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.2016 Apr 29;12(4):e1005606.
doi: 10.1371/journal.ppat.1005606. eCollection 2016 Apr.

Interrogating the Plasmodium Sporozoite Surface: Identification of Surface-Exposed Proteins and Demonstration of Glycosylation on CSP and TRAP by Mass Spectrometry-Based Proteomics

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Interrogating the Plasmodium Sporozoite Surface: Identification of Surface-Exposed Proteins and Demonstration of Glycosylation on CSP and TRAP by Mass Spectrometry-Based Proteomics

Kristian E Swearingen et al. PLoS Pathog..

Abstract

Malaria parasite infection is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver for infection. A promising approach to developing a malaria vaccine is the use of proteins located on the sporozoite surface as antigens to elicit humoral immune responses that prevent the establishment of infection. Very little of the P. falciparum genome has been considered as potential vaccine targets, and candidate vaccines have been almost exclusively based on single antigens, generating the need for novel target identification. The most advanced malaria vaccine to date, RTS,S, a subunit vaccine consisting of a portion of the major surface protein circumsporozoite protein (CSP), conferred limited protection in Phase III trials, falling short of community-established vaccine efficacy goals. In striking contrast to the limited protection seen in current vaccine trials, sterilizing immunity can be achieved by immunization with radiation-attenuated sporozoites, suggesting that more potent protection may be achievable with a multivalent protein vaccine. Here, we provide the most comprehensive analysis to date of proteins located on the surface of or secreted by Plasmodium falciparum salivary gland sporozoites. We used chemical labeling to isolate surface-exposed proteins on sporozoites and identified these proteins by mass spectrometry. We validated several of these targets and also provide evidence that components of the inner membrane complex are in fact surface-exposed and accessible to antibodies in live sporozoites. Finally, our mass spectrometry data provide the first direct evidence that the Plasmodium surface proteins CSP and TRAP are glycosylated in sporozoites, a finding that could impact the selection of vaccine antigens.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The 6-Cys protein p38 is a microneme protein.
Transgenic salivary gland sporozoites expressing p38 with an HA tag were subjected to an indirect fluorescence assay (IFA). p38 was localized using antibodies against the HA tag (green) and parasites were co-stained with antibodies specific for TRAP (A) or CSP (B). Scale bar is 5 microns.
Fig 2
Fig 2. Localization of the sugar transporter in sporozoites and exoerythrocytic stages.
(A) Schematic of the 12 transmembrane domains predicted to exist in the putative sugar transporter PY17X_0823700. Both N- and C-termini are predicted to be intracellular. An HA tag is appended to the C-terminus. (B) Transgenic salivary gland sporozoites were stained with antibodies against the HA tag (green) and co-stained with antibodies directed against the repeat region of CSP (red). Nucleic acids were labeled with DAPI. The differential interference contrast (DIC) image demonstrated overall sporozoite morphology. Scale bar is 5 microns. (C) Localization of sugar transporter in liver stage parasites at 2, 4, 8, and 16 h post-infection. Anti-HA staining (green) was adjacent and internal to anti-UIS4 staining (red). Scale bar is 5 microns.
Fig 3
Fig 3. The inner membrane complex proteins MTIP and GAP45 are surface-exposed in unpermeabilized salivary gland sporozoites.
Fluorescence microscopy ofP.falciparum salivary gland sporozoites allowed to glide for 20 min and then stained for GAP45 (A) or MTIP (B). Sporozoites were fixed with 4% paraformaldehyde and either left unpermeabilized or permeabilized with Triton-X100. Shown are representative fluorescent images with their paired differential interference contrast (DIC) images. Unpermeabilized sporozoites featured strong patches of staining at the anterior or posterior ends, or in the middle of the sporozoite. In contrast, antibodies stained the entire sporozoite in permeabilized specimens. Scale bar is 10 microns.
Fig 4
Fig 4. Ribbon diagrams of TRAP and CSP.
(A & B) Three-dimensional models of TRAP extracellular domains (A) and detail of its TSR domain (B) showing the location of the observed glycosylation modifications. View in B is rotated ~180° about an axis vertical in the page relative to (A). TRAP fromP.vivax, which is highly homologous to TRAP fromP.falciparum, is shown, with the open, putative high-affinity state of the Von Willebrand factor type A (VWA) domain with its bound Mg2+ ion as a gold sphere [60]. (C) A three-dimensional model of the TSR domain of CSP showing location of the glycosylation event observed by mass spectrometry [61]. For all panels, sticks show glycans where carbons are green and oxygens are red. Amino acid side chains to which they are attached are shown with silver carbons and blue nitrogens. Disulfide bonds are shown in yellow. Side chains are shown for both Trp in the WXXW motif. TSR ribbons are in cyan, and in B and C, peptide segments identified by mass spectrometry with glycans attached are colored in marine. C-termini are marked with “C”. Mannose attached to Trp is modeled from the TRAP homologue inToxoplasma gondii [62]; and the disaccharide attached to Thr in CSP is modeled from that in TRAP. Carbohydrate was added after superposition on the Trp or Thr side chain. The Trp and Thr residues have essentially identical orientations (rotamers) with and without the glycan. In both the TRAP structure and the CSP model, the glucose residue in the disaccharide has a similar role in burying the adjacent disulfide bond.
Fig 5
Fig 5. Mass spectral evidence for glycosylation of TRAP.
(A & B) Representative extracted ion chromatograms (XIC) of the doubly-charged ions of the glycosylated TRAP peptide TASCGVWDEWSPCSVTCGK fromP.falciparum salivary gland sporozoites. (A) XIC from an un-enriched sporozoite sample (data acquired from our previous work [18]). (B) XIC from a surface-labeled sporozoite sample (S1 Table). Traces are offset on the Y-axis for clarity. The mass-to-charge ratio (m/z) for each species is indicated. Representative precursor spectra from the species in (A) and (B) are shown in (C). The Orbitrap enabled measurement of peptide masses with high accuracy (<1 ppm mass error). The largest chromatographic peak (red) was produced by a species with a mass matching the peptide plus a hexose and a deoxyhexose, which we presume to be C-mannose and O-fucose. The peptide was also observed with a mass equal to two hexoses and a deoxyhexose (green), which we presume to be a C-mannose and an O-linked glucosylfucose disaccharide. A species was also observed with a mass matching the peptide modified only with C-mannose (blue). However, the peaks associated with this species co-eluted with the more heavily-modified peptides, indicating that they likely arose from loss of the O-linked glycan due to in-source fragmentation. (D) Representative collision-induced dissociation (CID) fragmentation spectra of the three peptide species. CID of the C-mannosylated peptide lacking O-fucose was obtained from (A), providing confirmation of the assignments of the fragment spectra obtained from the O-fucosylated species seen in (B). Fragment ions are annotated as b-ions (blue) and y-ions (red). The unfragmented peptide is designated M (green), with addition of protons (H) and neutral loss of hexose (Hex) and deoxyhexose (dHex). Ions are singly-charged unless indicated doubly-charged (++). Neutral loss of water (-18 Da) is indicated with (o). Loss of C4H8O4 (-120 Da) due to cross-ring cleavage of the C-mannose is indicated with (#). Fragmentation spectra of the O-fucosylated peptides show that the dominant product of CID fragmentation was the intact peptide that had lost the O-linked glycan but retained the C-mannose. The same species with partial loss of the C-mannose due to cross-ring fragmentation was the second most abundant species. The dominant peptide fragments retained the C-mannose, allowing confident localization of this glycan at the Trp of WSPC (indicated as W[Hex]). No peptide fragment spectra were observed with the O-fucose glycans intact, but based on the presence of the O-fucosylation motif, we presume that the Thr of CSVTCG is modified with O-fucose.
Fig 6
Fig 6. Mass spectral evidence for glycosylation of CSP.
(A) Representative extracted ion chromatograms (XIC) of the triply-charged ions of the glycosylated CSP peptide IQNSLSTEWSPCSVTCGNGIQVR acquired fromP.falciparum salivary gland sporozoites. (Data from an un-enriched sporozoite sample acquired in our previous work [18]). Traces are offset on the Y-axis for clarity. The mass-to-charge ratio (m/z) for each species is indicated. Representative precursor spectra from the species in (A) are shown in (B). The Orbitrap enabled measurement of peptide masses with high accuracy (<2 ppm mass error). The largest chromatographic peak (red) was produced by a species with a mass matching the peptide plus a deoxyhexose which we presume to be O-fucose. The peptide was also observed with a mass equal to a hexose and a deoxyhexose (green), which we presume to be an O-linked glucosylfucose disaccharide. Evidence for loss of O-linked glycans due to in-source fragmentation is seen as chromatographic peaks matching the unmodified peptide ion (blue) co-eluting with the O-fucosylated species. The unmodified peptide was also observed in a peak with distinct retention time, suggesting that the peptide was also present in the sample in unmodified form. (C) Representative collision-induced dissociation (CID) fragmentation spectra of the three peptide species are shown with annotated fragment b-ions (blue) and y-ions (red). The unfragmented peptide is designated M (green), with addition of protons (H) and neutral loss of hexose (Hex) and deoxyhexose (dHex). Ions are singly-charged unless indicated doubly-charged (++) or triply-charged (+++). Fragmentation spectra of the fucosylated peptides show that a primary product of CID fragmentation was the intact peptide that had lost the O-linked glycan. No peptide fragment spectra were observed with the O-fucose glycans intact, but based on the presence of the O-fucosylation motif, we presume that the Thr of CSVTCG is modified with O-fucose.
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