Plasmodium falciparum: Histidine-Rich Protein II Is Expressed during Gametocyte Development

Experimental Parasitology 96, 139–146 (2000) doi:10.1006/expr.2000.4557, available online at http://www.idealibrary.com on

Plasmodium falciparum: Histidine-Rich Protein II Is Expressed during Gametocyte Development

Rhian E. Hayward,*,1 David J. Sullivan,† and Karen P. Day*,2 *Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS. United Kingdom; and †Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205, U.S.A.

Hayward, R. E., Sullivan D. J., and Day, K. P. 2000. Plasmodium falciparum: Histidine-rich protein II is expressed during gametocyte development. Experimental Parasitology 96, 139–146. Both early gametocytes (I–II) and asexual trophozoite stages of Plasmodium falciparum digest hemoglobin and detoxify haem by polymerizing it into parasite pigment called hemozoin. The mechanism of polymerization is unclear but it has been proposed that histidine-rich protein II may facilitate transport of hemoglobin to the food vacuole and catalyze the polymerization in asexual stages. We describe the transcription of histidine-rich protein II in gametocytes by Northern blot analysis and the expression of the protein in these stages by immunoprecipitation and Western blotting. Localization of histidine-rich protein II within the gametocyte by immunofluorescence assay and immunoelectron microscopy clearly illustrated the presence of this molecule in the infected red cell cytosol in the early stages of gametocyte development and internalization in the later gametocyte as it matures. There is a strong correlation between the stage-specific trafficking of histidinerich protein II in gametocytes and the susceptibility of early but not late gametocytes to the antimalarial drug chloroquine. 䉷 2000 Academic Press Index Descriptors and Abbreviations: HRP, histidine-rich protein; Plasmodium falciparum; gametocytes; RBC, red blood cell; mAb, monoclonal antibody; IFA, indirect immunofluorescence assay; UTR, untranslated regions; TEM, transmission electron microscopy.

10–14 days (Garnham 1966; Ponnudurai et al. 1982). This period of growth has been divided into stages I–V (Hawking et al. 1971) based on the extensive morphological changes that occur as the parasite develops and finally matures into an infectious gametocyte which is transmitted to the mosquito vector. During the early stages of gametocyte development (stages I–IIA) a number of structural and biochemical features are common to gametocytes and asexual parasites (trophozoites). Both life cycle stages have knob structures at the RBC surface and express histidine-rich protein I (HRPI; Day et al. 1998), which is essential for knob formation (Crabb et al. 1997). In addition, early gametocytes as well as trophozoites express P. falciparum erythrocyte membrane protein 1 (PfEMP1), which mediates adhesion of both parasite stages to CD36 in vitro (Day et al. 1998). Identical var gene repertoires encode the PfEMP1 molecules that are expressed by early gametocytes and trophozoites of the same parasite isolate (Hayward et al. 1999) and a cross-stage, variant-specific humoral immunity can be detected as a result (Piper et al. 1999). Both developing gametocytes and trophozoites metabolize host hemoglobin and detoxify the resulting molecules of heme by polymerizing them into crystalline hemozoin (Scheibel and Sherman 1988). As they grow, gametocytes digest 70–80% of the host cell hemoglobin. Despite this fundamental similarity, the distribution of hemozoin or “parasite pigment” in each parasite stage is dramatically different. Gametocyte hemozoin is contained within multiple food vacuoles, giving a granular appearance of pigment which is characteristic of this life cycle stage (Sinden 1982). In the trophozoite, hemozoin collects in a single digestive food vacuole. The mechanism of hemozoin formation remains

INTRODUCTION Gametocytes of the malaria parasite Plasmodium falciparum develop within the human red blood cell (RBC) for 1 Present address: Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda MD 20892, U.S.A. 2 To whom reprint requests should be addressed. Fax: 44-1865281245. E-mail: [email protected].

0014-4894/00 $35.00 Copyright 䉷 2000 by Academic Press All rights of reproduction in any form reserved.

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140 unclear but this process occurs actively for some time during the gametocyte life span as parasite catabolism continues and nutrients are needed. Discontinuation of heme polymerization may be inferred from cell cycle experiments. Sinden and Smalley (1979) show that gametocyte RNA and protein synthesis persist until stage III of development (6 days post RBC invasion). This is closely followed by cessation of all but subsistence levels of protein synthesis between stages III and IV of growth. It is likely that hemoglobin digestion also halts at this time, in which case heme polymerization may also stop, if it is mediated by a parasite protein (Pandey and Tekwani 1996). Once gametocytes develop beyond stage III, and hemoglobin digestion/heme polymerization has stopped, they are coincidentally no longer affected by chloroquine. This is in contrast to early gametocytes and asexual stages, which are susceptible to the action of this drug (Smalley 1977). Histidine-rich protein II (HRPII; Howard et al. 1986) can promote the in vitro polymerization of monomeric heme into crystals of hemozoin in the trophozoite, and chloroquine inhibits this reaction (Sullivan et al. 1996a). HRP II is secreted into the infected RBC cytosol, trafficked back to the digestive food vacuole of the trophozoite with hemoglobin (Sullivan et al. 1996a), and also secreted out of the host erythrocyte, where it may also have a function (Howard et al. 1986). Based on the biochemical similarities between early gametocytes and trophozoites we hypothesized that gametocytes also express and utilize HRPII in hemoglobin digestion.

MATERIALS AND METHODS

Parasites. Parasites from the cloned line 3D7 (Walliker et al. 1987) and isolate 1776 (Cox et al. 1994) were maintained in in vitro culture as previously described (Trager and Jensen 1976). Trophozoites were enriched by Plasmagel flotation (Pasvol et al. 1978). Stage I–IIA gametocytes were purified as described by Hayward et al. (1999). Stage IIB–V gametocytes were purified from mixed cultures (containing asexual parasites) by 3 consecutive days of sorbitol treatment and discontinuous gradients of Percoll, as previously described (Day et al. 1998). The purity of gametocyte samples was confirmed by IFA with an antibody, IIC5-B10, raised against the gametocyte-specific protein Pfs48/45 (Rener et al. 1983). Northern blotting. Total RNA was extracted with Trizol reagent (Life Technologies). RNA (10 ␮g) from each parasite stage was electrophoresed in 1% agarose–formaldehyde gels and transferred to nylon membrane (Hybond N+, Amersham) by capillary blotting overnight (Sambrook et al. 1989). The HRPII DNA probe was generated by Nco1 restriction digestion of the gene (minus the secretory leader sequence; 834 bp) from an expression plasmid. The HRPI probe was

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generated by polymerase chain reaction with gene-specific primers which amplified a 500-bp fragment of the gene. Probes were labeled with [32P]dCTP by random priming (Feinberg and Vogelstein 1983, 1984). Filters were incubated with probe overnight at 42⬚C, washed at the same temperature in 0.1 ts SSC and 0.5% SDS for 30 min and then at 55⬚C for 30 min, and exposed to autoradiographic film (BioMax MR, Kodak) overnight. Western blotting and immunoprecipitation. Parasites (5 ⫻ 106 cells) were solubilized in 3 ⫻ SDS–sample buffer, fractionated by SDS–PAGE (Laemmli 1970), and transferred electrophoretically to nitrocellulose membrane (Towbin et al. 1979). Incubation and detection of bound antibody were carried out as previously described (Day et al. 1998). Metabolic labeling of developing gametocytes with 100 ␮Ci/ ml L-[3H] histidine and immunoprecipitation of extracted proteins was carried out as described by Howard and Barnwell (1984) and Howard et al. (1986). Gels of 5–15% acrylamide were used to fractionate labeled proteins and then soaked in Enhance (Amersham) and exposed to Hyperfilm (Amersham) after drying. Immunolocalization. Indirect immunofluorescence assays (IFA) were based on a previous protocol (Biggs et al. 1990) modified to include methanol fixation of air-dried, thin blood films for 45 min at ⫺20⬚C followed by incubation of the slides with human tonicity PBS/ 10% normal human serum for 45 min. Labeling with anti-HRPII mAb 2G12 and a fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody (Sigma) was viewed on an Olympus BX50 fluorescence microscope. Immunogold electron microscopy was carried out following the protocol of Aikawa and Atkinson (1990) using mAb 2G12 and 5nm gold-conjugated goat anti-mouse IgG secondary antibody (Sigma).

RESULTS

Transcription of HRPII in gametocytes. Northern blot analysis of total RNA from cloned parasite line 3D7 was performed using the HRPII gene (without the secretory signal peptide) as a probe. The probe hybridized to a 2.1-kb HRPII mRNA transcript from gametocytes (stages IIB–V), as well as asexual parasites (Fig. 1). As a control, additional Northern blot analysis of the same RNA samples was performed using a 500-bp fragment of the HRPI gene. This probe hybridized exclusively to asexual RNA samples, confirming that the gametocyte RNA preparation was free of asexual parasites, as detectable by this technique. Interestingly, the lack of HRPI transcript in stage IIB–V gametocytes suggests that only the early stages of gametocyte development (stage I–IIA) actively transcribe the HRPI gene (Day et al. 1998). HRPII expression. Western blot analysis using antiHRPII mAb 2G12 (Rock et al. 1987) was performed to determine whether HRPII was expressed by gametocytes. This technique demonstrated that mAb 2G12 recognizes a 68-kDa/66-kDa protein doublet characteristic of HRPII in

HISTIDINE-RICH PROTEIN II IN GAMETOCYTES

FIG. 1. Northern blot analysis of HRPII mRNA from 3D7 gametocytes. RNA (10 ␮g) from trophozoites and stage IIB–V gametocytes was electrophoresed in 1% agarose–formaldehyde gels, transferred to nylon membrane, and hybridized with a [32P]dCTP-labeled HRPII DNA probe (full-length minus the secretory signal peptide) excised from an expression plasmid. Blots were washed and exposed to autoradiographic film overnight. The blots were stripped and reprobed with a [32P]dCTP-labeled full-length HRPI probe generated by PCR.

all stages of gametocytes purified from sorbitol-treated cultures as well as trophozoite and schizont stage protein extracts (Fig. 2A). A higher molecular weight band (75 kDa) was also consistently reactive with the mAb in every infected RBC sample. Although this protein band is not the molecular weight diagnostic of other histidine-rich proteins that have been described, such as HRPIII (Wellems and Howard 1986) and HRPIV (Sullivan et al. 1996a), it is reasonable to suggest that it may represent an additional histidine-rich protein from a family of molecules related by their high histidine content, which is cross-reactive with mAb 2G12 under these experimental conditions. Synthesis of HRPII by gametocytes was confirmed by metabolic labeling experiments (Fig. 2B). [3H]histidine labeling was carried out on young gametocyte cultures (day 9 of culture without RBC replenishment), which were sorbitol treated for 3 days prior to labeling. Labeled gametocyte proteins were extracted with Triton X-100 and subsequently immunoprecipitated with anti-HRPII mAb 1D6 (Rock et al., 1987). A single labeled protein of 61 kDa was reactive with this mAb. This protein is of similar mass and solubility to

141 HRPII (Rock et al. 1987) and was also observed in radiolabeled trophozoite protein extracts. Localization of HRPII in gametocytes. HRPII expression was localized by IFA in all stages of gametocyte from 3D7 and field isolate 1776 (Fig. 3). As controls, trophozoiteinfected RBC were intensely labeled by mAb 2G12 near or at the infected cell surface membrane and discrete reactivity was observed within the permeabilized parasite (Fig. 3A). Sullivan et al. (1996a) have previously shown similar staining with mAb 2G12 to be the result of HRPII labeling in trophozoite food vacuoles. In contrast, gametocytes were labeled with a diffuse, granular pattern of staining (Figs. 3B and 3C). This labeling was strong in early gametocytes (stage I) and was clearly present throughout the host cell cytoplasm to the surface membrane. However, in the “D-shaped” IIB gametocytes, fluorescence was restricted to the parasite within the RBC. Similarly, in more mature gametocytes (stages III–V), reactivity appeared to be localized to the parasite alone. Antibody reactivity with a subcellular structure, such as a food vacuole, could not be confirmed using this technique. An isotype-matched, unrelated antibody did not react with trophozoites or gametocytes (data not shown) and uninfected RBC were not recognized by mAb 2G12. There was a small amount of labeling in zygote stages but we found no evidence (by IFA) for expression of HRPII protein in the parasite stages that develop in the mosquito midgut, i.e., ookinetes and oocysts (data not shown). We examined the ultrastructural localization of HRPII in gametocytes by immunogold electron microscopy. The localization of bound mAb 2G12 and thus HRPII in late gametocytes (stage IIB–V) was identified by the pattern of colloidal gold particles, after labeling of multiple thin sections for transmission electron microscopy (TEM), and a typical labeling is shown in Fig. 4A. By TEM, gametocytes were identified by a corset of microtubules, a three-membrane pellicular complex, Laveran’s bib, and multiple food vacuoles (Sinden 1982). Gold particles were found throughout the cytoplasm of all gametocytes as the IFA experiments predicted. Particles were associated with individual food vacuoles as well as the trilaminate membrane and occasional “packets” of gold particles, indicative of HRPII secretion (Howard et al. 1986), were observed within the infected RBC cytosol and in close proximity to the host cell membrane, as seen in trophozoite-infected RBC. There appeared to be no bias in the labeling of any organelles. In the absence of primary antibody, very few gold particles were associated with infected RBCs (Fig. 4B) and an isotype-matched control antibody did not react with infected RBCs (data not shown).

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FIG. 2. (A) Identification of HRPII from gametocytes by Western blotting. Trophozoites (T), schizonts (S), early gametocytes (stage I–IIA; EG), and late gametocytes (stage IIB–V; LG) were purified. Uninfected erythrocytes (RBC) were included as a control for antibody binding. Membranes were incubated with mAb 2G12 and peroxidase-conjugated anti-mouse IgG secondary antibody and reactivity was visualized using ECL chemiluminescence reagents (Amersham). (B) Immunoprecipitation of metabolically labeled HRPII from gametocytes; trophozoites (T), gametocytes (all stages; G), and uninfected RBC (RBC). Extracted proteins were precipitated with mAb 1D6 or an unrelated IgG control antibody. Samples were electrophoresed under reducing conditions and the dried gels exposed to autoradiographic film.

DISCUSSION P. falciparum gametocytes transcribe and translate HRPII. This raises the interesting question of the role of this protein in the sexual stage parasite. As proposed for the trophozoite (Sullivan et al. 1996a), HRPII may be involved in heme polymerization in gametocytes. Localization studies of HRPII in early gametocytes (stage I–IIA) indicate that the

molecule is trafficked to the red cell cytoplasm. Given this observation, HRPII may also be secreted by early gametocytes, as it is by trophozoites, to perform a similar function in both life cycle stages. Gametocytes have multiple food vacuoles. HRPII is localized in these compartments but also to the parasite cytoplasm in general, suggesting that the function of HRPII in gametocytes may not correlate strictly with biochemical events in

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FIG. 3. Indirect immunofluorescence assay of HRPII expression in 1776 gametocytes using mAb 2G12; (A) trophozoites (B and C) gametocytes. Bar, 10 ␮m.

the food vacuole. Further studies, such as the purification of early gametocyte food vacuoles, are needed to fully characterize HRPII compartmentalization. When the gametocyte matures, the distribution of HRPII to the host cell cytoplasm is limited and may be affected by the formation of the trilaminate membrane in stage IIA gametocytes and certainly a general reduction in protein synthesis and trafficking. A reduction in HRPII trafficking correlates with cessation of hemoglobin breakdown and subsequent heme processing as gametocytes mature and we can speculate that HRPII in the infected RBC of late gametocytes is residual protein from synthesis, translocation, and function in earlier stages. HRPII is expressed in a stage-specific manner similar to HRPI during gametocyte development. In early gametocytes (and trophozoites) HRPII and HRPI have markedly different patterns of localization, reflecting different functions. In the late gametocyte, HRPII and HRPI are restricted to the parasite body and patterns of labeling with antibodies in IFA are indistinguishable, presumably reflecting a loss of trafficking of proteins that are no longer required. HRPI localization to the gametocyte-infected RBC surface is lost after stage IIB of development, correlating with the disappearance of knobs from the erythrocyte surface (Day et al., 1998). In turn, stage-specific HRPII localization correlates with early gametocyte sensitivity to chloroquine and late gametocyte resistance to this drug (Smalley 1977). In addition, consistent

with the role of HRPII in hemoglobin processing in early but not late gametocytes, this molecule does not appear to be expressed in the mosquito stage parasites. HRPII shares 20% amino acid homology with HRPIII (Wellems and Howard 1986) and the 5⬘ UTR of HRPII is 85–90% identical to the 5⬘ UTR of HRPIII at the nucleotide level (Sullivan et al. 1996b). Despite these extensive homologies, a recent study of gene regulation during P. falciparum sexual development (Dechering et al. 1999) demonstrates that the 5⬘ UTR of HRPIII does not drive expression of the CAT reporter protein included in a transfection construct after the first week of gametocyte development. These data predict that the HRPIII protein will not be present in late gametocytes and that the promoter may not be appropriate for studying expression of transfected genes in the late sexual stages of the parasite life cycle. In contrast, the presence of HRPII transcripts and protein throughout gametocyte development suggests that the 5⬘ UTR of HRPII would drive expression of a selectable marker throughout asexual, early and late gametocyte development. The use of this 5⬘ UTR may allow investigation of the proposed switch to an alternate program of gene expression which may occur at commitment to sexual differentiation (Alano and Carter 1990) up to the point of gamete formation. To this end, expression of HRPII may aid our understanding of the poorly defined process of gametocytogenesis.

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FIG. 4. (A) Immunogold electron microscopy for ultrastructural localization of HRPII in the gametocyte. Gametocytes were identified by a corset of microtubules (M) and a three-membrane pellicular complex (P). Arrowheads mark gold particles. (B) Gametocyte labeling with secondary gold-conjugated antibody only. Bar, 500 nm.

ACKNOWLEDGMENTS The authors thank Professor Diane Taylor of Georgetown University for providing anti-HRPII mAbs 2G12 and 1D6, Mr. Mike Delannoy

for expert assistance in electron microscopy, which was performed at The Johns Hopkins University School of Hygiene and Public Health EM facility, and Drs. Karl Hoffmann and Thomas Templeton for critical review of the manuscript.

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FIG. 4—Continued

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