Total and putative surface proteomics of malaria parasite salivary gland sporozoites

Abstract

Malaria infections of mammals are initiated by the transmission of Plasmodium salivary gland sporozoites during an Anopheles mosquito vector bite. Sporozoites make their way through the skin and eventually to the liver, where they infect hepatocytes. Blocking this initial stage of infection is a promising malaria vaccine strategy. Therefore, comprehensively elucidating the protein composition of sporozoites will be invaluable in identifying novel targets for blocking infection. Previous efforts to identify the proteins expressed in Plasmodium mosquito stages were hampered by the technical difficulty of separating the parasite from its vector; without effective purifications, the large majority of proteins identified were of vector origin. Here we describe the proteomic profiling of highly purified salivary gland sporozoites from two Plasmodium species: human-infective Plasmodium falciparum and rodent-infective Plasmodium yoelii. The combination of improved sample purification and high mass accuracy mass spectrometry has facilitated the most complete proteome coverage to date for a pre-erythrocytic stage of the parasite. A total of 1991 P. falciparum sporozoite proteins and 1876 P. yoelii sporozoite proteins were identified, with >86% identified with high sequence coverage. The proteomic data were used to confirm the presence of components of three features critical for sporozoite infection of the mammalian host: the sporozoite motility and invasion apparatus (glideosome), sporozoite signaling pathways, and the contents of the apical secretory organelles. Furthermore, chemical labeling and identification of proteins on live sporozoites revealed previously uncharacterized complexity of the putative sporozoite surface-exposed proteome. Taken together, the data constitute the most comprehensive analysis to date of the protein expression of salivary gland sporozoites and reveal novel potential surface-exposed proteins that might be valuable targets for antibody blockage of infection.

Fig. 1.

One-dimensional SDS-PAGE fractionation of sporozoite proteins. The one-dimensional SDS-PAGE separation of a P. yoelii salivary gland sporozoite whole cell lysate (flanked on both sides by a molecular weight (MW) marker) is shown with a bar graph representing the total number of high-quality (false positive error rate < 1.0%) peptide spectrum matches found from a single injection of each fraction after in-gel tryptic digest. Bovine serum albumin (BSA) from the purification protocol can be seen as the large band in fractions 9 and 10. In every other fraction, P. yoelii proteins were the major component.

Fig. 2.

Components of the Plasmodium glideosome identified in sporozoite stages. The currently known components of Plasmodium's gliding motility and invasion machinery (the “glideosome”) mainly identified from asexual blood stages and orthologs from Toxoplasma gondii are illustrated in this model and are depicted based on their presence in the sporozoite proteome. Sporozoite proteins are colored to indicated whether they were detected in P. falciparum sporozoites only (red), P. yoelii sporozoites only (yellow), both species (orange), or neither species (gray). Two shades of orange were used to provide contrast between the intertwined tubulin and F-actin molecules. The anchoring of CSP in the sporozoite plasma membrane occurs via a glycosylphosphatidylinositol anchor, whereas invasins do so via a transmembrane domain and a cytoplasmic C-terminal tail that interacts with aldolase through a conserved penultimate tryptophan (this interaction for Thrombospondin Related Anonymous Protein (TRAP)-related protein has not been experimentally validated and is indicated by a question mark). Aldolase 2 and Actin 2 were previously thought to be present only in blood stage parasites but were confidently identified in these experiments. They are marked with question marks, as their involvement in gliding motility has not been experimentally validated.

Fig. 3.

Calcium signaling pathways identified in the sporozoite proteome. A composite model of known calcium-dependent signaling molecules and their related pathways mainly identified from asexual blood stages are illustrated in this model and depicted based on their presence in the sporozoite proteome. Sporozoite proteins are colored according to their presence in both P. falciparum and P. yoelii sporozoites (orange) or their absence from both species (gray). Two endoplasmic-reticulum-associated receptors (inositol trisphosphate receptor (IP₃R) and ryanodine receptor (RyR), in green) have no identified orthologous genes but have been detected based on the chemical inhibition of their functions. The triggering stimuli for rhoptry release and the directionality of the Non-SERCA calcium pump are not known and are denoted by question marks.

Fig. 4.

Proteins associated with the invasion organelles of Plasmodium sporozoites. A composite model of known proteins identified in the apical invasive organelles mainly identified from asexual Plasmodium blood stages is illustrated in this model. Proteins are listed if they were detected in sporozoites. Orthologs of invasion-related proteins from Toxoplasma gondii were also detected (supplemental Table S7).

Fig. 5.

Expression and localization of a representative low abundance sporozoite protein as detected via indirect immunofluorescence assay. The protein PY01024 (CCAAT-box DNA binding protein subunit B) was confidently identified in P. yoelii salivary gland sporozoites by means of mass spectrometry, but with poor sequence coverage (one unique peptide, four peptide spectrum matches) and low abundance (lower 15th percentile as estimated based on the normalized spectral abundance factor). To confirm the presence of the protein, a transgenic P. yoelii salivary gland sporozoite bearing a C-terminal 4xMyc tag on PY01024 was subjected to an indirect immunofluorescence assay with anti-c-myc (red) that confirmed the presence of the protein. Anti-PyCSP (circumsporozoite protein, a major surface protein; top, green) and the DNA label 4′,6-diamidino-2-phenylindole (DAPI) (blue) showed PY01024 to be located at the anterior apical end of the sporozoite. Anti-PyRON4 (Rhoptry Neck Protein 4, known to localize to the rhoptry neck; bottom, green) and DAPI indicated localization of PY01024 to the rhoptry organelles. Sporozoites are marked by dotted lines layered onto the differential interference contrast images on the lower panel for clarity. Scale bar = 10 μm.