A Role for Apical Membrane Antigen 1 during Invasion of Hepatocytes by Plasmodium falciparum Sporozoites
2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês
10.1074/jbc.m311331200
ISSN1083-351X
AutoresOlivier Silvie, Jean‐François Franetich, Stéphanie Charrin, Markus S. Mueller, Anthony Siau, Myriam Bodescot, Eric Rubinstein, L Hannoun, Yupin Charoenvit, Clemens H. M. Kocken, Alan W. Thomas, Geert‐Jan van Gemert, Robert W. Sauerwein, Michael J. Blackman, Robin F. Anders, Gerd Pluschke, Dominique Mazier,
Tópico(s)Mosquito-borne diseases and control
ResumoPlasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection. Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection. The apicomplexan parasite Plasmodium falciparum is a causative agent of malaria, one of the major human infectious diseases, responsible for more than 1 million deaths per year worldwide. Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade the liver of the mammalian host, where they undergo schizogony and differentiate into merozoites that subsequently invade erythrocytes and cause disease. Blocking sporozoite invasion of hepatocytes represents an attractive anti-malarial strategy because it would prevent malaria symptoms and parasite transmission to the mosquito, which both occur at erythrocytic stages. Sporozoites migrate through tissues and invade target cells using gliding motility (1Kappe S.H. Kaiser K. Matuschewski K. Trends Parasitol. 2003; 19: 135-143Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), a process involving secretory vesicles called micronemes. Upon microneme exocytosis, proteins are delivered onto the parasite surface and then redistributed from the anterior to the posterior end of the parasite, leading to its forward movement and penetration into a host cell (2Menard R. Cell Microbiol. 2001; 3: 63-73Crossref PubMed Scopus (101) Google Scholar). Only two sporozoite microneme proteins, namely circumsporozoite protein (CSP) 1The abbreviations used are: CSP, circumsporozoite protein; AMA-1, apical membrane antigen 1; DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PMSF, phenylmethylsulfonyl fluoride; TLCK, N-tosyl-L-lysine chloromethyl ketone; TRAP, thrombospondin-related adhesive protein; Z-GML-CH2Cl, N-benzyloxycarbonyl-Gly-Met-Leu-chloromethyl ketone.1The abbreviations used are: CSP, circumsporozoite protein; AMA-1, apical membrane antigen 1; DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PMSF, phenylmethylsulfonyl fluoride; TLCK, N-tosyl-L-lysine chloromethyl ketone; TRAP, thrombospondin-related adhesive protein; Z-GML-CH2Cl, N-benzyloxycarbonyl-Gly-Met-Leu-chloromethyl ketone. and thrombospondin-related adhesive protein (TRAP), have been extensively studied (3Menard R. Microbes Infect. 2000; 2: 633-642Crossref PubMed Scopus (55) Google Scholar). It is quite likely, however, that other microneme proteins are involved during the invasion process. Recently, Florens et al. (4Florens 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. Nature. 2002; 419: 520-526Crossref PubMed Scopus (1086) Google Scholar) reported a comprehensive view of the proteome of P. falciparum obtained with high resolution liquid chromatography and tandem mass spectrometry. Interestingly, some of the proteins detected in sporozoites had not been reported in this stage before, including apical membrane antigen 1 (AMA-1), a microneme protein involved in merozoite invasion of erythrocytes that is considered to be a leading candidate for inclusion in a vaccine against erythrocytic stages of P. falciparum (5Healer J. Crawford S. Ralph S. McFadden G. Cowman A.F. Infect. Immun. 2002; 70: 5751-5758Crossref PubMed Scopus (139) Google Scholar, 6Bannister L.H. Hopkins J.M. Dluzewski A.R. Margos G. Williams I.T. Blackman M.J. Kocken C.H. Thomas A.W. Mitchell G.H. J. Cell Sci. 2003; 116: 3825-3834Crossref PubMed Scopus (118) Google Scholar, 7Triglia T. Healer J. Caruana S.R. Hodder A.N. Anders R.F. Crabb B.S. Cowman A.F. Mol. Microbiol. 2000; 38: 706-718Crossref PubMed Scopus (254) Google Scholar). Here, we have analyzed AMA-1 expression in P. falciparum sporozoites and investigated its potential role during invasion of hepatocytes.EXPERIMENTAL PROCEDURESAntibodies and Inhibitors—We used previously characterized anti-P. falciparum AMA-1 (PfAMA-1) domain III monoclonal antibody (mAb) DV5 (8Mueller M.S. Renard A. Boato F. Vogel D. Naegeli M. Zurbriggen R. Robinson J.A. Pluschke G. Infect. Immun. 2003; 71: 4749-4758Crossref PubMed Scopus (83) Google Scholar), anti-PfAMA-1 prodomain mAb 5G8 (9Coley A.M. Campanale N.V. Casey J.L. Hodder A.N. Crewther P.E. Anders R.F. Tilley L.M. Foley M. Protein Eng. 2001; 14: 691-698Crossref PubMed Scopus (72) Google Scholar), anti-PfAMA-1 domain I mAb 1F9 (9Coley A.M. Campanale N.V. Casey J.L. Hodder A.N. Crewther P.E. Anders R.F. Tilley L.M. Foley M. Protein Eng. 2001; 14: 691-698Crossref PubMed Scopus (72) Google Scholar), anti-3D7 strain PfAMA-1 polyclonal rabbit IgG (10Hodder A.N. Crewther P.E. Anders R.F. Infect. Immun. 2001; 69: 3286-3294Crossref PubMed Scopus (274) Google Scholar), anti-FVO strain PfAMA-1 polyclonal rabbit IgG (11Kocken C.H. Withers-Martinez C. Dubbeld M.A. van der Wel A. Hackett F. Valderrama A. Blackman M.J. Thomas A.W. Infect. Immun. 2002; 70: 4471-4476Crossref PubMed Scopus (170) Google Scholar), anti-PfTRAP mAb SSP2.2 (12Charoenvit Y. Fallarme V. Rogers W.O. Sacci Jr., J.B. Kaur M. Aguiar J.C. Yuan L.F. Corradin G. Andersen E. Wizel B. Houghten R.A. Oloo A. De la Vega P. Hoffman S.L. Infect. Immun. 1997; 65: 3430-3437Crossref PubMed Google Scholar), and anti-PfCSP mAb E9 (13Stuber D. Bannwarth W. Pink J.R. Meloen R.H. Matile H. Eur. J. Immunol. 1990; 20: 819-824Crossref PubMed Scopus (50) Google Scholar). For liver schizont counting, we used anti-Plasmodium HSP-70 sera obtained from mice immunized with i72 recombinant protein (gift from D. Mattei, Institut Pasteur, Paris, France). Secondary antibodies were fluorescein isothiocyanate-conjugated goat anti-mouse IgG (GAM-FITC; Sigma), Alexa Fluor® 594 goat anti-rabbit IgG (GAR- Alexa Fluor® 594; Molecular Probes), peroxidase goat anti-mouse and peroxidase goat anti-rabbit IgG (Becton Dickinson). Cytochalasin D (500 μm stock in Me2SO) and protease inhibitors chymostatin (2 mg/ml stock in Me2SO), pepstatin A (1 mg/ml stock in Me2SO), leupeptin (1 mg/ml stock in water), phenylmethylsulfonyl fluoride (PMSF, 100 mm stock in ethanol), and N-tosyl-l-lysine chloromethyl ketone (TLCK, 20 mm stock in water) were from Sigma. The peptidyl chloromethylketone N-benzyloxycarbonyl-Gly-Met-Leu-CH2Cl (Z-GML-CH2Cl, 10 mm stock in Me2SO) was synthesized as described (14Howell S.A. Wells I. Fleck S.L. Kettleborough C. Collins C. Blackman M.J. J. Biol. Chem. 2003; 278: 23890-23898Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar).Isolation of P. falciparum Sporozoites—Anopheles stephensi adult females were infected with the NF54 strain of P. falciparum, using a membrane-based feeder system (15Ponnudurai T. Meuwissen J.H. Leeuwenberg A.D. Verhave J.P. Lensen A.H. Trans. R. Soc. Trop. Med. Hyg. 1982; 76: 242-250Abstract Full Text PDF PubMed Scopus (141) Google Scholar). After 14-21 days, mosquitoes were killed, and their salivary glands were aseptically dissected and disrupted by trituration in a glass tissue grinder, and the sporozoites were counted in a KovaSlide® chamber.Isolation and Culture of Human Hepatocytes—Primary human hepatocyte cultures were prepared as described (16Guguen-Guillouzo C. Campion J.P. Brissot P. Glaise D. Launois B. Bourel M. Guillouzo A. Cell Biol. Int. Rep. 1982; 6: 625-628Crossref PubMed Scopus (173) Google Scholar), with minor modifications. We used either fresh or cryopreserved hepatocytes, both susceptible to infection with P. falciparum sporozoites (17Meis J.F. Rijntjes P.J. Verhave J.P. Ponnudurai T. Hollingdale M.R. Yap S.H. Cell Biol. Int. Rep. 1985; 9: 976Crossref PubMed Scopus (13) Google Scholar, 18Mazier D. Beaudoin R.L. Mellouk S. Druilhe P. Texier B. Trosper J. Miltgen F. Landau I. Paul C. Brandicourt O. et al.Science. 1985; 227: 440-442Crossref PubMed Scopus (133) Google Scholar). In our hands, P. falciparum sporozoite infectivity is similar in fresh and cryopreserved hepatocytes. 2O. Silvie, unpublished observations. Briefly, the cells were isolated by collagenase (PAA Laboratories) perfusion of human liver fragments, used in agreement with the French ethical regulations, and further purified over a 40% Percoll gradient. The hepatocytes were cryopreserved in liquid nitrogen in fetal calf serum with 10% Me2SO; after thawing, viable hepatocytes were purified over a 40% Percoll gradient. The hepatocytes were seeded in eight-chamber plastic Lab-Tek slides or in 24-well culture plates (Nalge Nunc International) coated with rat tail collagen I (Becton Dickinson) at a density of 18 × 104 cells/cm2 (fresh hepatocytes) or 25 × 104 cells/cm2 (cryopreserved hepatocytes). The hepatocytes were cultured at 37 °C in 4% CO2, in William's E medium (Invitrogen) supplemented to a final concentration of 10% fetal calf serum (Invitrogen), 2% penicillin-streptomycin (100× stock solution; Invitrogen), 1% sodium pyruvate (100× stock solution; Invitrogen), 1% l-glutamine (100× stock solution; Invitrogen), 1% insulin-transferrin-selenium (100× stock solution; Invitrogen). After complete cell adherence (12-24 h), the culture medium was supplemented with 10-7m dexamethasone (Sigma) and 2% Me2SO to maintain hepatocyte differentiation (19Isom H.C. Secott T. Georgoff I. Woodworth C. Mummaw J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3252-3256Crossref PubMed Scopus (336) Google Scholar).Culture of P. falciparum Liver Stages—Human hepatocytes were cultured for at least 48 h before inoculation with P. falciparum sporozoites (18Mazier D. Beaudoin R.L. Mellouk S. Druilhe P. Texier B. Trosper J. Miltgen F. Landau I. Paul C. Brandicourt O. et al.Science. 1985; 227: 440-442Crossref PubMed Scopus (133) Google Scholar). After the removal of medium from the culture chambers, sporozoites in culture medium were added to the Lab-Tek wells (1 × 105 sporozoites/well) or plate wells (1 × 106 sporozoites/well) and incubated with the hepatocytes for 3 h. The cultures were then washed and further incubated in complete culture medium supplemented with 0.25% Me2SO. Culture medium was renewed every 2-3 days.Reverse Transcriptase-PCR—Total RNA was purified from P. falciparum NF54 sporozoites using the QIAamp RNA Blood Mini Kit (Qiagen), including a DNase treatment step. Reverse transcription was performed using the ProSTAR First-Strand reverse transcriptase-PCR kit (Stratagene). One μl of cDNA was PCR amplified with the sense primer 5′-TTCAAATACTACTTTTTTAACACCG-3′ and the antisense primer 5′-CTCTTTCGATTTCTTTCATTATTTC-3′, by use of 40 cycles of 45 s of denaturation at 94 °C, 45 s of annealing at 55 °C, and 60 s of elongation at 70 °C. The resulting products (890-base pair amplicon) were stained with ethidium bromide and run on a 1% agarose gel in Tris acetate-EDTA buffer.Immunofluorescence Assays—For detection of AMA-1 expression in P. falciparum sporozoites and liver stages, air-dried sporozoites and methanol-fixed infected hepatocyte cultures were blocked with 3% bovine serum albumin in phosphate-buffered saline for 30 min and then incubated with anti-PfAMA-1 mAb DV5 for 1 h at 37 °C, followed by incubation with GAM-FITC and 1 μg/ml DNA stain diamidino-phenylindole (DAPI) for 30 min before examination by fluorescence confocal microscopy. Surface staining was performed on nonpermeabilized sporozoites deposited on poly-l-lysine-coated multiwell glass slides, in a "wet" sporozoite assay (20Druilhe P. Pradier O. Marc J.P. Miltgen F. Mazier D. Parent G. Infect. Immun. 1986; 53: 393-397Crossref PubMed Google Scholar); sporozoites were fixed in 2% paraformaldehyde for 20 min at room temperature, and endogenous fluorescence was quenched with 0.1 m glycine. The parasites were blocked with 3% bovine serum albumin in phosphate-buffered saline for 1 h and then incubated with primary antibodies (anti-PfAMA-1 mAbs DV5, 1F9, or 5G8 or rabbit polyclonal anti-PfAMA-1 IgG, as indicated in the figure legends) for 2 h at 37 °C, followed by incubation with secondary antibodies (GAM-FITC for mAbs or GAR- Alexa Fluor® 594 for rabbit IgG) and DAPI for 1 h before examination by fluorescence microscopy.Western Blotting and Secretion Assays—For detection of parasite proteins in sporozoite lysates and supernatants, the parasites were dissolved in SDS-PAGE sample buffer and incubated at 70 °C for 5 min. The protein samples were subjected to 10% SDS-PAGE (1 × 105 parasites or equivalent/lane for AMA-1 and TRAP detection, 1 × 103 parasites or equivalent/lane for CSP detection) and transferred onto polyvinylidene membranes. These were probed with primary antibodies, followed by a peroxidase-conjugated secondary antibody, and antigens were revealed by enhanced chemiluminescence. To analyze proteins secreted in the parasite supernatant, sporozoites were resuspended in medium without serum and incubated for 2 h at 37 °C in the presence or absence of hepatocytes. The parasite suspension was then dissolved by adding SDS-PAGE sample buffer. In some experiments, the supernatants were separated from the sporozoites by centrifugation at 15,000 × g for 5 min, and the parasite pellets and supernatants were dissolved in SDS-PAGE sample buffer separately.Gliding Assays—To analyze sporozoite motility, 30,000 sporozoites were deposited on multispot glass slide wells precoated with anti-PfCSP mAb E9 (100 μg/ml 1 h at 37 °C) and incubated at 37 °C for 1 h. The slides were then washed, and the deposited CSP trails were fixed with 4% paraformaldehyde for 15 min. The trails were then labeled using the anti-PfCSP mAb E9 conjugated to Alexa Fluor® 488 and visualized under a fluorescence microscope.Inhibition of Sporozoite Invasion Assays—To analyze sporozoite invasion, triplicate hepatocyte cultures were inoculated with P. falciparum sporozoites (1 × 105/Lab-Tek well). After 3 h at 37 °C, the cultures were washed, further incubated in fresh medium for 3 days, and then fixed in methanol. Liver schizonts were stained using an anti-HSP-70 mouse serum followed by goat anti-mouse FITC conjugate and counted under a fluorescence microscope. To determine the effects of anti-AMA-1 antibodies on sporozoite infectivity, sporozoites were incubated with hepatocytes in the presence of increasing concentrations of anti-PfAMA-1 rabbit IgG. The percentage of inhibition was determined in comparison with control rabbit IgG. To determine the effects of protease inhibitors and cytochalasin D on sporozoite infectivity, P. falciparum sporozoites were incubated with these inhibitors (each from a 100× stock solution diluted directly in the parasite suspension) for 5 min at room temperature and then washed (except for cytochalasin D, the effects of which are reversible) before inoculation onto hepatocytes. The percentage of inhibition was determined in comparison with control sporozoites treated with solvents alone. Inhibition results were analyzed for statistical significance using the one-way analysis of variance followed by the Tukey multiple comparison test.RESULTSAMA-1 Is Expressed in P. falciparum Sporozoites—In a recent analysis of P. falciparum proteome, peptides corresponding to the merozoite antigen AMA-1 were identified in sporozoites. Because AMA-1 had never been reported in sporozoites before, we were interested in further characterizing its expression at this stage. Using reverse transcriptase-PCR, we could readily detect ama-1 transcripts in salivary gland sporozoites (Fig. 1A), thus confirming the expression of the gene at this stage. To test whether AMA-1 protein is also present in sporozoites, we performed immunofluorescence assays using the anti-PfAMA-1 domain III mAb DV5 (8Mueller M.S. Renard A. Boato F. Vogel D. Naegeli M. Zurbriggen R. Robinson J.A. Pluschke G. Infect. Immun. 2003; 71: 4749-4758Crossref PubMed Scopus (83) Google Scholar). All permeabilized sporozoites displayed a strong fluorescence (Fig. 1B), with a bipolar nucleus-sparing pattern similar to the immunofluorescence pattern observed with antibodies to TRAP, also known as sporozoite surface protein 2 (12Charoenvit Y. Fallarme V. Rogers W.O. Sacci Jr., J.B. Kaur M. Aguiar J.C. Yuan L.F. Corradin G. Andersen E. Wizel B. Houghten R.A. Oloo A. De la Vega P. Hoffman S.L. Infect. Immun. 1997; 65: 3430-3437Crossref PubMed Google Scholar). In contrast, surface labeling with the anti-AMA-1 mAb showed no or very little surface fluorescence on sporozoites (Fig. 1C). When detected, AMA-1 surface fluorescence was restricted to the apical extremity of the sporozoites, on the same side of the nucleus as the apicoplast, a DNA-containing organelle visualized as a DAPI-positive spot forward of the nucleus, as described in Toxoplasma (21Fichera M.E. Roos D.S. Nature. 1997; 390: 407-409Crossref PubMed Scopus (498) Google Scholar, 22Kohler S. Delwiche C.F. Denny P.W. Tilney L.G. Webster P. Wilson R.J. Palmer J.D. Roos D.S. Science. 1997; 275: 1485-1489Crossref PubMed Scopus (610) Google Scholar). A similar surface fluorescence pattern was observed using polyclonal anti-PfAMA-1 antibodies (see Fig. 5A). Western blotting of protein extracts from P. falciparum sporozoites probed with DV5 confirmed AMA-1 expression in sporozoites. Two proteins of ∼83 and ∼66 kDa were detected in both sporozoites and parasitized erythrocytes and were absent from noninfected mosquito salivary glands (Fig. 1D). In P. falciparum erythrocytic stages, these two bands have been shown to correspond respectively to a precursor protein that contains a prosequence and to a mature form of AMA-1 that lacks the prosequence (23Crewther P.E. Culvenor J.G. Silva A. Cooper J.A. Anders R.F. Exp. Parasitol. 1990; 70: 193-206Crossref PubMed Scopus (96) Google Scholar, 24Howell S.A. Withers-Martinez C. Kocken C.H. Thomas A.W. Blackman M.J. J. Biol. Chem. 2001; 276: 31311-31320Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). To confirm that the two bands observed in the sporozoite lysates also correspond to the precursor and mature forms, we performed Western blot analysis using the anti-AMA-1 mAbs 1F9, specific for P. falciparum AMA-1 domain I, and 5G8, specific for AMA-1 prosequence. The mAb 5G8 recognized only the upper ∼83-kDa band, whereas 1F9 recognized both the ∼83-kDa and ∼66-kDa proteins (Fig. 1E), thus confirming that in P. falciparum sporozoites these two bands correspond to the precursor and the mature form of AMA-1, respectively. The relative amount of precursor and mature proteins detected by Western blot varied between the different sporozoite preparations (Fig. 1 and see Fig. 4).Fig. 5Protease inhibitors and cytochalasin D do not prevent AMA-1 translocation to the sporozoite surface upon incubation at 37 °C. Immunofluorescence analysis of AMA-1 surface exposure using anti-AMA-1 polyclonal rabbit IgG (red) and DAPI (blue). A, untreated sporozoites kept at 4 °C after isolation from salivary glands. B, untreated sporozoites incubated at 37 °C for 1 h. C, sporozoites treated with 200 μm TLCK and incubated at 37 °C for 1 h. D, sporozoites treated with 100 μm Z-GML-CH2Cl and incubated at 37 °C for 1 h. E, sporozoites treated with 5 μm cytochalasin D and incubated at 37 °C for 1h. The arrow and the arrowhead indicate the sporozoite nucleus and the apicoplast, respectively. Bar, 5 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Upon incubation of sporozoites at 37 °C, P. falciparum AMA-1 and TRAP undergo proteolytic processing and shedding, which is sensitive to a subset of serine protease inhibitors but not to cytochalasin D.A, Western blotting of sporozoite lysates probed with anti-AMA-1 polyclonal rabbit IgG. The arrows indicate the 48 and 52 kDa AMA-1 cleavage products, respectively. B, Western blotting of sporozoite lysates probed with anti-TRAP mAb SSP2.2. The arrowhead indicates the TRAP cleavage product. C, Western blotting of pellet (P) and supernatant (SN) from sporozoites incubated at 37 °C, probed with anti-AMA-1 polyclonal rabbit IgG or with anti-TRAP mAb SSP2.2. The arrows and the arrowhead indicate the cleavage products of AMA-1 and TRAP, respectively. D, Western blotting of supernatants from sporozoites incubated at 37 °C, run under reducing (with dithiothreitol, DTT +) or non reducing (without dithiothreitol, DTT -) conditions and probed with anti-AMA-1 polyclonal rabbit IgG. E, Western blotting of pellet (P) and supernatant (SN) from sporozoites kept at 4 °C or incubated at 37 °C, in the absence (-hep) or presence (+hep) of hepatocytes, run under reducing conditions, and probed with anti-AMA-1 polyclonal rabbit IgG or with anti-TRAP mAb SSP2.2. The arrow and the arrowhead indicate the 48-kDa AMA-1 and the TRAP cleavage products, respectively. F, Western blotting of supernatants from sporozoites treated with protease inhibitors TLCK and Z-GML-CH2Cl (ZGML) before incubation at 37 °C. The samples were run under reducing conditions and probed with anti-AMA-1 polyclonal rabbit IgG, anti-TRAP mAb SSP2.2, or anti-CSP mAb E9. The arrows and the arrowhead indicate the cleavage products of AMA-1 and TRAP, respectively. G, Western blotting of supernatants from sporozoites treated with PMSF or cytochalasin D and incubated at 37 °C. The samples were run under reducing conditions and probed with anti-AMA-1 polyclonal rabbit IgG or anti-TRAP mAb SSP2.2. The arrows and the arrowhead indicate the cleavage products of AMA-1 and TRAP, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)AMA-1 Is Lost after Invasion of Hepatocytes and Is Re-expressed in Liver Merozoites—To assess the fate of AMA-1 after sporozoite invasion of hepatocytes, we analyzed AMA-1 expression in primary human hepatocytes infected with sporozoites (18Mazier D. Beaudoin R.L. Mellouk S. Druilhe P. Texier B. Trosper J. Miltgen F. Landau I. Paul C. Brandicourt O. et al.Science. 1985; 227: 440-442Crossref PubMed Scopus (133) Google Scholar). We consistently failed to detect the protein in early liver stages by immunofluorescence assay (Fig. 2), suggesting that it was lost after invasion of hepatocytes. AMA-1 was only re-expressed in mature P. falciparum liver schizonts, with a punctate pattern likely corresponding to nascent liver merozoites (Fig. 2).Fig. 2AMA-1 is lost after invasion of hepatocytes and re-expressed in liver merozoites.P. falciparum liver schizonts (arrowheads) stained with the anti-AMA-1 mAb DV5 (green) and DAPI (blue), 3, 8, or 10 days after infection of human hepatocyte cultures with sporozoites. N, hepatocyte nucleus. Bars, 5 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Anti-AMA-1 Antibodies Inhibit the Invasion of Human Hepatocytes by P. falciparum Sporozoites—The expression of AMA-1 in P. falciparum sporozoites and the fact that AMA-1 was not detected in liver schizonts until the differentiation of liver merozoites suggested that AMA-1 could be involved in the process of invasion of hepatocytes by sporozoites. Therefore, we tested whether antibodies to AMA-1 could inhibit the invasion of hepatocytes by P. falciparum sporozoites. Polyclonal IgG raised in rabbits immunized with a recombinant AMA-1 protein from the P. falciparum 3D7 clone (10Hodder A.N. Crewther P.E. Anders R.F. Infect. Immun. 2001; 69: 3286-3294Crossref PubMed Scopus (274) Google Scholar) inhibited invasion of human hepatocytes by NF54 (homologous strain) P. falciparum sporozoites in a concentration-dependent manner (Fig. 3). Relatively high concentrations were necessary to achieve significant inhibition, but equivalently high concentrations of IgG from rabbits immunized with a reduced and alkylated form of the recombinant 3D7 PfAMA-1 protein did not inhibit sporozoite invasion (Fig. 3), indicating that inhibition is mediated by antibodies directed to conformational epitopes. IgG from rabbits immunized with a recombinant AMA-1 from the P. falciparum heterologous strain FVO (11Kocken C.H. Withers-Martinez C. Dubbeld M.A. van der Wel A. Hackett F. Valderrama A. Blackman M.J. Thomas A.W. Infect. Immun. 2002; 70: 4471-4476Crossref PubMed Scopus (170) Google Scholar) also inhibited NF54 P. falciparum sporozoite invasion (Fig. 3).Fig. 3Anti-AMA-1 polyclonal antibodies inhibit P. falciparum sporozoite invasion of human hepatocytes in vitro. Human hepatocytes were inoculated with P. falciparum NF54 sporozoites in the presence of IgG from rabbits immunized with a 3D7 clone PfAMA-1 recombinant protein (gray bars), with a reduced and alkylated form of this 3D7 PfAMA-1 recombinant protein (white bar), or with a FVO strain PfAMA-1 recombinant protein (black bar). The results are expressed as the means of the percentage of inhibition of at least two independent experiments, each done in triplicate wells (± S.D.).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Upon Microneme Exocytosis, AMA-1 and TRAP Are Translocated to the Sporozoite Surface and Shed as Soluble Forms after Proteolytic Processing—Only a minority of salivary gland sporozoites (∼5-10%) displayed detectable amounts of AMA-1 on their apical surface (Fig. 1C and see Fig. 5A), suggesting that most of the protein is sequestered within the sporozoite micronemes, as observed with TRAP (25Gantt S. Persson C. Rose K. Birkett A.J. Abagyan R. Nussenzweig V. Infect. Immun. 2000; 68: 3667-3673Crossref PubMed Scopus (87) Google Scholar). Upon incubation at 37 °C, which induces microneme exocytosis (26Bhanot P. Frevert U. Nussenzweig V. Persson C. Mol. Biochem. Parasitol. 2003; 126: 263-273Crossref PubMed Scopus (26) Google Scholar, 27Carruthers V.B. Giddings O.K. Sibley L.D. Cell Microbiol. 1999; 1: 225-235Crossref PubMed Scopus (278) Google Scholar), most of the sporozoites (∼60-80%) expressed AMA-1 on their surface, distributed either over the whole surface or as a posterior cap (see Fig. 5B). Sporozoite surface AMA-1 reacted with 1F9 but not with 5G8 mAb, indicating that only the mature 66-kDa protein translocates to the parasite surface (see Fig. 6, C and D). As expected, both 5G8 and 1F9 mAbs labeled air-dried permeabilized sporozoites (see Fig. 6, A and B) with a fluorescent pattern similar to that observed with DV5. In P. falciparum merozoites, AMA-1 translocates to the surface upon microneme exocytosis and is proteolytically cleaved and shed as soluble fragments (14Howell S.A. Wells I. Fleck S.L. Kettleborough C. Collins C. Blackman M.J. J. Biol. Chem. 2003; 278: 23890-23898Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 24Howell S.A. Withers-Martinez C. Kocken C.H. Thomas A.W. Blackman M.J. J. Biol. Chem. 2001; 276: 31311-31320Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). To determine whether similar processing events also occur in P. falciparum sporozoites, we analyzed by Western blot lysates from sporozoites incubated at 37 °C. Indeed, an AMA-1 cleavage product of ∼48 kDa, associated with a less abundant product of ∼52 kDa, was found in lysates from sporozoites incubated at 37 °C but not in lysates from sporozoites kept at 4 °C upon isolati
Referência(s)