Leishmaniasis, a vector-borne worldwide-distributed parasitic disease, is caused by dimorphic protozoan flagellates of the genus Leishmania
. This organism alternates between two different stages: the promastigote lives extracellular in its vector, the phlebotomine sandfly (Diptera, Psychodidae), and the amastigote resides within the phagolysosome of mononuclear macrophages of mammalian hosts. The disease is characterized by diversity and complexity, presenting a wide spectrum of clinical forms in humans, ranging from cutaneous leishmaniasis (CL) to fatal visceral leishmaniasis (VL) [1
Recent studies have reported the diffusion of new “hot spots” of canine leishmaniosis in previously non-endemic areas of the northern United States and some provinces of southern Canada and northern Europe [3
]. Leishmaniosis seems to spread because of a combination of factors: environmental/climatic changes, factors related to the immune status of the host and drug resistance [6
]. Therefore, over the last decades, considerable efforts have been directed towards the research into new proteinacious targets for the development of an effective therapy against human VL.
Phosphorylcholine (PC, Figure 1
) has been recognized as a common antigenic determinant in many important disease-causing parasites, such as, gastrointestinal and filarial nematodes [8
] but also in protozoa like Trypanosoma
PC-bearing antigens have been found to possess immunomodulatory capacity and to interfere with key proliferative signaling pathways in B- and T-cells, dendritic cell maturation and mast cell degranulation, thus facilitating the survival of parasites in their hosts [8
]. Therefore, these effects could contribute to the observed modulated cytokine levels and impairment of lymphocyte proliferation. Detailed data on the different types of PC-carrying biomolecules as well as their biosynthesis, however, are limited and have only been reported in the last few years in nematodes [8
Structural analyses of nematode-derived molecules with PC epitopes focused, so far, on glycolipids and glycoprotein glycans. It could be shown that glycosphingolipids of the pig parasitic nematode, Ascaris suum
, are characterized by the presence of a phosphodiester-bound PC-substituent which has been assigned to C-6 of the central N
-acetylglucosamine (GlcNAc) residue of an arthro-series carbohydrate core [22
]. Furthermore, some glycolipid species were found to carry phosphorylethanolamine linked to C-6 of an adjacent mannose residue in addition to PC [23
]. Comparable glycosphingolipids have been verified in different nematodes, including Nippostrongylus brasiliensis
], Litomosoides sigmodontis
], Onchocerca volvulus
and Setaria digitata
], indicating that arthro-series glycosphingolipids carrying, in part, PC substituents represent highly conserved glycolipid markers within the nematode phylum. A biosynthetic route homologous to A. suum
glycosphingolipids was also confirmed for the free-living nematode Caenorhabditis elegans
Analogous analyses of the PC-substituted glycoprotein ES-62, an excretory/secretory (ES) product of Acanthocheilonema viteae
, indicated that the zwitterionic substituent is linked via N
-glycans to the polypeptide backbone [30
]. Mass spectrometric analysis of the respective N
-linked glycans revealed the presence of trimannosyl N
-glycan variants, carrying between one and four terminal GlcNAc residues [32
]. Only this type of glycans was found to be substituted with PC-moieties which could be again assigned to C-6 of terminal GlcNAc residues [33
]. Comparative studies of N
-glycans present in extracts of A. viteae
, Onchocerca gibsoni
and O. volvulus
confirmed a high conservation of such PC-substituted N
-glycans within filarial parasites [35
]. For C. elegans
, two types of PC-substituted N
-glycans have been reported so far: (1) a pentamannosyl-core structure carrying up to three PC-residues [36
] and (2) trimannosyl-core species elongated by GlcNAc residues substituted at C-6 with PC [33
]. Furthermore, combinations of both types of structural motifs [19
] as well as the occurrence of extended glycan structures with composition of PCho1-2
have been reported [18
]. For A. suum
hybrid-type bi- and triantennary N
-glycans substituted with PC have been described [21
PC-modified proteins were also detected in different developmental stages of the malarial blood parasite Plasmodium falciparum
]. Fourteen putative proteins carrying the PC modification were identified, among them, proteins that are located on the surface of the parasite, or are involved in metabolism. Erythrocyte membrane protein 1 (EMP1, a member of the var
gene family), together with the heat shock protein 70 (HSP-70), was detected in every P. falciparum
stage of the erythrocyte pathway. In P. falciparum
genes encode adhesive proteins that are transported to the surface of infected erythrocytes, thereby acting as major virulence determinants for immune evasion [38
]. There is increasing evidence that HSP-70 could play an important role in the life cycle of P. falciparum
both as a chaperone and as immunogen [40
]. In the merezoite-stage, only one surface protein (EMP1, P154varH), was detected.
The eukaryotic elongation factor-1α (eEF1α) is an enzyme that catalyzes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein synthesis and is involved in the capture of deacylated tRNA [41
]. Furthermore, eEF1α was found to serve as a central hub in protein networks with hundreds of interacting partners [43
eEF1α was found to bind and activate the Src-homology 2 domain containing protein tyrosine phosphatase-1 (SHP-1), a protein known to be involved in the macrophage inactivation pathogenesis of leishmaniasis [41
]. Additionally, eEF1α was found in Leishmania
exosomes and identified as an important factor for immunosuppression and priming host cells for Leishmania
In this study, we identified eEF1α as the only PC-positive protein found in Leishmania infantum MON-1 by a 2D-gel proteomic approach. Furthermore, we confirmed the presence of PC modifications by quantitative determination of its choline content. Additionally, we localized the PC epitopes within procyclic and stationary phase promastigotes by confocal microscopy. Finally, we were able to demonstrate that the interaction of L. infantum EF1α and human SHP-1 is dependent on the PC modification of EF1α.
2. Materials and Methods
2.1. Cultivation of Leishmania infantum Promastigotes
zymodeme MON-1 promastigotes were cultured in Tobie–Evans modified medium at 24 °C [46
]. Promastigotes at day 3 or 10 of culture were used. The liquid phases were centrifuged at 1000× g
for 5 min at 4 °C. Supernatant was then discarded and the pellet was resuspended in phosphate buffered saline (PBS) pH 7.2, and washed 3 times by centrifugation at 1000× g
for 10 min.
L. infantum promastigotes were washed twice in PBS before fixation in 200 µL of 1% formaldehyde in PBS for 30 min at room temperature (RT). After a PBS wash, the cells were permeabilized by resuspension in 200 µL of 0.1% Triton X-100 in PBS for 10 min. Following an additional PBS wash, the cells were resuspended in 200 µL of 0.1 M glycine in PBS and incubated for a further 10 min at RT before being washed in PBS. Glass slides were washed with 70% ethanol and coated with a 0.01% solution of poly-l-lysine (0.1% stock; Sigma Alrich, Taufkirchen, Germany), and the fixed, permeabilized cells were then left to sediment and adhere to the surfaces of these polylysine-coated slides for 15 min at RT.
Monoclonal mouse TEPC-15 (Sigma Aldrich, Taufkirchen, Germany) diluted 1:1000 in TB buffer (0.1% (v/v) Triton X-100, 0.1% (w/v) bovine serum albumin (BSA) in PBS), was added to the slide and incubated with the cells overnight at 4 °C. After a 10-mL PBS wash, cells were incubated in the dark for 1 h at RT with FITC-conjugated secondary antibodies (LifeTechnologies, Darmstadt, Germany) diluted 1:500 in TB buffer. Unbound secondary antibody was washed away with 1.5 mL PBS (3-times 0.5 mL).
The cells were then covered with 10 µL of Vectashield mounting medium with DAPI for staining of the cellular DNA (Vector Laboratories, Burlingame, CA, USA).
Preparations were examined with a Zeiss LSM 710 confocal microscope using a 488 nm laser for FITC and a 405 nm laser for DAPI. The instrument control and image analysis were done with the software ZEN (version 2012, blue edition; Zeiss, Wetzlar, Germany).
2.3. Protein Isolation
Parasites were prepared as described in section “Cultivation of Leishmania infantum
promastigotes”. Approximately 10 mg of pelleted promastigotes were homogenized in 30 μL 0.2% (w
) sodium dodecylsulfate (SDS; Roth, Karlsruhe, Germany) and boiled for 5 min at 95 °C. After cooling on ice, the sample was extracted with 370 µL lysis buffer consisting of 6 M urea (Sigma, Taufkirchen, Germany), 2 M thiourea (Sigma, Taufkirchen, Germany), 1% Triton X-100 (Fluka, Selze, Germany), 65 mM dithiothreitol (DTT; Fluka, Selze, Germany), 0.5% IPG-buffer pH 3–10 (GE Healthcare, Freiburg, Germany), 0.1 mM phenylmethylsulfonylfluoride (PMSF; Sigma, Taufkirchen, Germany), and Protease Inhibitor Cocktail for general use (Sigma Alrich, Taufkirchen, Germany). Sample homogenization was done by four pulses of 30 s in a bullet blender using 1.4 mm stainless steel beads (Next Advance, New York, NY, USA) with intermediate cooling on ice and centrifugation (20,000× g
, 1 h at 4 °C). To remove lipid contaminants and SDS proteins were precipitated with chloroform/methanol (1:4 by v
). For isoelectric focusing, the protein pellet was dissolved in 100 μL lysis buffer containing 4% 3-3′-(Cholamidopropyl)-3,3-dimethylammoniumpropylsulfat (CHAPS; Roth, Karlsruhe, Germany) instead of Triton X-100 and 2% IPG-buffer pH 3–10 [47
2.4. Two-Dimensional Gel Electrophoresis and Detection of PC-Modified Proteins
Two-dimensional separation, Western blotting of proteins and detection of PC-modified proteins was performed as described in [47
]. For preparative gels 0.4–0.5 mg, and for Western blot analysis 25 µg of the extracted protein were loaded.
eEF1α was detected by the specific antibody PA5-17213 (Thermo Scientific, Dreieich, Germany, 1:2000 dilution) with horseradish peroxidase conjugated anti-rabbit Ig (DakoCytomation, Glostrup, Denmark, 1:2000) as secondary antibody. Proteins recognized by the antibodies were visualized by enhanced chemiluminescence using the ECL SuperSignal kit (GE Healthcare, Solingen, Germany). The corresponding protein spots were excised from preparative gels with the ExQuest™ Spot Cutter (Bio-Rad, Munich, Germany) and transferred into 96-well plates (Greiner Bio-One, Frickenhausen, Germany).
2.5. Tryptic in-Gel Digestion of Proteins, Matrix-Assisted Laser-Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOFMS) and Database Search
Performed as described in [47
2.6. Choline Quantification
For the quantitation of choline-substitution of eEF1α, 400 µg of total protein from L. infantum
were separated on a 2D-gel as described above and stained with FlamingoTM
according to the instructions of the manufacturer (BioRad, München, Germany). The absolute amount of eEF1α was determined by densitometric analysis using Quantity One 4.6.2 (Bio-Rad, Munich, Germany) using lysozyme (14.3 kDa, 5 µg), β-lactoglobulin (18.4 kDa, 3 µg), aldolase (36 kDa, 2 µg), and bovine serum albumin (66 kDa, 1 µg; all from Sigma Aldrich, Taufkirchen, Germany) as standards (see results). The spots containing 1.9 and 2.1 respectively µg of EF1α were cut from the gels and tryptic digestion was performed as described above. PC residues were removed from the peptides by cleavage of the phosphodiester bonds with hydrogen fluoride: one sixth (6.26/7.25 pmol) of the tryptic digest of eEF1α was lyophilized, dissolved in 50 µL of hydrofluoric acid (48%, Merck, Darmstadt, Germany) and incubated on ice overnight. The sample was dried under a stream of nitrogen, dissolved in 500 µL of water and lyophilized. Choline was measured by HPLC according to published methods [48
2.7. Co-Purification of Leishmania EF1α with Human SHP-1
Approximately 50 mg of pelleted promastigotes were extracted with 1 ml RIPA buffer (25 mM TRIS-HCl pH 7.6, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS), half of the extract was supplemented either with 2 µg of GST-SHP-1 (Jena Bioscience, Jena, Germany) alone or in presence of phosphorylcholine (5 mM final concentration; Sigma Aldrich, Taufkirchen, Germany). To get rid of residual glutathione from the preparation of GST-SHP-1, the buffer was exchanged three times by ultrafiltration (10 kDa cutoff; Amicon, Darmstadt, Germany). After an incubation on ice for three hours, the solutions were separately passed two times through 50 µL glutathione agarose (Jena Bioscience, Jena, Germany), pre-incubated with RIPA or RIPA/PC respectively. Proteins were then eluted with 80 µL of twofold concentrated Laemmli sample buffer and subjected to SDS-PAGE. Western blotting and detection was performed as described above using 3% BSA in TBS-T instead of Roti-Block.
2.8. Staining of Proteins on Western Blot Membranes
To visualize all the proteins transferred to the PVDF membranes these were incubated with black Fount India Ink (Pelikan, Hannover, Germany; diluted 1:1000 in PBS-T) for at least five hours, washed five times with pure water and dried.