SSP3 Is a Novel Plasmodium yoelii Sporozoite Surface Protein with a Role in Gliding Motility Anke Harupa,a,b Brandon K. Sack,a Viswanathan Lakshmanan,a Nadia Arang,a Alyse N. Douglass,a Brian G. Oliver,a Andrew B. Stuart,a D. Noah Sather,a Scott E. Lindner,a* Kevin Hybiske,c Motomi Torii,d Stefan H. I. Kappea,e

Plasmodium sporozoites develop within oocysts in the mosquito midgut wall and then migrate to the salivary glands. After transmission, they embark on a complex journey to the mammalian liver, where they infect hepatocytes. Proteins on the sporozoite surface likely mediate multiple steps of this journey, yet only a few sporozoite surface proteins have been described. Here, we characterize a novel, conserved sporozoite surface protein (SSP3) in the rodent malaria parasite Plasmodium yoelii. SSP3 is a putative type I transmembrane protein unique to Plasmodium. By using epitope tagging and SSP3-specific antibodies in conjunction with immunofluorescence microscopy, we showed that SSP3 is expressed in mosquito midgut oocyst sporozoites, exhibiting an intracellular localization. In sporozoites derived from the mosquito salivary glands, however, SSP3 localized predominantly to the sporozoite surface as determined by immunoelectron microscopy. However, the ectodomain of SSP3 appeared to be inaccessible to antibodies in nonpermeabilized salivary gland sporozoites. Antibody-induced shedding of the major surface protein circumsporozoite protein (CSP) exposed the SSP3 ectodomain to antibodies in some sporozoites. Targeted deletion of SSP3 adversely affected in vitro sporozoite gliding motility, which, surprisingly, impacted neither their cell traversal capacity, host cell invasion in vitro, nor infectivity in vivo. Together, these data reveal a previously unappreciated complexity of the Plasmodium sporozoite surface proteome and the roles of surface proteins in distinct biological activities of sporozoites.

M

alaria is a devastating global health problem that accounted for 627,000 deaths in 2012 (1). The disease is caused by the protozoan parasites of the genus Plasmodium, and infection is initiated by the sporozoite forms of the parasite, which are transmitted through bites of infected Anopheles mosquitoes. Sporozoites develop within oocysts in the mosquito midgut wall, actively egress into the hemocoel, and enter the salivary glands (2). Upon transmission to the mammalian host by mosquito bite, sporozoites become highly motile and actively traverse the dermal tissue to invade a blood vessel. The blood circulation carries the sporozoites to the liver, where they leave the bloodstream by traversing the sinusoidal endothelium, infect hepatocytes, and commence intracellular development as liver stages. The intrahepatocytic liverstage parasite undergoes growth and differentiation to form tens of thousands of exoerythrocytic merozoites (3, 4). The complex sporozoite journey from the mosquito midgut to the mammalian liver likely involves sporozoite surface proteins, but few have been identified to date. The first identified surface protein was the circumsporozoite protein (CSP), which covers the entire sporozoite surface. CSP is the most clinically advanced malaria vaccine candidate (5), affording significant but limited protection against malaria. One important finding that provided the rationale for clinical testing of CSP was that antibodies against it block sporozoite motility and inhibit invasion of hepatocytes (6, 7). Thus, identifying novel surface proteins could potentially provide new targets for blocking sporozoite infection. A second sporozoite protein, thrombospondin-related anonymous protein (TRAP), also known as sporozoite surface protein 2 (SSP2), is essential for sporozoite motility, mosquito salivary gland invasion, and hepatocyte infection (8–11). TRAP is released from micronemes and anchors into the sporozoite plasma membrane,

November 2014 Volume 82 Number 11

where it becomes part of the glideosome, a unique actomyosinbased motor complex which powers motility and invasion. The actomyosin motor is located in the space between the plasma membrane and the underlying inner membrane complex (IMC), which is made up of flattened vesicles that are connected to the parasite cytoskeleton. Myosin is anchored to the IMC, while actin is indirectly linked to the cytoplasmic tail of TRAP, which in turn interacts with the substrate or target cell via its extracellular adhesive domains. As the stationary myosin pulls on actin filaments, TRAP is displaced toward the posterior end of the sporozoite, resulting in forward movement (12). While several other micronemal proteins have been shown to associate with the sporozoite surface (13, 14), it is unclear whether additional surface proteins are present and important for motility. Using chemical labeling and mass spectrometry, we have recently identified several novel putative surface-exposed proteins in the rodent malaria parasite Plasmodium yoelii and in the human

Received 19 March 2014 Returned for modification 14 April 2014 Accepted 9 August 2014 Published ahead of print 25 August 2014 Editor: J. H. Adams Address correspondence to Stefan H. I. Kappe, [email protected]. * Present address: Scott E. Lindner, Department of Biochemistry & Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.01800-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.01800-14

Infection and Immunity

p. 4643– 4653

iai.asm.org

4643

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

Seattle Biomedical Research Institute, Seattle, Washington, USAa; Institute of Biology, Freie Universitaet Berlin, Berlin, Germanyb; Department of Medicine, University of Washington, Seattle, Washington, USAc; Department of Molecular Parasitology, Ehime University, Toon, Ehime, Japand; Department of Global Health, University of Washington, Seattle, Washington, USAe

Harupa et al.

MATERIALS AND METHODS Experimental mice, parasites, and mosquitoes. Six- to 8-week-old female BALB/cJ or Swiss Webster (SW) mice from the Jackson Laboratory (Bar Harbor, ME) were used for production of transgenic parasites and for mosquito feedings. Female BALB/cJ mice (6 to 8 weeks old) from The Jackson Laboratory (Bar Harbor, ME) were used for parasite infectivity assays, i.e., patency experiments and determination of liver-stage burden. Wild-type (WT) P. yoelii 17XNL (nonlethal strain) clone 1.1 and transgenic parasites were cycled between SW mice and Anopheles stephensi mosquitoes. Mosquitoes were maintained on sugar water at a temperature of 24.5°C and 70% humidity and with a photoperiod of 12.5 h of light and 11.5 h of dark. All animal work was conducted in accordance with Institutional Animal Care and Use Committee-approved protocols. Recombinant SSP3 expression and antiserum production. The protein-coding sequence of P. yoelii SSP3 (PySSP3) was derived from PlasmoDB (http://plasmodb.org; accession no. PY01796). The putative endogenous leader peptide (amino acid residues 1 to 22) was removed and replaced with the human tissue plasminogen signal peptide (MDAM KRGLCCVLLLCGAVFVSPSAS) to optimize protein expression in mammalian cells. Additionally, the predicted transmembrane domain and the putative cytoplasmic tail were removed, leaving a final expression construct containing amino acid positions 23 to 386. For purification purposes, a glycine-serine linker, 6-histidine tag, and AviTag (SGHHHHH HGLNDIFEAQKIEWHE) were added to the C terminus of the expressed sequence. The resultant sequence was codon optimized for expression in mammalian cell culture and cloned into the pTT3 vector (17), placing the coding sequence under the control of the cytomegalovirus (CMV) promoter (18). The soluble protein was produced in HEK293F cells adapted for suspension culture under serum-free conditions, as previously described (19). PySSP3-encoding DNA was transfected into HEK293F cells using 293-Free transfection reagent (Life Technologies). For one liter of production culture, we mixed 500 ␮g of plasmid DNA with 1 ml 293-Free transfection reagent in 40 ml phosphate-buffered saline (PBS), which was allowed to complex for 15 min at 25°C. The DNA/293-Free mixture was added to 1 liter of 293F cells at a density of 1 ⫻ 106 cells/ml in FreeStyle 293 expression medium (Life Technologies). The culture was allowed to grow at 37°C with shaking for 6 days. After harvest, the cells were removed by centrifugation and imidazole was added to the supernatant at a final concentration of 10 mM. Protein was purified by a two-step chromatography protocol. First, the supernatant was passed over a HisTrap FF nickel affinity column (GE Healthcare), and the His-tagged proteins were eluted in 20 mM sodium phosphate– 0.5 M sodium chloride– 0.5 M imidazole. The proteins were separated by size exclusion chromatography (SEC) on a HiLoad 16/60 Superdex 200 column (GE Healthcare) (see Fig. S4A in the supplemental material). The fractions containing monomeric PySSP3 were collected and concentrated for final storage in PBS at pH 7.4. The final purified protein products were analyzed by native PAGE and SDS-PAGE (see Fig. S4B in the supplemental material) for size and purity. For production of specific antiserum, 25 ␮g of purified PySSP3 was

4644

iai.asm.org

suspended in TiterMax Gold adjuvant (Sigma-Aldrich) and subcutaneously injected into BALB/cJ mice. Two boosters of 25 ␮g each in TiterMax Gold adjuvant were given at 2 and 6 weeks after the first immunization. Two weeks after the second boost, blood was collected and serum was isolated by centrifugation of blood at 9,600 rpm for 10 min at 4°C. Generation of ssp3ⴚ parasites. P. yoelii 17XNL genomic DNA (gDNA) was used to amplify a 500-bp fragment of the 5= untranslated region (UTR) and a 530-bp fragment of the 3= UTR of PySSP3 (accession no. PY01796, available at http://PlasmoDB.org). The two PCR products were fused by sequence overlap extension PCR (SOE PCR) (20). The product was digested with KpnI and NotI, gel purified (Qiagen gel extraction kit), and cloned into a modified version of plasmid pL0005 (MR4; MRA-774) containing GFPmut2 under the control of the constitutive elongation factor 1 alpha promoter of Plasmodium berghei. The final plasmid was linearized with SbfI. Transfection of Py17XNL parasites using the Amaxa Nucleofector device (Lonza), and selection of resistant parasites was conducted as described elsewhere (21, 22). The presence of knockout parasites was assessed by genotyping PCR, and limiting dilution was used to isolate clones. Primer sequences are listed in Table S1 in the supplemental material. Epitope tagging of SSP3. The tagging construct was designed to replace the endogenous locus with the tagged version of PySSP3 by doublecrossover homologous recombination. We used a modified version of plasmid pL0005 (MR4; MRA-774), pL0005-HA-cMyc, which allows simultaneous tagging of proteins with a C-terminal quadruple Myc (4⫻Myc) tag and an internal triple hemagglutinin (3⫻HA) tag. For double tagging of PySSP3, the open reading frame excluding the signal peptide was amplified and cloned in frame into the plasmid between the 3⫻HA and the 4⫻Myc tags. Then, the 5= UTR of SSP3 plus the start codon and the predicted signal peptide as well as the 3= UTR were amplified, gel purified (Qiagen gel extraction kit), and fused by SOE PCR (20). The resulting PCR product was digested at the 5= and 3= ends via exogenous restriction sites designed in the primers, gel purified, and inserted into the plasmid in frame upstream of the 3⫻HA tag. The final plasmid was linearized with EagI. Transfection, selection, and cloning of transgenic parasites were conducted as described above. Primer sequences are listed in Table S1 in the supplemental material. Southern blot analysis. Genomic DNA from wild-type parasites and from the ssp3⫺ clone 1 was digested with the restriction enzymes HindIII and XbaI. The resulting DNA fragments were separated by gel electrophoresis on a 0.8% agarose gel, followed by transfer to a nylon membrane (Roche). DNA probes specific to the 5= and 3= flanking regions were digoxigenin (DIG) labeled by PCR (Roche). Hybridization of the probe and chemiluminescent detection were conducted according to the manufacturer’s instructions (Roche). Phenotypic analysis of parasite development in mosquitoes. Anopheles stephensi mosquitoes were allowed to feed on infected SW mice harboring mature gametocytes for 30 min. Ten days after the infectious blood meal, mosquitoes were dissected to evaluate midgut infection. On day 14 or 15 after the blood meal, salivary gland sporozoite numbers per mosquito were determined by counting sporozoites isolated from at least 30 mosquitoes on a hemocytometer at a magnification of ⫻400. Immunofluorescence assay (IFA). Isolated oocyst or salivary gland sporozoites were fixed with 4% paraformaldehyde (PFA) for 10 min. Sporozoites were washed twice with PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 3% bovine serum albumin (BSA) in PBS for at least 45 min. Sporozoites were stained with either CSP monoclonal antibody (MAb) (2F6), TRAP MAb (F3B5), myosin A tailinteracting protein (MTIP) polyclonal antibody (PAb), c-Myc MAb (9E10; Roche), c-Myc PAb (SC-789; Santa Cruz), HA MAb (12C5A; Roche), HA PAb (Y-11; Santa Cruz), or anti-SSP3 antibodies for 1 h. Sporozoites were washed and incubated with Alexa Fluor-conjugated secondary antibodies specific to rabbit and mouse IgGs for 30 min. In live stainings, sporozoites were simultaneously incubated with CSP MAb and HA PAb or c-Myc PAb in 3% BSA–RPMI for 1 h. Sporozoites were then

Infection and Immunity

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

malaria parasite Plasmodium falciparum (15). One potential surface protein detected in this screen was the putative type I transmembrane protein PY01796 (also denoted S23), which was previously identified in a screen for sporozoite-specific transcripts in P. yoelii (16). In this study, we characterized P. yoelii PY01796 and analyzed its role in sporozoite biology. Using epitope tagging and specific antibodies, we confirmed surface localization of PY01796 by immunoelectron microscopy (IEM) and therefore named this protein sporozoite surface protein 3 (SSP3). We furthermore generated gene knockout parasites and found that the lack of SSP3 leads to a defect in gliding motility.

Novel Plasmodium Sporozoite Surface Protein

November 2014 Volume 82 Number 11

reference cDNA sample for PCR amplification. Relative transcript quantification was determined using the 2⫺⌬⌬CT method. Western blotting. Sporozoite lysates were prepared using M-PER buffer (Pierce) and protease inhibitor cocktail tablets (cOmplete; Roche). A nonreducing lane marker (5⫻; Pierce) and 5% beta-mercaptoethanol (BME) were added to lysates, and samples were boiled for 5 min at 95°C, unless samples were run under nonreducing conditions. Proteins were separated by SDS-PAGE (4 to 20% gels; Pierce), blotted onto polyvinylidene difluoride (PVDF) membranes, and blocked with 5% nonfat skim milk in 0.1% Tween 20 in PBS for at least 1 h at RT. Primary and secondary antibodies were diluted in blocking buffer, and blots were incubated with antibodies for at least 1 h at RT. Analysis was carried out using the Odyssey infrared imaging system (Li-Cor Biosciences).

RESULTS

SSP3 is a predicted type I transmembrane protein. We previously analyzed the putative sporozoite surface proteomes of P. yoelii 17XNL (nonlethal) and P. falciparum NF54. We biotinylated surface-exposed proteins in live sporozoites, enriched the biotinylated proteins using streptavidin beads, and analyzed them by mass spectrometry (15). One of the potential surface proteins identified by this method was SSP3. It was also detected in an earlier screen for sporozoite-specific transcripts, using suppression subtractive cDNA hybridization of P. yoelii salivary gland sporozoites and merozoites, and was thus previously named sporozoite-expressed gene 23 (S23) (16). SSP3 is annotated as a hypothetical protein of unknown function with a predicted molecular mass of 50.4 kDa. It is predicted to be a surface-exposed type I transmembrane protein. Homology searches using BLASTP identified orthologs in all Plasmodium species (see Fig. S1 in the supplemental material) but did not yield any potential orthologs in other apicomplexan or nonapicomplexan organisms. SSP3 contains 13 highly conserved cysteine residues, 12 of which are within the predicted ectodomain and one within the predicted transmembrane domain (Fig. 1A). Structural homology searches using the HHpred web server (http://toolkit .tuebingen.mpg.de/hhpred) suggested that the N-terminal region of SSP3 is similar to the s48/45 domain of Plasmodium 6-Cys proteins (97% confidence; PDB accession no. 2YMO) (Fig. 1B). The 6-Cys family includes several micronemal and surface proteins that are expressed in a stage-specific manner and have been implicated in cell-cell interactions (23–25). For example, P48/45 is a gamete surface protein and plays a crucial role for male gametes in attachment to females, and the sporozoite-expressed proteins P52 and P36 localize to micronemes and are important for hepatocyte infection (24, 26, 27). The region upstream of the predicted transmembrane domain of SSP3 has some similarity to the fourth thrombospondin type 1 repeat (TSR) of F-spondin (55% confidence; PDB accession no. 1VEX) and to that of Plasmodium TRAP (64% confidence; PDB accession no. 4HQO). TSR domains are ⬃60 amino acids in length and have adhesive properties thought to be mediated by the motif WxxWSxCSxTCGxGxxxRxR (where “x” can be any amino acid), which is partially conserved in SSP3 (Fig. 1C) (28). PySSP3 shows peripheral localization in salivary gland sporozoites. Since SSP3 was identified in the putative sporozoite surface proteome (15), we sought to confirm its localization on the sporozoite surface. To monitor expression and analyze the localization of SSP3 in vivo, we generated transgenic P. yoelii 17XNL (nonlethal) parasites that express SSP3 N-terminally tagged with a triple hemagglutinin (HA) tag and simultaneously

iai.asm.org 4645

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

washed, fixed with 4% PFA for 10 min, washed twice, and incubated with secondary antibodies. Nuclei were stained with 4=,6=-diamidino-2-phenylindole (DAPI) for 5 min. Sporozoites were applied to glass slides and mounted with the antifade reagent VectaShield (Vector Laboratories). All steps were performed at room temperature (RT). Images were acquired using Olympus IX70 DeltaVision microscopy equipped with deconvolution software. Immunoelectron microscopy. Mosquito salivary glands infected with P. yoelii SSP3nHA-cMyc were fixed in 1% PFA– 0.2% glutaraldehyde in PBS and embedded in LR White resin (Polysciences). Sections were blocked for 30 min in PBS-milk-Tween 20, incubated overnight at 4°C in PBS-milk-Tween 20 containing mouse anti-HA or mouse anti-SSP3 (both at a 1:25 dilution), and then incubated for 1 h in PBS-milk-Tween 20 containing goat anti-mouse IgG conjugated with gold particles (15-nm diameter; British BioCell International). Sections were examined with a JEM-1230 electron microscope (JEOL, Japan) after staining with 2% uranyl acetate and lead citrate. Gliding motility assay. Coverslips were precoated with 10 ␮g/ml CSP MAb (2F6) in PBS overnight at RT and then washed three times with PBS. P. yoelii sporozoites were isolated from mosquito salivary glands in RPMI. Sporozoites were pelleted for 1 min at 8,000 rpm, resuspended in 20% FBS–RPMI, and added to the precoated coverslips. Sporozoites were allowed to glide for 1 h at 37°C, the medium was then removed, and the sporozoites were fixed with 4% PFA for 10 min, washed, and blocked with 3% BSA–PBS for 45 min. To visualize the CSP-containing trails, coverslips were incubated with Alexa Fluor 488-conjugated CSP MAb for 30 min. Trails were counted using a fluorescence microscope. Cell traversal and invasion assay. Isolated salivary gland sporozoites were activated for 20 min in 20% FBS–PBS at RT. Sporozoites (1 ⫻ 105 per well) were added to 3 ⫻ 105 Hepa 1-6 cells in the presence of 1 mg/ml Alexa Fluor 488-labeled dextran (10,000 molecular weight [MW], lysine fixable; Invitrogen). Sporozoites were allowed to invade for 1.5 h at 37°C. Cells were then washed with PBS, trypsinized, and fixed with Cytofix/ Cytoperm buffer (BD Biosciences). Cells were blocked with Cytoperm/ Cytowash buffer (BD Biosciences) supplemented with 2% BSA for 10 min. Cells were then stained with Alexa Fluor 647-conjugated CSP MAb in Cytoperm/Cytowash buffer for 1 h, washed, and analyzed by flow cytometry using a BD-LSRII flow cytometer (BD Biosciences). Analysis of sporozoite infectivity. Sporozoites were isolated from mosquito salivary glands at day 14 or day 15 after the infectious blood meal. Sporozoites were injected either intradermally (i.d.) or intravenously (i.v.) into the tail veins of BALB/cJ mice (n ⫽ 5 per group). The time to blood-stage patency (defined as ⬎1 infected erythrocyte/10,000 erythrocytes) was monitored microscopically with Giemsa-stained thin blood smears. For delivery of sporozoites via infectious mosquito bite, BALB/cJ mice (n ⫽ 5 per group) were anesthetized and individually exposed to the bites of 12 to 15 sporozoite-infected mosquitoes. Mosquitoes were allowed to feed for a total of 7.5 min, rotating the mice between five mosquito cages every 1.5 min. Salivary glands of blood-fed mosquitoes were then isolated to ensure the presence of sporozoites and to quantify them. The time to blood-stage patency was monitored microscopically with Giemsa-stained thin blood smears. Quantitative analysis of liver-stage burden in vivo. Female BALB/cJ mice (n ⫽ 5) were intravenously injected with 1 ⫻ 105 salivary gland sporozoites. Livers were harvested at 44 h postinjection and homogenized in TRIzol reagent (Invitrogen). Total RNA was extracted using the Directzol RNA MiniPrep kit (Zymo Research). cDNA synthesis was performed using the QuantiTect reverse transcription kit (Qiagen) according to the manufacturer’s instructions. Quantitative PCR was carried out using the POWER SYBR green master mix (Applied Biosystems) and the ABI thermocycler 7300. Parasite RNA was detected using primers specific to the Plasmodium 18S rRNA gene. Primers specific to the mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene were used as internal control primers. A standard curve was generated using 1:4 dilutions of a

Harupa et al.

C-terminally tagged with a quadruple Myc tag (P. yoelii SSP3nHacMyc) (Fig. 2A). Transgenic parasites were generated via doublecrossover homologous recombination to replace the endogenous open reading frame with the tagged version of SSP3 (see Fig. S2 in the supplemental material). Expression of SSP3nHA-cMyc was controlled by its endogenous promoter and the commonly used 3= regulatory elements of the P. berghei DHFR/TS gene. We verified expression of double-tagged SSP3 in salivary gland sporozoites by Western blotting using Myc- and HA-specific antibodies (Fig. 2B). A single protein species of the expected size (⬃70 kDa [50.4-kDa SSP3 plus the tags]) representing full-length double-tagged SSP3 was uniquely detected in transgenic sporozoites, suggesting that SSP3 is not proteolytically processed. SSP3 was not detected in lysates of mixed blood stages (see Fig. S4E in the supplemental material), which is in agreement with the lack of SSP3 in the previously published proteomes of asexual and sexual blood stages of P. falciparum and P. berghei (29–33). Because SSP3 contains 13 conserved cysteine residues, we tested whether these residues form disulfide bonds by analyzing the electrophoretic mobility of SSP3 under reducing and nonreducing (omitting the disulfide-reducing agent BME and with no boiling) conditions. Indeed, we did observe a mobility shift (Fig. 2C), indicating the presence of intramolecular disulfide bonds. We next performed immunofluorescence assays (IFAs) on sporozoites to analyze the localization of SSP3. IFA of fixed and permeabilized oocyst sporozoites isolated from mosquito midguts revealed an internal localization of SSP3 that partially overlapped with the luminal endoplasmic reticulum (ER) protein binding immunoglobulin protein (BiP) (Fig. 2D). The observed signal for SSP3 varied from faint to readily detectable, possibly marking sporozoites of different stages of maturation, potentially due to the asynchronous development of oocysts (34). In mosquito sali-

4646

iai.asm.org

vary glands, 75% of sporozoites showed a strong circumferential signal for SSP3 that colocalized with that of the surface protein CSP, whereas the remaining 25% of sporozoites exhibited SSP3 expression within the sporozoite (Fig. 2E, top panels). This might be caused by differences in maturation or could be due to a contamination with hemolymph sporozoites, for which we observed mainly a strong internal, heterogenous localization for SSP3 (see Fig. S3 in the supplemental material). Importantly, the number of P. yoelii SSP3nHa-cMyc sporozoites per mosquito was similar to the wild-type (WT) level (see Table S2 in the supplemental material), indicating that the addition of the epitope tags had no gross impact on parasite fitness. Overall, these data suggest that SSP3 localization changes from internal to the periphery of sporozoites during sporozoite maturation. To further characterize the subcellular localization of doubletagged SSP3 and determine the orientation within the plasma membrane, we performed IFAs on nonpermeabilized salivary gland sporozoites. Antibody staining with anti-Myc antibodies did not yield a signal (Fig. 2F), indicating that the Myc-tagged C terminus of SSP3 might face the cytoplasm, which would be in agreement with its predicted topology. Unexpectedly however, only a few sporozoites stained positive when using antibodies against the N-terminal HA tag of SSP3 (Fig. 2F). Thus, it appears that the N terminus of SSP3 is not readily accessible to antibodies in nonpermeabilized sporozoites. To further substantiate this finding, we generated polyclonal antibodies against the putative ectodomain of SSP3 (see Fig. S4 in the supplemental material) and performed IFAs on SSP3nHa-cMyc sporozoites. Again, SSP3 was not readily detected in nonpermeabilized sporozoites (see Fig. S4C in the supplemental material), whereas permeabilized sporozoites showed mainly a peripheral staining that colocalized with

Infection and Immunity

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

FIG 1 Schematic of the predicted protein structure of PySSP3 and sequence alignments. (A) Cartoon of SSP3 protein structure and amino acid (aa) sequence identities to its orthologs in P. berghei (Pb), P. falciparum (Pf), and P. vivax (Pv). Black bars indicate conserved cysteine (Cys) residues. Boxes below mark regions of structural similarity to the indicated domains identified through HHpred search. SP, signal peptide; TM, transmembrane domain. (B and C) Sequence alignments of PySSP3 and PfSSP3 with the s48/45 domain 1 of two representative members of the 6-Cys protein family (B) and with TSRs of PfTRAP and F-spondin (C). Conserved cysteine residues in SSP3 are marked by an asterisk. Identical residues are boxed gray. Invariant residues in most TSRs are highlighted in bold.

Novel Plasmodium Sporozoite Surface Protein

matic representation and predicted topology of SSP3 tagged with an N-terminal triple HA tag and a C-terminal quadruple Myc tag. Transgenic P. yoelii parasites expressing double-tagged SSP3 were generated by double-crossover homologous recombination (see Fig. S2 in the supplemental material). (B) Two-color Western blot analysis of lysate from P. yoelii SSP3nHA-cMyc salivary gland sporozoites (lane 1) probed with anti-HA and anti-Myc shows expression of both tags. WT sporozoites (lane 2) were used as a negative control. Analysis was carried out using the Odyssey infrared imaging system. The blot is displayed for each channel in grayscale as well as in color when both channels are merged (HA in green and Myc in red). Note that anti-Myc antibody detects a nonspecific band at ⬃55 kDa. (C) Lysate of P. yoelii SSP3nHAcMyc sporozoites was run under reducing (⫹ BME) and nonreducing (⫺ BME) conditions and revealed a mobility shift of SSP3. Incubation of the same blot with antibodies against myosin A tail-interacting protein (MTIP) served as a loading control. (D) IFA of oocyst sporozoites shows partial colocalization of double-tagged SSP3 with the ER marker BiP. (E) IFA of salivary gland sporozoites. The top two panels show representative images of the different SSP3 staining patterns observed (internal versus circumferential) in costaining with the micronemal marker TRAP. The percentage indicates the prevalence of each pattern, determined by analysis of 467 sporozoites. The majority of sporozoites displayed a peripheral pattern which overlapped with the surface protein CSP (third row from top). The bottom row confirms expression and colocalization of the epitope tags. Insets show zoomed-in views of the indicated regions. (F) IFA of nonpermeabilized (non-perm.) salivary gland sporozoite. All scale bars are 5 ␮m.

November 2014 Volume 82 Number 11

iai.asm.org 4647

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

FIG 2 Expression and localization of SSP3 in P. yoelii sporozoites. (A) Sche-

the antibody staining of the SSP3 tags (see Fig. S4D, middle panel, in the supplemental material). IEM determines surface localization of SSP3. To further analyze the precise subcellular localization of SSP3, we performed immunoelectron microscopy (IEM). Sections of P. yoelii SSP3nHa-cMyc sporozoite-infected salivary glands were stained with anti-HA antibodies or with anti-SSP3 antibodies followed by secondary antibodies conjugated to gold particles. SSP3 localized predominantly to the sporozoite surface, with gold particles closely associated with the plasma membrane. Interestingly, SSP3 appeared to be nonhomogenously distributed as indicated by the frequent clustering of gold particles (Fig. 3). A small amount of SSP3 was detected intracellularly within the sporozoite and might represent SSP3 trafficking through the secretory pathway to the periphery (Fig. 3D and E). In addition to being detected on the sporozoite surface, SSP3 was less frequently localized to the space between the plasma membrane and the inner membrane complex (Fig. 3E). SSP3 is not shed following sporozoite activation. Sporozoite surface proteins such as CSP and TRAP are shed in trails during gliding motility on a solid substrate (35, 36), and sporozoite microneme secretion and motility can be activated in vitro by incubating sporozoites at 37°C in the presence of serum (13, 37). To investigate whether SSP3 is also shed in trails, P. yoelii SSP3nHacMyc sporozoites were allowed to glide on glass coverslips in the presence of serum for 1 h at 37°C and were then stained with anti-CSP and anti-HA or anti-Myc antibodies. Gliding trails were clearly visible based on CSP staining; however, SSP3 was not detected in trails as determined by the lack of staining of HA or Myc (Fig. 4A). To corroborate the finding that SSP3 is not released upon gliding motility activation, we analyzed the supernatants from preparations that contained activated sporozoites by Western blotting. We detected SSP3 not in the supernatant but only in the sporozoite pellet, whereas the micronemal protein TRAP was secreted and detected in the supernatant (Fig. 4B). These results suggest that SSP3 is not released following sporozoite activation and is not shed during gliding motility. Accessibility to SSP3 on the sporozoite surface might be masked by CSP. SSP3 clearly localized to the surface of salivary gland sporozoites as determined by IEM; however, we were unable to reliably detect SSP3 on the surface in nonpermeabilized sporozoites using IFA. One reason for this could be that SSP3 is inaccessible to antibodies, potentially due to the dense coat of CSP that entirely covers the sporozoite surface. CSP is a putatively glycosylphosphatidylinositol (GPI)-anchored protein that is shed when cross-linked by antibodies, a process known as the CSP precipitation reaction (38). To test whether SSP3 is accessible to antibodies upon shedding of CSP, we incubated live P. yoelii SSP3nHa-cMyc sporozoites with anti-CSP antibodies as well as anti-HA or antiMyc to label SSP3. Sporozoites that had shed CSP were readily distinguished from those that had not either through the presence of the precipitate or through weaker fluorescence intensity of the CSP signal remaining on the sporozoite surface (Fig. 4C). While the majority of sporozoites that had shed CSP still did not stain positive for SSP3, we observed a strong circumferential signal for the N-terminal HA tag in ⬃ 20% of P. yoelii SSP3nHa-cMyc sporozoites that shed CSP (Fig. 4C). As shown (Fig. 2F), the Cterminal Myc tag was never detected in these experiments (Fig. 4C), further indicating that the C terminus of SSP3 faces the sporozoite interior. Overall, these results suggest that SSP3 might

Harupa et al.

cMyc sporozoites were incubated either with anti-HA antibodies (A and B) or with anti-SSP3 antisera (C to E) followed by secondary antibodies conjugated to gold particles. IMC, inner membrane complex; Mn, microneme; Mt, microtubule; N, nucleus; PM, plasma membrane; Rh, rhoptry. Scale bars in panels A, D, and E are 500 nm; those in panels B and C are 250 nm.

be masked by CSP and is exposed and accessible to antibodies once CSP is shed. SSP3-deficient parasites are viable but are defective in substrate attachment and gliding motility. To determine whether SSP3 is important for progression through the life cycle of P. yoelii, we generated gene knockout parasites (P. yoelii ssp3⫺) using standard methodology (Fig. 5A) (22). Recombinant parasites were cloned by serial dilution, and the gene knockout was confirmed by PCR genotyping and Southern blot analysis (Fig. 5B and C). The successful deletion of SSP3 in P. yoelii showed that it does not play a role in the growth of blood-stage parasites, which is in good agreement with the lack of SSP3 expression in these stages. To determine whether the deletion of SSP3 affects the parasite life cycle stages within the mosquito, we analyzed oocyst development and quantified sporozoite numbers in midguts and salivary glands. WT and ssp3⫺ parasites generated comparable numbers of oocyst and salivary gland sporozoites (see Table S2 in the supplemental material). These results demonstrate that the deletion of SSP3 does not affect sporogony, sporozoite egress from oocysts, or invasion of salivary glands. We next wanted to test whether the sporozoites’ gliding locomotion is affected by the lack of SSP3. Productive gliding motility depends on adhesion to the substrate, which is mediated by surface proteins, including CSP and TRAP proteins (39, 40). Adhesion is a multistep process that involves an initial attachment with either the apical or the posterior end of the sporozoite, followed by attachment of the other end and finally adhesion of the central part (41). Sporozoites then start to move and leave trails of cleaved CSP and TRAP behind. To analyze motility of ssp3⫺ parasites, we added salivary gland sporozoites to glass coverslips precoated with anti-CSP antibodies and visualized the trails using Alexa Fluor 488-conjugated anti-CSP antibodies. We observed a significantly

4648

iai.asm.org

reduced number of trails left behind by ssp3⫺ sporozoites compared to WT sporozoites (Fig. 5D). WT sporozoites showed typical circular gliding trails, whereas the majority of ssp3⫺ sporozoites did not produce any trails. We then performed video microscopy on live sporozoites. WT sporozoites exhibited continuous circular gliding motility, whereas ssp3⫺ sporozoites were often attached to the glass slide with only one end (see Movies S1 and S2 in the supplemental material). It appeared that the adhesion steps following the initial attachment were unsuccessful and thus did not allow the sporozoite to initiate gliding. These results suggest that the motility defect in ssp3⫺ sporozoites might result from the failure to properly attach to the substrate. SSP3 is not required for cell traversal and invasion. We next analyzed whether the observed motility defect of ssp3⫺ sporozoites translates into a defect in cell traversal and hepatocyte invasion by using a flow cytometry-based cell wounding and invasion assay (42–44). Sporozoites were added to hepatoma cells in the presence of cell-impermeable Alexa Fluor 488-conjugated dextran and incubated for 90 min. Traversing sporozoites migrate through cells by disrupting the membrane, which leads to uptake of dextran that gets trapped inside the cell once the membrane is resealed (42). Intracellular sporozoites were detected by staining of infected cells with CSP antibody. Surprisingly, WT and ssp3⫺ sporozoites showed no significant differences in their capacity to traverse and invade hepatoma cells in vitro (Fig. 6A and B). We next investigated whether the lack of SSP3 affected the transmission of sporozoites from mosquito to mouse in vivo and whether ssp3⫺ sporozoites are able to infect the liver and ultimately establish blood-stage infection. We injected 1 ⫻ 103 ssp3⫺ sporozoites intravenously into BALB/cJ mice and monitored for the onset of blood-stage infection (patency) by analyzing Giemsastained thin blood smears. To analyze the ability of ssp3⫺ sporo-

Infection and Immunity

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

FIG 3 Localization of PySSP3 in sporozoites by immunoelectron microscopy. Ultrathin sections of mosquito salivary glands infected with P. yoelii SSP3nHA-

Novel Plasmodium Sporozoite Surface Protein

stained with anti-HA and Alexa Fluor 488-conjugated anti-CSP to visualize trails. (B) To test whether SSP3 is shed into the supernatant upon activation, sporozoites were incubated in the presence of serum for 1 h on ice (control) and at 37°C. Supernatants (S) and pellets (P) were subjected to SDS-PAGE, blotted, and incubated with anti-HA; anti-MTIP and anti-TRAP served as controls. (C) Live P. yoelii SSP3nHA-cMyc sporozoites were incubated for 1 h with anti-CSP antibodies to induce shedding of CSP and simultaneously incubated with anti-HA or anti-Myc to label SSP3. Sporozoites were then washed, fixed, and stained with secondary antibodies. Explanatory cartoons for the observed staining patterns are shown on the left. Scale bars are 5 ␮m.

zoites to exit the dermal tissue, we inoculated mice with 1 ⫻ 103 or 1 ⫻ 104 ssp3⫺ sporozoites intradermally (i.d.) and via infectious mosquito bite. All mice became blood-stage patent on day 3 postinfection (Table 1) except for mice that received the low dose i.d. One of five mice injected with 1 ⫻ 103 ssp3⫺ sporozoites i.d. did not develop blood-stage parasitemia, while the other mice of this group become patent on day 3 postinfection. A similar result was obtained in the respective control group (2/5 mice remained patency negative). This suggests that i.d. injection of 1 ⫻ 103 sporozoites is below the 100% infectious dose. Overall, the results demonstrate that ssp3⫺ sporozoites are fully capable of infecting mice and that SSP3 is not critical for traversal of dermal tissue and for invasion of hepatocytes. Additionally, we analyzed the liver-stage burden by quantitative reverse transcription-PCR. BALB/cJ mice were intravenously injected with 1 ⫻ 105 sporozoites, and livers were harvested at 44 h postinjection. Livers of mice infected with WT and ssp3⫺ sporozoites displayed no significant differences in parasite burden (Fig. 6C). DISCUSSION

Plasmodium sporozoites are remarkably versatile. Once transmitted to the mammalian host by a mosquito, they experience a dramatic change in environment and have to cross numerous cellular barriers in order to reach the liver, where they infect hepatocytes. Tissue traversal, host cell recognition, and invasion require the action of sporozoite surface and secreted proteins (45). However, to date, only a few sporozoite surface proteins have been described. The most studied ones are CSP and TRAP, both of which

November 2014 Volume 82 Number 11

are important for gliding motility, salivary gland infection, and recognition and invasion of hepatocytes (39). Other sporozoite surface proteins include AMA-1, which relocates from micronemes to the surface upon sporozoite activation but is dispensable for hepatocyte infection (46, 47), a secreted phospholipase involved in cell traversal (48), rhomboid protease 4, which cleaves TRAP (36), sporozoite invasion-associated protein 1 with a role in sporozoite motility (49, 50), and a few surface antigens with unknown function (51–54). Here, we characterized a novel sporozoite surface transmembrane protein, SSP3, which we previously identified in the putative surface proteome of P. yoelii and P. falciparum salivary gland sporozoites (15). Localization of SSP3 changes as the sporozoites develop within the mosquito. In oocyst sporozoites, SSP3 partially colocalized with the ER marker BiP. Hemolymph sporozoites exhibited heterogenous intracellular staining patterns for SSP3, while most salivary gland sporozoites showed a peripheral staining that overlapped with the surface protein CSP. Indeed, IEM analysis clearly determined the presence of SSP3 on the salivary gland sporozoite surface. These data and the fact that SSP3 contains a predicted signal peptide suggest that SSP3 is routed to the sporozoite surface via the secretory pathway during sporozoite maturation, which culminates in its predominant surface localization in infectious sporozoites. In an effort to determine the orientation of SSP3 in the sporozoite plasma membrane, we epitope tagged both the N and C termini and analyzed the accessibility of the tags to antibodies. Interestingly, neither tag was accessible to antibodies in fixed, nonpermeabilized sporozoites, which was un-

iai.asm.org 4649

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

FIG 4 SSP3 is not shed during sporozoite gliding motility. (A) Sporozoites were allowed to glide on CSP-coated glass coverslips for 1 h, fixed, permeabilized, and

Harupa et al.

expected as IEM localized SSP3 on the sporozoite surface. One explanation is that the dense coat of CSP on the sporozoite surface might prevent antibodies from gaining access to SSP3. CSP has a predicted rod-like structure and is attached to the sporozoite plasma membrane by a putative GPI anchor (27). Based on apparent molecular mass, the putative ectodomain of SSP3 is not smaller than CSP but of similar size. However, SSP3 likely has a

complex structure due to intramolecular disulfide bonds. It therefore seems reasonable to assume that SSP3 could be masked by CSP and thus shielded from antibodies. Indeed, when shedding of CSP was induced in live sporozoites, the SSP3 N-terminal epitope tag more frequently become accessible to antibodies, but the Cterminal tag never does so. This suggests that the C terminus faces the cytoplasm and that SSP3 is, as predicted, a type I transmem-

FIG 6 Phenotypic analysis of P. yoelii ssp3⫺ sporozoites. (A and B) In vitro traversal and infection assay. Salivary gland sporozoites were added to Hepa 1-6 cells in the presence of cell-impermeative Alexa Fluor 488-dextran. Cells were trypsinized at 1.5 h postinfection (p.i.), fixed, stained with Alexa Fluor 647-conjugated anti-CSP antibodies, and subjected to flow cytometry analysis. Error bars represent SD of technical triplicates. (C) Quantification of liver-stage burden in vivo. BALB/cJ mice (n ⫽ 5) were injected intravenously with 1 ⫻ 105 salivary gland sporozoites. Liver-stage burden was monitored 44 h p.i. using quantitative reverse transcription-PCR. Parasite RNA was detected using primers specific to the P. yoelii 18S rRNA gene. Transcript expression levels were normalized to mouse GAPDH expression levels and are shown as mean ⫾ SD. No significant differences in phenotypes between WT and ssp3⫺ sporozoites were found in any assay.

4650

iai.asm.org

Infection and Immunity

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

FIG 5 P. yoelii ssp3⫺ sporozoites show a gliding defect in vitro. (A) Schematic of targeting strategy used to generate P. yoelii ssp3⫺ parasites. The SSP3 open reading frame was replaced with the positive selectable marker human dihydrofolate reductase (hDHFR) by double-crossover homologous recombination using the 5= and 3= untranslated regions (UTRs) of SSP3. (B) PCR genotyping of gDNA from two ssp3⫺ clones with primers (bars in panel A) specific to the wild-type locus (wt) and to the transgenic locus (test 1 and test 2) shows proper integration of the construct. (C) Southern blot analysis of ssp3⫺ clone 1. Digestion of ssp3⫺ gDNA with HindIII and XbaI (“H” and “X” in panel A) results in the expected band shift compared to WT gDNA using probes (dumbbell bars in panel A) specific to the 5= and 3= UTRs of SSP3. Sizes of the expected fragments are shown in panel A. (D) In vitro gliding motility assay. Salivary gland sporozoites were allowed to glide on CSP-coated glass coverslips, and trails were visualized using Alexa Fluor 488-conjugated anti-CSP antibodies. The number of circles per trail associated with a sporozoite was counted for at least 100 trails per experiment. ssp3⫺ sporozoites showed significantly fewer trails that consist of more than 10 circles (*, P ⬍ 0.001 compared to WT [unpaired t test]). Error bars represent standard deviations (SD) of three biological replicates for WT and ssp3⫺ clone 1 and three technical replicates for ssp3⫺ clone 2.

Novel Plasmodium Sporozoite Surface Protein

TABLE 1 P. yoelii ssp3⫺ sporozoites successfully establish liver infection and transition to blood-stage infection in mice P. yoelii genotype

Injected sporozoitesa

⫺

ssp3 clone 1 1 ⫻ 103 i.v. WT 1 ⫻ 103 i.v.

No. of infected BALB/cJ Day of mice/total patencyb 3 3

ssp3⫺ clone 1 Mosquito bite (31,355/mosquito) 5/5 WT Mosquito bite (43,536/mosquito) 5/5

3 3

ssp3⫺ clone 1 1 ⫻ 103 i.d. WT 1 ⫻ 103 i.d.

4/5 3/5

3 3

ssp3⫺ clone 1 1 ⫻ 104 i.d. WT 1 ⫻ 104 i.d.

5/5 5/5

3 3

a Sporozoites were delivered intravenously (i.v.), intradermally (i.d.), or by mosquito bite. Mice were individually exposed to the bites of 12 to 15 mosquitoes for 7.5 min, rotating the mice between mosquito cages every 1.5 min. Salivary gland sporozoites of blood-fed mosquitoes were quantified; numbers are shown in parentheses. b The time to blood stage patency was monitored microscopically with Giemsa-stained thin blood smears.

brane protein. However, since only some CSP-shed sporozoites stained HA positive, other factors must play a role. Proteolytic processing might be one factor. Cleavage and shedding of surface proteins involved in motility and invasion constitute a key feature of apicomplexan parasites (55). It serves either to expose adhesive domains such as TSRs or to disengage adhesive interactions (36, 55, 56). SSP3 contains a TSR-like fold upstream of the predicted transmembrane domain. However, we did not detect SSP3 in sporozoite gliding trails by IFA or in the supernatant of sporozoites incubated under motility-activating conditions. It therefore seems unlikely that the lack of tag detection in live sporozoites is due to a cleavage event. However, this cannot be ruled out, as cleaved fragments may be unstable and quickly degraded or too small to detect. It is also possible that the N-terminal tag is buried inside the ectodomain of SSP3 and may thus be difficult for antibodies to access even when CSP is shed. To investigate the importance of SSP3 in sporozoites, we deleted the encoding gene in the P. yoelii genome. ssp3⫺ parasites showed no significant difference in their capacity to establish mosquito colonization compared to WT parasites. However, ssp3⫺ sporozoites displayed a gliding motility defect. WT sporozoites showed typical circular gliding trails, whereas the vast majority of ssp3⫺ sporozoites did not leave any trails behind. Video microscopy suggested that ssp3⫺ sporozoites are able to attach to the substrate with one end but fail to establish subsequent adhesion sites and thus cannot initiate gliding motility. SSP3 might therefore act as an adhesin. However, it does not contain motifs that suggest its linkage to the actomyosin motor, i.e., the stretches of negatively charged residues in the cytoplasmic tail and a subterminal tryptophan residue which are features of TRAP family proteins (40). It is also possible that the lack of SSP3 on the sporozoite surface might alter the distribution or functionality of other proteins important for attachment and/or gliding in vitro. It has been shown that TRAP and S6, another TRAP family protein, are necessary for initial adhesion to glass slides (33). TRAP proteins are released from micronemes onto the sporozoite surface and are redistributed from the front to the back of the sporozoite. This or

November 2014 Volume 82 Number 11

ACKNOWLEDGMENTS We thank Lynett Robertson and Patrick McDougall for intradermal injections and the insectary team for providing us with mosquitoes. This work was partially funded by a Bill and Melinda Gates Foundation grant (grant no. OPP1067687).

REFERENCES 1. World Health Organization. 2013. World malaria report. World Health Organization, Geneva, Switzerland. 2. Aly AS, Matuschewski K. 2005. A malarial cysteine protease is necessary for Plasmodium sporozoite egress from oocysts. J. Exp. Med. 202:225– 230. http://dx.doi.org/10.1084/jem.20050545. 3. Vanderberg JP, Frevert U. 2004. Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int. J. Parasitol. 34:991–996. http://dx .doi.org/10.1016/j.ijpara.2004.05.005. 4. Amino R, Thiberge S, Martin B, Celli S, Shorte S, Frischknecht F, Ménard R. 2006. Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nat. Med. 12:220 –224. http://dx.doi.org/10.1038 /nm1350. 5. Regules JA, Cummings JF, Ockenhouse CF. 2011. The RTS,S vaccine candidate for malaria. Expert Rev. Vaccines 10:589 –599. http://dx.doi.org /10.1586/erv.11.57. 6. Yoshida N, Nussenzweig RS, Potocnjak P, Nussenzweig V, Aikawa M. 1980. Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science 207:71–73. http://dx.doi.org /10.1126/science.6985745. 7. Stewart MJ, Nawrot RJ, Schulman S, Vanderberg JP. 1986. Plasmodium berghei sporozoite invasion is blocked in vitro by sporozoiteimmobilizing antibodies. Infect. Immun. 51:859 – 864. 8. Sultan AA, Thathy V, Frevert U, Robson KJ, Crisanti A, Nussenzweig V, Nussenzweig RS, Ménard R. 1997. TRAP is necessary for gliding motility and infectivity of plasmodium sporozoites. Cell 90:511–522. http: //dx.doi.org/10.1016/S0092-8674(00)80511-5. 9. Wengelnik K, Spaccapelo R, Naitza S, Robson KJ, Janse CJ, Bistoni F, Waters AP, Crisanti A. 1999. The A-domain and the thrombospondinrelated motif of Plasmodium falciparum TRAP are implicated in the invasion process of mosquito salivary glands. EMBO J. 18:5195–5204. http: //dx.doi.org/10.1093/emboj/18.19.5195.

iai.asm.org 4651

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

5/5 3/3

a similar process may be affected in ssp3⫺ sporozoites, leading to suboptimal attachment and/or gliding motility. Surprisingly, despite their defect in gliding motility, ssp3⫺ sporozoites exhibited normal cell traversal activity and hepatocyte invasion rates that were similar to WT. Furthermore, transmission of ssp3⫺ sporozoites via mosquito bite led to blood-stage infection in mice without a delay in patency, suggesting that the observed gliding motility defect does not impact infectivity. The current model for host cell invasion suggests that the gliding motility is critical for cell traversal and that the same machinery drives the invasion process. Our results would argue that this model is in need of revision, and they are intriguing in the context of recent studies in Toxoplasma demonstrating that gliding motility is not essential for cell invasion (57). However, it is important to note that gliding on an artificial substrate likely poses different requirements on the sporozoite than gliding over cells or through tissues. Thus, another explanation for these results could be that the in vitro gliding defect is substrate dependent and can be compensated for by the presence of host cell ligands. In summary, we show that SSP3 is a novel sporozoite surface protein. SSP3 plays a role in gliding motility but is dispensable for the parasites’ life cycle progression. SSP3 reveals a previously unappreciated complexity of the sporozoite surface beyond CSP and warrants further investigation into the molecular composition of the surface of this intriguing invasive stage of the malaria parasite.

Harupa et al.

4652

iai.asm.org

28.

29.

30.

31.

32.

33.

34. 35.

36.

37. 38.

39.

40. 41. 42.

43.

44.

45.

SHI. 2007. Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection. Infect. Immun. 75:3758 –3768. http://dx.doi.org/10 .1128/IAI.00225-07. Adams JC, Tucker RP. 2000. The thrombospondin type 1 repeat (TSR) superfamily: diverse proteins with related roles in neuronal development. Dev. Dyn. 218:280 –299. http://dx.doi.org/10.1002/(SICI)1097-0177 (200006)218:2⬍280::AID-DVDY4⬎3.3.CO;2-S. Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates JR, Carucci DJ. 2002. A proteomic view of the Plasmodium falciparum life cycle. Nature 419:520 – 526. http://dx.doi.org/10.1038/nature01107. Treeck M, Sanders JL, Elias JE, Boothroyd JC. 2011. The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites’ boundaries. Cell Host Microbe 10:410 – 419. http://dx.doi.org/10.1016/j.chom.2011.09.004. Pease BN, Huttlin EL, Jedrychowski MP, Talevich E, Harmon J, Dillman T, Kannan N, Doerig C, Chakrabarti R, Gygi SP, Chakrabarti D. 2013. Global analysis of protein expression and phosphorylation of three stages of Plasmodium falciparum intraerythrocytic development. J. Proteome Res. 12:4028 – 4045. http://dx.doi.org/10.1021/pr400394g. Khan SM, Franke-Fayard B, Mair GR, Lasonder E, Janse CJ, Mann M, Waters AP. 2005. Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology. Cell 121:675– 687. http://dx.doi.org/10.1016/j.cell.2005.03.027. Silvestrini F, Lasonder E, Olivieri A, Camarda G, van Schaijk B, Sanchez M, Younis Younis S, Sauerwein R, Alano P. 2010. Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum. Mol. Cell. Proteomics 9:1437–1448. http://dx .doi.org/10.1074/mcp.M900479-MCP200. Vanderberg JP. 1975. Development of infectivity by the Plasmodium berghei sporozoite. J. Parasitol. 61:43–50. http://dx.doi.org/10.2307 /3279102. Stewart MJ, Vanderberg JP. 1991. Malaria sporozoites release circumsporozoite protein from their apical end and translocate it along their surface. J. Protozool. 38:411– 421. http://dx.doi.org/10.1111/j.1550-7408 .1991.tb01379.x. Ejigiri I, Ragheb DRT, Pino P, Coppi A, Bennett BL, Soldati-Favre D, Sinnis P. 2012. Shedding of TRAP by a rhomboid protease from the malaria sporozoite surface is essential for gliding motility and sporozoite infectivity. PLoS Pathog. 8:e1002725. http://dx.doi.org/10.1371/journal .ppat.1002725. Vanderberg JP. 1974. Studies on the motility of Plasmodium sporozoites. J. Protozool. 21:527–537. http://dx.doi.org/10.1111/j.1550-7408.1974 .tb03693.x. Vanderberg J, Nussenzweig R, Most H. 1969. Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. V. In vitro effects of immune serum on sporozoites. Mil. Med. 134:1183– 1190. Aly ASI, Vaughan AM, Kappe SHI. 2009. Malaria parasite development in the mosquito and infection of the mammalian host. Annu. Rev. Microbiol. 63:195–221. http://dx.doi.org/10.1146/annurev.micro .091208.073403. Morahan BJ, Wang L, Coppel RL. 2009. No TRAP, no invasion. Trends Parasitol. 25:77– 84. http://dx.doi.org/10.1016/j.pt.2008.11.004. Hegge S, Münter S, Steinbüchel M, Heiss K, Engel U, Matuschewski K, Frischknecht F. 2010. Multistep adhesion of Plasmodium sporozoites. FASEB J. 24:2222–2234. http://dx.doi.org/10.1096/fj.09-148700. Mota MM, Pradel G, Vanderberg JP, Hafalla JC, Frevert U, Nussenzweig RS, Nussenzweig V, Rodríguez A. 2001. Migration of Plasmodium sporozoites through cells before infection. Science 291:141–144. http://dx .doi.org/10.1126/science.291.5501.141. Kaushansky A, Rezakhani N, Mann H, Kappe SHI. 2012. Development of a quantitative flow cytometry-based assay to assess infection by Plasmodium falciparum sporozoites. Mol. Biochem. Parasitol. 183:100 –103. http://dx.doi.org/10.1016/j.molbiopara.2012.01.006. Kaushansky A, Metzger PG, Douglass AN, Mikolajczak SA, Lakshmanan V, Kain HS, Kappe SH. 2013. Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis. Cell Death Dis. 4:e762. http://dx.doi.org/10.1038/cddis.2013.286. Ejigiri I, Sinnis P. 2009. Plasmodium sporozoite-host interactions from

Infection and Immunity

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

10. Ghosh AK, Devenport M, Jethwaney D, Kalume DE, Pandey A, Anderson VE, Sultan AA, Kumar N, Jacobs-Lorena M. 2009. Malaria parasite invasion of the mosquito salivary gland requires interaction between the Plasmodium TRAP and the Anopheles saglin proteins. PLoS Pathog. 5:e1000265. http://dx.doi.org/10.1371/journal.ppat.1000265. 11. Robson KJ, Frevert U, Reckmann I, Cowan G, Beier J, Scragg IG, Takehara K, Bishop DH, Pradel G, Sinden R. 1995. Thrombospondinrelated adhesive protein (TRAP) of Plasmodium falciparum: expression during sporozoite ontogeny and binding to human hepatocytes. EMBO J. 14:3883–3894. 12. Keeley A, Soldati D. 2004. The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa. Trends Cell Biol. 14:528 –532. http://dx.doi.org/10.1016/j.tcb.2004.08.002. 13. Silvie O. 2004. A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites. J. Biol. Chem. 279: 9490 –9496. http://dx.doi.org/10.1074/jbc.M311331200. 14. Steinbuechel M, Matuschewski K. 2009. Role for the Plasmodium sporozoite-specific transmembrane protein S6 in parasite motility and efficient malaria transmission. Cell. Microbiol. 11:279 –288. http://dx.doi.org/10 .1111/j.1462-5822.2008.01252.x. 15. Lindner SE, Swearingen KE, Harupa A, Vaughan AM, Sinnis P, Moritz RL, Kappe SHI. 2013. Total and putative surface proteomics of malaria parasite salivary gland sporozoites. Mol. Cell. Proteomics 12:1127–1143. http://dx.doi.org/10.1074/mcp.M112.024505. 16. Kaiser K, Matuschewski K, Camargo N, Ross J, Kappe SHI. 2004. Differential transcriptome profiling identifies Plasmodium genes encoding pre-erythrocytic stage-specific proteins. Mol. Microbiol. 51:1221– 1232. http://dx.doi.org/10.1046/j.1365-2958.2003.03909.x. 17. Durocher Y, Perret S, Kamen A. 2002. High-level and high-throughput recombinant protein production by transient transfection of suspensiongrowing human 293-EBNA1 cells. Nucleic Acids Res. 30:E9. http://dx.doi .org/10.1093/nar/30.2.e9. 18. Sellhorn G, Caldwell Z, Mineart C, Stamatatos L. 2009. Improving the expression of recombinant soluble HIV envelope glycoproteins using pseudo-stable transient transfection. Vaccine 28:430 – 436. http://dx.doi .org/10.1016/j.vaccine.2009.10.028. 19. Carbonetti S, Oliver BG, Glenn J, Stamatatos L, Sather DN. 2014. Soluble HIV-1 envelope immunogens derived from an elite neutralizer elicit cross-reactive V1V2 antibodies and low potency neutralizing antibodies. PLoS One 9:e86905. http://dx.doi.org/10.1371/journal.pone .0086905. 20. Mikolajczak SA, Aly ASI, Dumpit RF, Vaughan AM, Kappe SHI. 2008. An efficient strategy for gene targeting and phenotypic assessment in the Plasmodium yoelii rodent malaria model. Mol. Biochem. Parasitol. 158: 213–216. http://dx.doi.org/10.1016/j.molbiopara.2007.12.006. 21. Jongco AM, Ting L-M, Thathy V, Mota MM, Kim K. 2006. Improved transfection and new selectable markers for the rodent malaria parasite Plasmodium yoelii. Mol. Biochem. Parasitol. 146:242–250. http://dx.doi .org/10.1016/j.molbiopara.2006.01.001. 22. Lindner SE, Llinás M, Keck JL, Kappe SHI. 2011. The primase domain of PfPrex is a proteolytically matured, essential enzyme of the apicoplast. Mol. Biochem. Parasitol. 180:69 –75. http://dx.doi.org/10 .1016/j.molbiopara.2011.08.002. 23. Van Schaijk BCL, van Dijk MR, van de Vegte-Bolmer M, van Gemert G-J, van Dooren MW, Eksi S, Roeffen WFG, Janse CJ, Waters AP, Sauerwein RW. 2006. Pfs47, paralog of the male fertility factor Pfs48/45, is a female specific surface protein in Plasmodium falciparum. Mol. Biochem. Parasitol. 149:216 –222. http://dx.doi.org/10.1016/j.molbiopara .2006.05.015. 24. Ishino T, Chinzei Y, Yuda M. 2005. Two proteins with 6-cys motifs are required for malarial parasites to commit to infection of the hepatocyte. Mol. Microbiol. 58:1264 –1275. http://dx.doi.org/10.1111/j.1365-2958 .2005.04801.x. 25. Sanders PR, Gilson PR, Cantin GT, Greenbaum DC, Nebl T, Carucci DJ, McConville MJ, Schofield L, Hodder AN, Yates JR, III, Crabb BS. 2005. Distinct protein classes including novel merozoite surface antigens in Raft-like membranes of Plasmodium falciparum. J. Biol. Chem. 280: 40169 – 40176. http://dx.doi.org/10.1074/jbc.M509631200. 26. Van Dijk MR, Janse CJ, Thompson J, Waters AP, Braks JA, Dodemont HJ, Stunnenberg HG, van Gemert GJ, Sauerwein RW, Eling W. 2001. A central role for P48/45 in malaria parasite male gamete fertility. Cell 104: 153–164. http://dx.doi.org/10.1016/S0092-8674(01)00199-4. 27. Labaied M, Harupa A, Dumpit RF, Coppens I, Mikolajczak SA, Kappe

Novel Plasmodium Sporozoite Surface Protein

46.

47.

49.

50.

51.

November 2014 Volume 82 Number 11

52.

53.

54.

55. 56.

57.

gen, STARP. Mol. Biochem. Parasitol. 64:219 –232. http://dx.doi.org/10 .1016/0166-6851(94)00012-3. Bottius E, BenMohamed L, Brahimi K, Gras H, Lepers JP, Raharimalala L, Aikawa M, Meis J, Slierendregt B, Tartar A, Thomas A, Druilhe P. 1996. A novel Plasmodium falciparum sporozoite and liver stage antigen (SALSA) defines major B, T helper, and CTL epitopes. J. Immunol. 156: 2874 –2884. Chattopadhyay R, Rathore D, Fujioka H, Kumar S, de la Vega P, Haynes D, Moch K, Fryauff D, Wang R, Carucci DJ, Hoffman SL. 2003. PfSPATR, a Plasmodium falciparum protein containing an altered thrombospondin type I repeat domain is expressed at several stages of the parasite life cycle and is the target of inhibitory antibodies. J. Biol. Chem. 278:25977–25981. http://dx.doi.org/10.1074/jbc.M300865200. Currà C, Di Luca M, Picci L, de Sousa Silva Gomes dos Santos C, Siden-Kiamos I, Pace T, Ponzi M. 2013. The ETRAMP family member SEP2 is expressed throughout Plasmodium berghei life cycle and is released during sporozoite gliding motility. PLoS One 8:e67238. http://dx .doi.org/10.1371/journal.pone.0067238. Carruthers VB, Blackman MJ. 2005. A new release on life: emerging concepts in proteolysis and parasite invasion. Mol. Microbiol. 55:1617– 1630. http://dx.doi.org/10.1111/j.1365-2958.2005.04483.x. Coppi A, Natarajan R, Pradel G, Bennett BL, James ER, Roggero MA, Corradin G, Persson C, Tewari R, Sinnis P. 2011. The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host. J. Exp. Med. 208:341–356. http://dx.doi.org/10.1084/jem.20101488. Meissner M, Ferguson DJP, Frischknecht F. 2013. Invasion factors of apicomplexan parasites: essential or redundant? Curr. Opin. Microbiol. 16:438 – 444. http://dx.doi.org/10.1016/j.mib.2013.05.002.

iai.asm.org 4653

Downloaded from http://iai.asm.org/ on February 3, 2015 by NYU MEDICAL CENTER LIBRARY

48.

the dermis to the hepatocyte. Curr. Opin. Microbiol. 12:401– 407. http: //dx.doi.org/10.1016/j.mib.2009.06.006. Giovannini D, Späth S, Lacroix C, Perazzi A, Bargieri D, Lagal V, Lebugle C, Combe A, Thiberge S, Baldacci P, Tardieux I, Ménard R. 2011. Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa. Cell Host Microbe 10:591– 602. http://dx.doi.org/10.1016/j.chom.2011.10.012. Bargieri DY, Andenmatten N, Lagal V, Thiberge S, Whitelaw JA, Tardieux I, Meissner M, Ménard R. 2013. Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion. Nat. Commun. 4:2552. http://dx.doi.org/10.1038 /ncomms3552. Bhanot P, Schauer K, Coppens I, Nussenzweig V. 2005. A surface phospholipase is involved in the migration of plasmodium sporozoites through cells. J. Biol. Chem. 280:6752– 6760. http://dx.doi.org/10.1074 /jbc.M411465200. Engelmann S, Silvie O, Matuschewski K. 2009. Disruption of Plasmodium sporozoite transmission by depletion of sporozoite invasionassociated protein 1. Eukaryot. Cell 8:640 – 648. http://dx.doi.org/10.1128 /EC.00347-08. Siau A, Silvie O, Franetich J-F, Yalaoui S, Marinach C, Hannoun L, van Gemert G-J, Luty AJF, Bischoff E, David PH, Snounou G, Vaquero C, Froissard P, Mazier D. 2008. Temperature shift and host cell contact up-regulate sporozoite expression of Plasmodium falciparum genes involved in hepatocyte infection. PLoS Pathog. 4:e1000121. http://dx.doi .org/10.1371/journal.ppat.1000121. Fidock DA, Bottius E, Brahimi K, Moelans II, Aikawa M, Konings RN, Certa U, Olafsson P, Kaidoh T, Asavanich A. 1994. Cloning and characterization of a novel Plasmodium falciparum sporozoite surface anti-