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Total and Putative Surface Proteomics of Malaria Parasite Salivary Gland Sporozoites*

  • Scott E. Lindner
    Footnotes
    Affiliations
    Malaria Program, Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109
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  • Kristian E. Swearingen
    Footnotes
    Affiliations
    Institute for Systems Biology, 401 Terry Ave N, Seattle, Washington 98109
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  • Anke Harupa
    Correspondence
    To whom correspondence should be addressed: Dr. Stefan H. Kappe, Tel.: +1-206-256-7205, Fax: +1-206-256-7229, E-mail: [email protected]; Dr. Robert L. Moritz, Tel.: +1-206-732-1200, E-mail: [email protected]
    Affiliations
    Malaria Program, Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109

    Institute of Biology, Freie Universitaet Berlin, Takustrasse 6, 14195 Berlin, Germany
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  • Ashley M. Vaughan
    Affiliations
    Malaria Program, Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109
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  • Photini Sinnis
    Affiliations
    Johns Hopkins University, 615 North Wolfe St., Suite E4626, Baltimore, Maryland 21205
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  • Robert L. Moritz
    Correspondence
    To whom correspondence should be addressed: Dr. Stefan H. Kappe, Tel.: +1-206-256-7205, Fax: +1-206-256-7229, E-mail: [email protected]; Dr. Robert L. Moritz, Tel.: +1-206-732-1200, E-mail: [email protected]
    Affiliations
    Institute for Systems Biology, 401 Terry Ave N, Seattle, Washington 98109
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  • Stefan H.I. Kappe
    Correspondence
    To whom correspondence should be addressed: Dr. Stefan H. Kappe, Tel.: +1-206-256-7205, Fax: +1-206-256-7229, E-mail: [email protected]; Dr. Robert L. Moritz, Tel.: +1-206-732-1200, E-mail: [email protected]
    Affiliations
    Malaria Program, Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109

    Department of Global Health, University of Washington, Seattle, Washington 98195
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  • Author Footnotes
    * This investigation was supported by the National Institutes of Health under a Ruth L. Kirschstein National Research Service Award (F32GM083438) to S.E.L., by the NIGMS (Center for Systems Biology Grant No. 2P50 GM076547) to R.L.M., by the NIGMS (Grant No. GM087221 to R.L.M.), and by the NIAID (Grant No. R01 AI053709-07A2 to S.H.I.K.). This investigation was also supported by the National Science Foundation MRI (Grant No. 0923536 to R.L.M.) and by the Bill and Melinda Gates Foundation (Grant No. OPP1067687 to S.E.L., R.L.M., P.S., and S.H.I.K.).
    This article contains supplemental material.
    § The authors contributed equally to this work.
Open AccessPublished:January 16, 2013DOI:https://doi.org/10.1074/mcp.M112.024505
      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.
      Malaria, a disease caused by eukaryotic parasites of the genus Plasmodium, causes hundreds of millions of clinical cases and kills approximately 1 million people annually WHO. World malaria report. 2011. http://www.who.int (last accessed 11 December 2012). Plasmodium is transmitted to humans by infected female anopheline mosquitoes seeking a blood meal, which results in the release of mosquito saliva and sporozoites into the skin (
      • Sinnis P.
      • Zavala F.
      The skin stage of malaria infection: biology and relevance to the malaria vaccine effort.
      ). Sporozoites leave the bite site, enter the blood stream, and are transported to the liver where they invade hepatocytes. Sporozoites transform into liver stages that grow, mature, and release exo-erythrocytic merozoites, which enter the blood stream and invade erythrocytes (
      • Baer K.
      • Klotz C.
      • Kappe S.H.
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      • Frevert U.
      Release of hepatic Plasmodium yoelii merozoites into the pulmonary microvasculature.
      ,
      • Lindner S.E.
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      Malaria parasite pre-erythrocytic infection: preparation meets opportunity.
      ). The erythrocytic stages of the infection lead to all clinical symptoms and disease. The sporozoite and liver stages of infection are asymptomatic, constitute population bottlenecks in the life cycle, and thus are perceived as important drug and vaccine targets (
      • Kappe S.H.
      • Vaughan A.M.
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      That was then but this is now: malaria research in the time of an eradication agenda.
      ). Indeed, irradiated and genetically attenuated sporozoites (lacking genes critical for liver stage development) are powerful immunogens that confer sterile protection against malaria infection (
      • Vaughan A.M.
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      • Kappe S.H.
      Genetically engineered, attenuated whole-cell vaccine approaches for malaria.
      ). Animal model data show that these attenuated sporozoites exhibit normal behavior in that they infect the liver, but then they arrest during liver stage development. Exposure to extracellular sporozoites provokes antibody responses, and the subsequent intrahepatocytic liver stages are thought to result in the presentation of intracellular antigens to the host immune system that elicit a CD8+ T cell response against infected hepatocytes (
      • Reyes-Sandoval A.
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      ,
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      ,
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      ,
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      ). However, achieving complete sterile protection against malaria infection with sporozoite and liver-stage-based subunit vaccines has so far proven an elusive goal.
      The subunit vaccine candidate RTS,S, which contains part of the major sporozoite surface protein, circumsporozoite protein (CSP),
      The abbreviations used are:
      CDPK
      calcium-dependent protein kinase
      CSP
      circumsporozoite protein
      IMC
      inner membrane complex
      PSM
      peptide spectrum match
      NSAF
      normalized spectral abundance factor
      TRAP
      thrombospondin-related adhesion protein
      UIS
      up-regulated in infectious sporozoites.
      1The abbreviations used are:CDPK
      calcium-dependent protein kinase
      CSP
      circumsporozoite protein
      IMC
      inner membrane complex
      PSM
      peptide spectrum match
      NSAF
      normalized spectral abundance factor
      TRAP
      thrombospondin-related adhesion protein
      UIS
      up-regulated in infectious sporozoites.
      has been tested in phase III trials and has shown partial efficacy (
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      ,
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      A Phase 3 trial of RTS,S/AS01 malaria vaccine in African infants.
      ). The RTS,S trial results highlight the need to identify additional antigen targets for inclusion in a next-generation malaria vaccine. Recently, a virally vectored platform of immunization has shown that recombinant chimpanzee adenovirus and modified vaccinia virus Ankara encoding thrombospondin-related adhesion protein (TRAP), a sporozoite- and early liver stage parasite-expressed antigen, can induce potent immune responses (
      • Reyes-Sandoval A.
      • Wyllie D.H.
      • Bauza K.
      • Milicic A.
      • Forbes E.K.
      • Rollier C.S.
      • Hill A.V.
      CD8+ T effector memory cells protect against liver-stage malaria.
      ). In preclinical studies, this prime-boost platform induced substantial protection that depended on the induction of a potent CD8+ T cell effector memory response directed against liver stage parasites (
      • Reyes-Sandoval A.
      • Wyllie D.H.
      • Bauza K.
      • Milicic A.
      • Forbes E.K.
      • Rollier C.S.
      • Hill A.V.
      CD8+ T effector memory cells protect against liver-stage malaria.
      ). The partial success of sporozoite protein subunit vaccine candidates and the sterilizing protection model of whole sporozoite vaccination can be further built upon in order to ultimately achieve complete sterilizing protection against malaria infection with a multivalent subunit vaccine. To aid this, a thorough assessment of the Plasmodium sporozoite protein arsenal is needed so that the best candidates can be identified and evaluated.
      Proteomic analysis via mass spectrometry, although a well-established and routine technique, has yet to define complete proteomes in single experiments. However, given a sufficient sample and access to state-of-the-art instrumentation, upwards of 50% complete proteomes (e.g. 68% of yeast (
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      Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation.
      ) and 51% of human (
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      The quantitative proteome of a human cell line.
      ,
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      Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins.
      ,
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      ) proteomes) can be determined with a reasonable amount of effort. A major obstacle to proteomic analysis of the Plasmodium parasite pre-erythrocytic stages is the difficulty of separating the parasites from vector or host material. The presence of excessive contaminating protein can overwhelm chromatographic column capacity and mass analyzer duty cycles, thereby limiting the number of parasite proteins that can be observed in this large dynamic range of host versus parasite protein abundance. In a study of P. falciparum mosquito stages, Lasonder et al. (
      • Lasonder E.
      • Janse C.J.
      • van Gemert G.J.
      • Mair G.R.
      • Vermunt A.M.
      • Douradinha B.G.
      • van Noort V.
      • Huynen M.A.
      • Luty A.J.
      • Kroeze H.
      • Khan S.M.
      • Sauerwein R.W.
      • Waters A.P.
      • Mann M.
      • Stunnenberg H.G.
      Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity.
      ) reported that mosquito proteins made up between 65% and 89% of the samples analyzed. Despite significant effort, only 728 mosquito stage parasite proteins were identified (71.8% with multiple peptides), which constitutes ∼13% of the total protein coding capacity of P. falciparum's genome. Florens et al. (
      • Florens L.
      • Washburn M.P.
      • Raine J.D.
      • Anthony R.M.
      • Grainger M.
      • Haynes J.D.
      • Moch J.K.
      • Muster N.
      • Sacci J.B.
      • Tabb D.L.
      • Witney A.A.
      • Wolters D.
      • Wu Y.
      • Gardner M.J.
      • Holder A.A.
      • Sinden R.E.
      • Yates J.R.
      • Carucci D.J.
      A proteomic view of the Plasmodium falciparum life cycle.
      ) reported 1048 P. falciparum salivary gland sporozoite protein identifications (∼19% of P. falciparum ORFs), but only 30.0% of those were identified by multiple peptides. Florens et al. did not discuss the presence of vector material in the sample, but they did report requiring the combined protein content of five separate sporozoite preparations. A proteomic study of P. berghei by Hall et al. (
      • Hall N.
      • Karras M.
      • Raine J.D.
      • Carlton J.M.
      • Kooij T.W.
      • Berriman M.
      • Florens L.
      • Janssen C.S.
      • Pain A.
      • Christophides G.K.
      • James K.
      • Rutherford K.
      • Harris B.
      • Harris D.
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      • Quail M.A.
      • Ormond D.
      • Doggett J.
      • Trueman H.E.
      • Mendoza J.
      • Bidwell S.L.
      • Rajandream M.A.
      • Carucci D.J.
      • Yates 3rd, J.R.
      • Kafatos F.C.
      • Janse C.J.
      • Barrell B.
      • Turner C.M.
      • Waters A.P.
      • Sinden R.E.
      A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.
      ) reported only 134 salivary gland sporozoite protein identifications (78.3% identified by multiple peptides), despite finding a total of 1836 proteins across the P. berghei life cycle. Although the success of any proteomic profiling experiment is directly linked to the purification technique used and access to state-of-the-art mass analyzers, the limited success of the above-described efforts suggests that more complete proteomic characterization of Plasmodium salivary gland sporozoites will depend even more heavily upon optimized sample preparation and purification.
      In this work we employed a high-resolution LTQ Orbitrap Velos mass analyzer coupled to nano–liquid chromatography (nanoLC) for the identification of proteins present in highly purified salivary gland sporozoites of both the human-infective P. falciparum and rodent-infective P. yoelii species. In addition to surveying the total salivary gland sporozoite proteome, we also identified factors and features of parasite motility and invasion processes and labeled live salivary gland sporozoites to capture and identify putative proteins present on their surface. Using our improved purification techniques for the removal of mosquito debris, we have achieved the most complete proteome coverage yet reported for a pre-erythrocytic stage of Plasmodium, and herein we identify novel, putative sporozoite surface protein candidates.

      DISCUSSION

      Determining the presence of gene products at the various stages of the Plasmodium life cycle is critical to understanding the parasite’s mechanisms of infection (for the ookinete, sporozoite, and merozoite) and replication (the midgut oocyst, the liver stage schizont, and the blood stage schizont). To this end, we present here key findings for the most complete proteome coverage to date of the invasive salivary gland sporozoite stage for two species of the malaria parasite, P. falciparum and P. yoelii. Although a lack of mass spectrometric evidence for a protein does not necessarily imply that protein’s absence from a sample, positive identification of a protein confers strong evidence of its presence, especially if the identification is corroborated by multiple unique PSMs, as was possible with >86% of the identified proteins. The high-confidence identification of proteins via mass spectrometry has enabled us to assess various features of the salivary gland sporozoite and reliably detect components of pathways that have been characterized only in other forms of the malaria parasite, which are more easily accessible for biochemical characterization. In the absence of any internal standards for absolute or relative quantification, we have used normalized spectral abundance factors to estimate relative protein abundance for our characterizations of these data. Proteins that were expected to be highly expressed and secreted in sporozoites were found to be among the proteins identified with the largest NSAFs. Furthermore, we have experimentally validated this approach through our successful detection of a low abundance protein of interest (PY01024) in the apical rhoptry organelles of P. yoelii salivary gland sporozoites.
      Careful sample preparation and improved purification protocols combined with mass spectrometric analysis using an LTQ Orbitrap Velos enabled the most complete proteome coverage to date of Plasmodium salivary gland sporozoites. Employing one-dimensional SDS-PAGE for whole cell fractionation, we were able to obtain 1991 P. falciparum salivary gland sporozoite protein identifications (86.6% identified by multiple peptides) from a single preparation of 107 purified sporozoites. Florens et al. (
      • Florens L.
      • Washburn M.P.
      • Raine J.D.
      • Anthony R.M.
      • Grainger M.
      • Haynes J.D.
      • Moch J.K.
      • Muster N.
      • Sacci J.B.
      • Tabb D.L.
      • Witney A.A.
      • Wolters D.
      • Wu Y.
      • Gardner M.J.
      • Holder A.A.
      • Sinden R.E.
      • Yates J.R.
      • Carucci D.J.
      A proteomic view of the Plasmodium falciparum life cycle.
      ) employed the MudPIT fractionation strategy (
      • Washburn M.P.
      • Wolters D.
      • Yates 3rd, J.R.
      Large-scale analysis of the yeast proteome by multidimensional protein identification technology.
      ) to identify 1048 proteins from P. falciparum salivary gland sporozoites, of which only 30.0% were identified by multiple peptides. The authors of that study did not discuss the extent of contamination by mosquito vector proteins, but it is notable that five separate preparations of 107 sporozoites were required in order to obtain the reported protein identifications. In their analysis of P. falciparum salivary gland sporozoites, Lasonder et al. (
      • Lasonder E.
      • Janse C.J.
      • van Gemert G.J.
      • Mair G.R.
      • Vermunt A.M.
      • Douradinha B.G.
      • van Noort V.
      • Huynen M.A.
      • Luty A.J.
      • Kroeze H.
      • Khan S.M.
      • Sauerwein R.W.
      • Waters A.P.
      • Mann M.
      • Stunnenberg H.G.
      Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity.
      ) divided lysates of 107 cells into soluble and insoluble fractions, each of which was then fractionated via one-dimensinoal SDS-PAGE. Despite running up to four LC-MS/MS technical replicates, they were able to identify only 477 Plasmodium proteins (72.5% identified by multiple peptides) from the sporozoite sample. Their work clearly illustrates how host material contamination impedes the discovery of parasite proteins; only 31% of the proteins identified from their salivary gland sporozoite samples were of parasite origin, with the remainder being primarily contaminating mosquito proteins.
      As illustrated here, the fractionation of complex samples prior to analysis, combined with mass analyzers with rapid duty cycle and high mass accuracy, enables increased protein identification. However, the success of protein discovery experiments is equally, if not predominantly, determined by the quality of the sample preparation. Sample preparation is a unique factor of the work presented here, as parasites were first manually isolated from infected mosquitoes, which presented opportunities for extensive contamination. With large variations in the dynamic range of host versus parasite proteins, the presence of contaminating proteins can obscure parasite analytes of interest. Furthermore, as the mass analyzer has a defined duty cycle, peptide spectra generated for contaminating protein tryptic peptides represent wasted instrument time and possible lost parasite protein identifications. In proteomic analyses of salivary gland sporozoites by others, as well as in our own initial attempt to analyze P. falciparum, a large proportion of mosquito protein in the sample resulted in significantly reduced parasite protein discovery relative to what was achieved with highly purified samples. The purification protocols used here (combined with a careful micro-dissection technique) facilitated a much more effective investigation of mosquito-stage parasites than has previously been reported.
      Many components of the Plasmodium glideosome have been described only with respect to the asexual blood stages of infection. We have discovered that the vast majority of these glideosome proteins are also present in salivary gland sporozoites and are likely utilized in similar ways to generate a directional locomotive force toward the posterior of the sporozoite (Fig. 2). Absent from our proteome datasets are only two proteins associated with the inner membrane complex (IMCb and IMCd) and another involved in actin dynamics (Formin 1). The amino acid sequences of these proteins contain domains that should result in detectable tryptic peptides, suggesting that these proteins are either of very low abundance or actually absent from the sporozoite glideosome. Interestingly, two proteins previously characterized as being stage-restricted to sexual and asexual parasites (Actin 2 and Aldolase 2, respectively) were confidently detected in relatively high abundance in sporozoites, along with the expected homologs. All together, the major components of the locomotive machinery and its supporting molecular infrastructure (microtubules, filamentous actin, and assembly/disassembly proteins) are present in our datasets.
      As the sporozoite must properly detect and respond to various extracellular stimuli, it expresses a variety of plasma-membrane-associated sensor proteins. In the current version of PlasmoDB (Version 9.2), there currently are only one G-protein-coupled receptor, one rhodopsin-like receptor, and four serpentine-receptor-like proteins (SR1, SR10, SR12, and SR25) annotated for Plasmodium species (
      • Madeira L.
      • Galante P.A.
      • Budu A.
      • Azevedo M.F.
      • Malnic B.
      • Garcia C.R.
      Genome-wide detection of serpentine receptor-like proteins in malaria parasites.
      ). We have detected a subset of these receptors in salivary gland sporozoites (SR1, SR10, rhodopsin-like receptor, G-protein-coupled receptor), which the parasite might use to sense key attributes about its ambient environment, such as specific cues to indicate whether it remains in the mosquito vector or has been transmitted to the mammalian host. In conjunction with these receptors, both adenylyl and guanylyl cyclases are expressed in Plasmodium to produce cAMP and cGMP secondary messengers for downstream signaling events, which we have also detected in our sporozoite proteomes (
      • Carucci D.J.
      • Witney A.A.
      • Muhia D.K.
      • Warhurst D.C.
      • Schaap P.
      • Meima M.
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      • Kelly J.M.
      • Baker D.A.
      Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum.
      ). These putative sensors can then feed into intracellular signaling cascades to stimulate the parasite to respond appropriately to its environment (e.g. maintain preparations for vector/host transmission, initiate motility/traversal/invasion programs). Interestingly, phospholipase C, a common enzyme in this signaling pathway, was not detected in either species. It is also noteworthy that tyrosine kinases, which have also been implicated with phospholipase C in producing inositol 1,4,5-trisphosphate in other eukaryotes, are apparently absent from Plasmodium genomes (
      • Ward P.
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      Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote.
      ). Finally, we detected a non-SERCA type calcium pump (also called ATPase 4) that also likely affects this signaling pathway and has been reported to traffic to punctate foci near the parasite periphery in infected RBCs (
      • Dyer M.
      • Jackson M.
      • McWhinney C.
      • Zhao G.
      • Mikkelsen R.
      Analysis of a cation-transporting ATPase of Plasmodium falciparum.
      ). However, little is known about its functions, including into what intracellular space it pumps calcium.
      Plasmodium parasites are able to store calcium at least in part because of a SERCA-type calcium pump (also called ATPase 6 or ATP6, detected in both species) that drives free calcium ions from the cytoplasm to the endoplasmic reticulum. Several reports have implicated ATP6 as a target of the front-line artemisinin family of antimalarials (
      • Eckstein-Ludwig U.
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      • East J.M.
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      • Bray P.G.
      • Ward S.A.
      • Krishna S.
      Artemisinins target the SERCA of Plasmodium falciparum.
      ), but recent efforts have demonstrated that this is not the case (
      • Cardi D.
      • Pozza A.
      • Arnou B.
      • Marchal E.
      • Clausen J.D.
      • Andersen J.P.
      • Krishna S.
      • Moller J.V.
      • le Maire M.
      • Jaxel C.
      Purified E255L mutant SERCA1a and purified PfATP6 are sensitive to SERCA-type inhibitors but insensitive to artemisinins.
      ,
      • Cui L.
      • Wang Z.
      • Jiang H.
      • Parker D.
      • Wang H.
      • Su X.Z.
      Lack of association of the S769N mutation in Plasmodium falciparum SERCA (PfATP6) with resistance to artemisinins.
      ). However, these calcium stores can be released from the endoplasmic reticulum to the cytosol by increased levels of inositol trisphosphate, likely via a yet-to-be-identified inositol trisphosphate receptor or ryanodine receptor associated with the endoplasmic reticulum (
      • Moreno S.N.
      • Ayong L.
      • Pace D.A.
      Calcium storage and function in apicomplexan parasites.
      ).
      Upon receiving external stimuli (such as highly sulfated heparan sulfate proteoglycans, albumin, or bicarbonate) that indicate that the sporozoite has arrived in the mammalian host (reviewed in Ref.
      • Lindner S.E.
      • Miller J.L.
      • Kappe S.H.
      Malaria parasite pre-erythrocytic infection: preparation meets opportunity.
      ), the parasite engages its gliding motility machinery to make its way through the skin, into the vasculature, and finally into the liver. Therein it undergoes a rapid dedifferentiation process that is associated with a rapid increase of intracellular calcium levels (
      • Doi Y.
      • Shinzawa N.
      • Fukumoto S.
      • Okano H.
      • Kanuka H.
      Calcium signal regulates temperature-dependent transformation of sporozoites in malaria parasite development.
      ). This free calcium not only induces secretion by the micronemes during motility and invasion, but also can be utilized by calmodulin in remodeling itself structurally in order to act upon downstream effector molecules, such as calcium-dependent protein kinases (CDPKs). Several recent studies have shown that CDPKs are important for parasite infectivity and transmission, and not surprisingly, at least two substrates of CDPK1 (GAP45 and MTIP) are members of the glideosome apparatus required for both processes (
      • Ojo K.K.
      • Pfander C.
      • Mueller N.R.
      • Burstroem C.
      • Larson E.T.
      • Bryan C.M.
      • Fox A.M.
      • Reid M.C.
      • Johnson S.M.
      • Murphy R.C.
      • Kennedy M.
      • Mann H.
      • Leibly D.J.
      • Hewitt S.N.
      • Verlinde C.L.
      • Kappe S.
      • Merritt E.A.
      • Maly D.J.
      • Billker O.
      • Van Voorhis W.C.
      Transmission of malaria to mosquitoes blocked by bumped kinase inhibitors.
      ,
      • Green J.L.
      • Rees-Channer R.R.
      • Howell S.A.
      • Martin S.R.
      • Knuepfer E.
      • Taylor H.M.
      • Grainger M.
      • Holder A.A.
      The motor complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase.
      ). Interestingly, only CDPKs 1, 4, and 7 were detected in both sporozoite datasets, with CDPK6 being solely identified in P. falciparum as well. The estimated abundances of these proteins were sufficiently high to make the absence of CDPKs 2, 3, and 5 especially noticeable (supplemental Table S6). Lastly, another sporozoite sensor of host arrival is the detection of increased external bicarbonate levels (
      • Hegge S.
      • Kudryashev M.
      • Barniol L.
      • Frischknecht F.
      Key factors regulating Plasmodium berghei sporozoite survival and transformation revealed by an automated visual assay.
      ), and this has been implicated as a key stimulus triggering the sporozoite to become invasive. We have detected that both P. falciparum and P. yoelii express a cytosolic carbonic anhydrase (type II) that might function in this process, as it converts CO2 and H2O to bicarbonate and hydrogen ions. The balance of these molecules might play a role in responding to external stimuli.
      The end result of responses to the host environment is the ordered activation and secretion of components of the invasion organelles (micronemes, rhoptry necks, rhoptries, and then dense granules/exonemes) in order to productively infect the host hepatocyte (reviewed in Ref.
      • Baum J.
      • Gilberger T.W.
      • Frischknecht F.
      • Meissner M.
      Host-cell invasion by malaria parasites: insights from Plasmodium and Toxoplasma.
      ). The protein composition of these organelles has been scantly studied in sporozoites but has been better characterized in the blood stages. Using this information in aggregate, we have determined which apical organelle proteins are detected in the sporozoite (supplemental Table S7). In addition to the canonical proteins known to be abundant in and secreted from sporozoites (e.g. CSP, TRAP, AMA1, SPECT, SPECT2, CelTOS), we have also identified proteins not previously identified in Plasmodium sporozoites (e.g. SUB2, Pf34, RON6, RAP1, CLAG3.2, RhopH2, RhopH3, RAMA, RALP1, and ASP/RON1). The precise subcellular localization of the latter proteins in sporozoites awaits further validation, but our data set will serve as a good starting point for further characterizing apical secreted proteins. For instance, whereas sporozoite rhoptries are easily identified based on morphology, micronemes and dense granules have so far not been clearly distinguished, and published claims have placed some proteins (e.g. SUB2) in one or the other organelle (
      • Barale J.C.
      • Blisnick T.
      • Fujioka H.
      • Alzari P.M.
      • Aikawa M.
      • Braun-Breton C.
      • Langsley G.
      Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1–42 maturase.
      ,
      • Hackett F.
      • Sajid M.
      • Withers-Martinez C.
      • Grainger M.
      • Blackman M.J.
      PfSUB-2: a second subtilisin-like protein in Plasmodium falciparum merozoites.
      ). Thus, our data set will also enhance the clarification of sporozoite cell biology. Moreover, we uncovered a novel putative rhoptry-neck-localized protein (PY01024) through our efforts to experimentally validate our approach of using spectral counting as a general proxy for protein abundance in these analyses. Also, we have identified several secreted proteins that are present in our analyses of P. yoelii sporozoites but absent in those from P. falciparum sporozoites. Most noteworthy of these is UIS3, which was also absent in a previous proteomic effort to characterize the P. falciparum sporozoite (
      • Lasonder E.
      • Janse C.J.
      • van Gemert G.J.
      • Mair G.R.
      • Vermunt A.M.
      • Douradinha B.G.
      • van Noort V.
      • Huynen M.A.
      • Luty A.J.
      • Kroeze H.
      • Khan S.M.
      • Sauerwein R.W.
      • Waters A.P.
      • Mann M.
      • Stunnenberg H.G.
      Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity.
      ). Understanding the similarity in the roles these secreted proteins play during infection across malaria species, or whether they have been adapted for additional sporozoite-specific roles, will improve our knowledge of how the parasite navigates its transmission to the mammalian host. Moreover, it can guide our efforts to strategically drive vaccination efforts.
      The combination of our improved sporozoite purification methods with careful mass-spectrometer-based identification and informatics analysis have now allowed the biochemical labeling and proteomic assessment of targeted protein groups. For instance, the identification of additional surface-exposed proteins could prove exceptionally useful in aiding the design of antibody-based vaccines to prevent infection by sporozoites, and thus malaria. Although CSP induces an immune response against sporozoites, recent studies have demonstrated that antibodies to other sporozoite proteins play a large role in limiting infection (
      • Mauduit M.
      • Gruner A.C.
      • Tewari R.
      • Depinay N.
      • Kayibanda M.
      • Chavatte J.M.
      • Franetich J.F.
      • Crisanti A.
      • Mazier D.
      • Snounou G.
      • Renia L.
      A role for immune responses against non-CS components in the cross-species protection induced by immunization with irradiated malaria sporozoites.
      ,
      • Mauduit M.
      • Tewari R.
      • Depinay N.
      • Kayibanda M.
      • Lallemand E.
      • Chavatte J.M.
      • Snounou G.
      • Renia L.
      • Gruner A.C.
      Minimal role for the circumsporozoite protein in the induction of sterile immunity by vaccination with live rodent malaria sporozoites.
      ). Because of the medical relevance of such a finding, we chose to biotinylate putative surface-exposed sporozoite proteins on live sporozoites, enrich for them from whole parasite lysates by virtue of the tight interaction of biotin with streptavidin-conjugated Dynabeads, and then detect and identify even very low abundance proteins. Because we were able to observe the biotinylation of CSP and other proteins via our initial metric (western blotting), we subjected these samples to mass spectrometric analysis. In addition to the “gold standard” proteins that have been shown to traffic to the sporozoite surface prior to (e.g. CSP, hexose transporter (supplemental Fig. S3)) or following sporozoite activation (e.g. TRAP, thrombospondin-related sporozoite protein, apical membrane antigen-1), we have identified several novel surface-exposed proteins as well. Many of these proteins exhibit signal peptides and either transmembrane domains or a glycosylphosphatidylinositol anchor sequence and additionally are predicted to have functions consistent with a surface localization, such as the active transport of small molecules into and out of the sporozoite (Table II, supplemental Table S1). Moreover, several other putative surface-exposed proteins that we have also identified bear no predictable functional domains and have not been previously characterized. Altogether, these proteins warrant further investigation to determine their roles in sporozoite biology, to provide additional experimental validation of their localization in sporozoites, and ultimately to determine whether targeting them with antibodies would inhibit sporozoite motility, cell traversal, and invasion capabilities and thus block sporozoite infection.
      It is important to point out that within the putative sporozoite surface proteomes we also observed proteins associated with the glideosome, which is a subsurface complex. These proteins were also detected by Wass and colleagues in their efforts to determine the ookinete surface proteome using similar approaches (
      • Wass M.N.
      • Stanway R.
      • Blagborough A.M.
      • Lal K.
      • Prieto J.H.
      • Raine D.
      • Sternberg M.J.
      • Talman A.M.
      • Tomley F.
      • Yates J.
      • Sinden R.E.
      Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls.
      ). In their studies, the detection of these glideosome proteins was experimentally explained by the partial permeability of the parasite plasma membrane to the cross-linking agents. This hypothesis was confirmed by the increasing accessibility of propidium iodide to the parasite under the progressive conditions used in the cross-linking process. We believe a similar phenomenon is occurring in our hands with sporozoites as well. Because of this, we have placed stronger confidence in the designation of proteins as surface localized if they are predicted to have a signal peptide and either a transmembrane domain(s) or a predicted glycosylphosphatidylinositol anchor (Table II), as proteins detected from the glideosome do not fit these criteria. However, as this is not a strict requirement for surface localization, it will be critical to further characterize and validate the localization of these proteins.
      In conclusion, the sample preparation and LC-MS/MS techniques described here for uncovering the total and putative surface proteome of Plasmodium sporozoites have demonstrated that careful sample preparation and purification techniques are required in order to produce high-quality proteomic data, especially when sample amounts are limiting. In anticipation of the great effect that further proteomics efforts will have on our understanding of the malaria parasite, we have made our high-mass-accuracy peptide detection data freely available in PeptideAtlas, which we hope will expedite high-confidence peptide identification in future efforts. Not only have these data sets yielded a valuable resource for ongoing experimentation with pre-erythrocytic stages of the parasite and for constructing more comprehensive models of their functions, but they also have produced potentially novel targets for producing humoral immunity. Currently the most advanced malaria vaccine (RTS,S, GlaxoSmithKline) induces an immune response to a single recombinant fusion protein containing a repeat domain from the major surface-exposed sporozoite coat protein (CSP) by fusing it to a highly immunogenic hepatitis B antigen. The initial results of an ongoing Phase 3 trial of this vaccine indicate that this approach provides only moderate levels of protection (
      • Agnandji S.T.
      • Lell B.
      • Soulanoudjingar S.S.
      • Fernandes J.F.
      • Abossolo B.P.
      • Conzelmann C.
      • Methogo B.G.
      • Doucka Y.
      • Flamen A.
      • Mordmuller B.
      • Issifou S.
      • Kremsner P.G.
      • Sacarlal J.
      • Aide P.
      • Lanaspa M.
      • Aponte J.J.
      • Nhamuave A.
      • Quelhas D.
      • Bassat Q.
      • Mandjate S.
      • Macete E.
      • Alonso P.
      • Abdulla S.
      • Salim N.
      • Juma O.
      • Shomari M.
      • Shubis K.
      • Machera F.
      • Hamad A.S.
      • Minja R.
      • Mtoro A.
      • Sykes A.
      • Ahmed S.
      • Urassa A.M.
      • Ali A.M.
      • Mwangoka G.
      • Tanner M.
      • Tinto H.
      • D'Alessandro U.
      • Sorgho H.
      • Valea I.
      • Tahita M.C.
      • Kabore W.
      • Ouedraogo S.
      • Sandrine Y.
      • Guiguemde R.T.
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      • Otieno K.
      • Awino N.
      • Omoto J.
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      • Gondi S.
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      • Ogutu B.
      • Wasuna R.
      • Owira V.
      • Jones D.
      • Onyango A.A.
      • Njuguna P.
      • Chilengi R.
      • Akoo P.
      • Kerubo C.
      • Gitaka J.
      • Maingi C.
      • Lang T.
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      • Tsofa B.
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      • Asante K.P.
      • Osei-Kwakye K.
      • Boahen O.
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      • Schellenberg D.
      • Sillman M.
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      First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children.
      ,
      • RTS,S Clinical Trials Partnership
      • Agnandji ST
      • Lell B
      • Fernandes JF
      • Abossolo BP
      • Methogo BG
      • Kabwende AL
      • Adegnika AA
      • Mordmüller B
      • Issifou S
      • Kremsner PG
      • Sacarlal J
      • Aide P
      • Lanaspa M
      • Aponte JJ
      • Machevo S
      • Acacio S
      • Bulo H
      • Sigauque B
      • Macete E
      • Alonso P
      • Abdulla S
      • Salim N
      • Minja R
      • Mpina M
      • Ahmed S
      • Ali AM
      • Mtoro AT
      • Hamad AS
      • Mutani P
      • Tanner M
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      • Sorgho H
      • Valea I
      • Bihoun B
      • Guiraud I
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      • Guiguemdé RT
      • Ouédraogo JB
      • Hamel MJ
      • Kariuki S
      • Oneko M
      • Odero C
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      • Awino N
      • McMorrow M
      • Muturi-Kioi V
      • Laserson KF
      • Slutsker L
      • Otieno W
      • Otieno L
      • Otsyula N
      • Gondi S
      • Otieno A
      • Owira V
      • Oguk E
      • Odongo G
      • Woods JB
      • Ogutu B
      • Njuguna P
      • Chilengi R
      • Akoo P
      • Kerubo C
      • Maingi C
      • Lang T
      • Olotu A
      • Bejon P
      • Marsh K
      • Mwambingu G
      • Owusu-Agyei S
      • Asante KP
      • Osei-Kwakye K
      • Boahen O
      • Dosoo D
      • Asante I
      • Adjei G
      • Kwara E
      • Chandramohan D
      • Greenwood B
      • Lusingu J
      • Gesase S
      • Malabeja A
      • Abdul O
      • Mahende C
      • Liheluka E
      • Malle L
      • Lemnge M
      • Theander TG
      • Drakeley C
      • Ansong D
      • Agbenyega T
      • Adjei S
      • Boateng HO
      • Rettig T
      • Bawa J
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      • Sarfo A
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      • Nkomo R
      • Tembo T
      • Tegha G
      • Tsidya M
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      • Chawinga C
      • Ballou WR
      • Cohen J
      • Guerra Y
      • Jongert E
      • Lapierre D
      • Leach A
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      • Ofori-Anyinam O
      • Olivier A
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      • Carter T
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      • Radford A
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      A Phase 3 trial of RTS,S/AS01 malaria vaccine in African infants.
      ). We propose that antibody-based vaccine efficacy could perhaps be significantly improved by producing a multivalent malaria subunit vaccine. Should one or more of these newly characterized surface-exposed antigens prove to be an effective antigen, it could be combined with CSP to induce a broader immune response to Plasmodium parasites and provide greater levels of protection. Taken together, the salivary gland sporozoite proteomes of P. falciparum and P. yoelii have provided further insights into critical aspects of basic sporozoite biology, and have also identified potentially targetable components of large multi-protein complexes necessary in order for the sporozoite to infect within a new host.

      Acknowledgments

      We thank the members of the Kappe and Moritz Laboratories for both technical assistance and critical discussion of this work. We also thank Mark Kennedy (Seattle BioMed) for advanced access to a density gradient sporozoite purification method. S.E.L., K.E.S., A.H., and A.M.V. performed research; P.S. contributed critical reagents; S.E.L., K.E.S., R.L.M., and S.H.I.K. designed research, analyzed and interpreted data, and wrote the manuscript.

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