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Fig. 1. Western blot analysis of purified LPGs. Immunoblotting of purified LPG (10 μg per lane) from late-log forms of L. major (FV1 — lane 1), L. turanica (BZ18 — lane 2) and L. donovani (LD4 — lane 3) strains probed with the monoclonal antibodies WIC 79.3, CA7AE and 3F12.

Please cite this article as: Volf P, et al, Structural comparison of lipophosphoglycan from Leishmania turanica and L. major, two species transmitted by Phlebotomus papatasi, Parasitology International (2014), http://dx.doi.org/10.1016/j.parint.2014.05.004

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While the midgut attachment is a critical point in Leishmania development at the end of bloodmeal digestion, in the late-stage infection parasites need to detach from the midgut epithelium and produce free-swimming metacyclic forms in order to colonize the thoracic midgut and stomodeal valve and to produce a transmissible infection [1,3]. In L. major metacyclogenesis, the procyclic LPG is replaced by the metacyclic one, which has increased numbers of PG repeats and side-chain galactose residues masked by the addition of terminal arabinose [17]. Thus, the modified metacyclic form of L. major LPG no longer binds to the P. papatasi midgut [5]. As terminal arabinose residues were found also in LPG isolated from late-log promastigotes of L. turanica, our experiments suggest that a similar molecular mechanism of detachment may occur during metacyclogenesis of L. turanica. Interestingly, despite the structural similarities between the LPG of L. major and L. turanica, these two Leishmania species represent welldefined species [23]. The similarity of L. major and L. turanica LPGs seems to be results of convergent coevolution of different parasites with the same specific vector, P. papatasi. Coevolution of Leishmania LPG with local sand flies was previously demonstrated in L. tropica. Two zoonotic foci of cutaneous leishmaniases in northern Israel were found to differ with respect to both L. tropica strains and sand fly species; specific vector P. sergenti or permissive vector P. arabicus were involved in L. tropica circulation [24]. While isolates from P. sergenti focus possessed “typical” L. tropica LPG with side chains capped with glucose, LPG of isolates from P. arabicus focus had abundant terminal β-galactose residues on the side chains [21]. Clearly, the LPG evolution is driven by the vector.

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presence a certain amount of arabinose in the L. turanica repeat units. This amount of arabinose was probably too low to be recognized by 3F12 antibodies in western blots (Fig. 1, 3F12). In P. papatasi, the molecule crucial for midgut binding of L. major was identified as galectin PpGalec [8]; this 35 kDa molecule containing two non-identical carbohydrate recognition domains is strongly upregulated in adult females, appears to be midgut-specific and efficiently binds to galactose side-chains of L. major LPG [8]. Similar galactose sidechains we detected also in L. turanica; most frequently the phosphodisaccharide units are substituted by one or two galactose residues. As this structure fully corresponds with the side chains of L. major FV1 [17,21] we can speculate that L. turanica and L. major bind to the P. papatasi midgut via the same receptor PpGalec. Due to this binding L. turanica survives defecation of bloodmeal remains and successfully competes with L. major during coinfections in the P. papatasi midgut [16].

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Fig. 2. FACE analysis of dephosphorylated LPG repeat units. Lane 1, maltooligomer ladder represented by 1–7 glucose residues (G1–G7); lane 2, repeat units of L. major (FV1 strain) and L. turanica (BZ18 strain). On the right are represented the known side-chain structures of the control strain L. Major (FV1) [17,21]. Legend: Ara, arabinose; Gal, galactose.

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Fig. 3. FACE analysis of dephosphorylated LPG repeat units from L. major FV1 and L. turanica incubated in the presence (+) or absence (−) of β-galactosidase. Lane 1, malto-oligomer ladder represented by G1–G7; lane 2, untreated repeat units of FV1 or BZ18 strains and lane 3, repeat units of FV1 (A) and BZ18 (B) treated with β-galactosidase. Upper arrows indicate arabinosylated repeat units and lower arrows indicate monosaccharide content. Legend: Ara, arabinose; Gal, galactose.

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2. Conclusion

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Our data indicate that the LPGs of L. major and L. turanica are similar, with the latter being less arabinosylated than L. major's. We suggest that both Leishmania species that attach to the P. papatasi midgut using the same mechanism as high galactose content present in L. turanica LPG could be recognized by PpGalec in the midgut of this specific vector. This study provides the first evidence that two distinct Leishmania species possess similar, highly modified LPG which enables them to develop in the same sand fly vector.

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Acknowledgements

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PV and JM are supported by the FP7 project EDENext 2011-261504 227 (paper is catalogued as EDENextXXX). RPS is supported by CNPq 228 Q11 (#301597/2013-8) and PAPES VI (407438/2012-2). 229

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Please cite this article as: Volf P, et al, Structural comparison of lipophosphoglycan from Leishmania turanica and L. major, two species transmitted by Phlebotomus papatasi, Parasitology International (2014), http://dx.doi.org/10.1016/j.parint.2014.05.004

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