Metamorphosis of the malaria parasite in the liver is associated with organelle clearance

1. The formation of a protrusion in the middle of the parasite marks the beginning of sporozoite differentiation into trophozoite

From about 4-h p.i. onwards, Plasmodium sporozoites undergo spectacular phenotypic changes in liver cells. Microscopic examination was performed to scrutinize the sequential developmental transformations of Plasmodium berghei sporozoites into trophozoites in vitro. During the remodeling processes, the ‘C’- or ‘S’-shaped sporozoite became progressively round (Fig. 1A). The initiation of metamorphosis is marked by the appearance of a protruding area in the middle of the parasite, originating on either the outer convex or concave edge. Sporozoites maintained in axenic conditions for ~9-h also developed into early liver forms and were morphologically similar to their intracellular counterparts (Fig. 1B) [12], suggesting that triggers for sporozoite differentiation are not host cell-dependent. The same basic steps of sporozoite remodeling were observed for Plasmodium yoelii (Fig. S1). In order to decipher the 3-D ultrastructural features of converting sporozoites, we capitalized on the fact that sporozoites can convert extracellularly to perform scanning electron microscopy (EM) imaging on axenic parasites. These studies revealed that the outer surface of the median bulbous area appeared wrinkled (Fig. 1B), presumably as a result of local indentations of the plasma membrane. The diameter of the bulge was about 1.5 and 3 μm at 4-h and 8-h, respectively. As the bulbous region of the parasite expanded, the two distal ends of the sporozoite gradually retracted and eventually disappeared (see arrows in Fig. 1B). This results in the smoothening of the surface and complete sphericalization, without any change in cell surface.

2. Nucleus protrusion occurs as a result of a loss in the membrane cytoskeleton rigidity

To determine the fate of the membrane cytoskeleton during sporozoite conversion, we performed immunofluorescence assays using antibodies against the myosin A tail domain interacting protein (MTIP) [21] associated with various proteins anchored to the IMC [3, 22]. MTIP is part of the invasion machinery of the parasite. The presence of parasite Hsp70, which is highly expressed in liver stage parasites compared to sporozoites [23], was used as a marker for viability and stage conversion in our culture system. While the IMC was continuous along the length of the sporozoite, at 7-h p.i., we consistently observed the disruption of the MTIP-labeled membranes at the central protruding area corresponding to the position of the nucleus (Fig. 2A-B) as previously reported [8, 12, 21]. As the sporozoite rounds up, the IMC was found to retract along with the parasite extremities, and after complete transformation, MTIP staining appeared restricted to a small area at the parasite periphery.

The precise morphological changes occurring during sporozoite conversion into trophozoite were then inspected by EM on axenically cultured P. berghei and P. yoelii. In contrast to salivary gland sporozoites in which a tight connection is maintained between the plasma membrane and the IMC (labeled with anti-MTIP antibodies; Fig. 3A), the continuity of the IMC was significantly impaired in converting parasites. Clearly points of IMC disruption were visible along the length of this organelle (Fig. 3B, panels a-b) as evidenced by a lack of gold particles (against MTIP antibodies) under the plasma membrane (Fig. 3, panel c). This detachment of the IMC from the parasite surface resulted in local cytoplasm infiltration, inducing plasma membrane distention. The gradual dismantling of the IMC on both sides of the nucleus then provoked the protrusion of this organelle (Fig. 3C). The loss of the IMC and microtubules from the nuclear area contributed to the wrinkled aspect of the surface membrane as observed by scanning EM (Fig. 1B and inset in Fig. 3C, panel a). The intracellular dissociation of the IMC from the plasma membrane was concomitant to organelle redistribution in converting parasites (Fig. S2). The vast majority of secretory organelles were engulfed in the portion of the cytoplasm delineated by the retracting IMC (Fig. 3C, panel b) while the nucleus, mitochondria and endoplasmic reticulum (ER) largely remained in the cytoplasmic area lacking the IMC. Some sections illustrated the dislocation of the anterior ring to the middle of the parasite (Fig. S3).

Following its dissociation from the plasma membrane at multiple foci (as seen in the serial sections depicted in Fig. S4A), the IMC is progressively compacted in the cytoplasm (Fig. S4B). Interestingly, membranous structures forming regular whorls with up to 10 layers were frequently observed in the cytoplasm next to the points of IMC breakdown (Figs. 3C, panel b, 4A and S4C). Immunogold staining of parasites with anti-MTIP-antibodies confirmed that the membranous whorls within the parasite were derived from the IMC (Fig. 4A, panel b). Similar packaged membranes consisting of membranous sheaths surrounded by the plasma membrane were also observed externally, either budding from the parasite or appended to the surface (Figs. 4B-C and S4D) as confirmed with MTIP staining (panels b and c in Figs. 4B and 4C). Free MTIP-labeled membranes were also detected between parasites (Fig. 4B, panel d), suggesting that residual IMC is eventually expelled by the parasite. Therefore, it is tempting to propose that the reduction of mechanical constraints due to the removal of the IMC together with the loss of polarity of the parasite may lead to the round shape of the trophozoite form.

3. Sporozoite development progresses more rapidly within a PV than axenically or extracellularly between cells

We then examined the peculiarities of P. yoelii sporozoite differentiation in intracellular conditions. After invasion, P. yoelii was most commonly found to lie in a narrow PV (Fig. 5A), preferentially located in the host perinuclear area [15] (Fig. 1A). Within 1-h p.i., the PVM showed close association with host rough ER (Fig. 5B). Host ribosomes were restricted to the face of the ER not associated with the PV membrane. The identity of intracellular parasites at early time points p.i. was confirmed by their elongated crescent shape and the presence of i) the IMC beneath the plasma membrane, ii) subpellicular microtubules and iii) secretory organelles. Major changes in these three structures and composition were visible in parasites 4-h p.i. As observed for axenic parasites, the area of the parasite containing the nucleus was enlarged and round (Fig. 5C) and in some regions, the IMC was detached, leaving the plasma membrane bare. Secretory organelles were encased in the extremities still delineated by the IMC. By 12-h p.i., the nuclear area of the parasites enlarged further, concomitant with a reduction in length of the distal ends. These 12-h forms were characterized by the presence of several membrane whorls in the cytoplasm (Figs 5E-F and S4) and the accumulation of osmiophilic material in the PV (Figs. 5F-G and S5), e.g. packaged membranes likely derived from the IMC. By comparison, the 12-h forms that developed intracellularly appeared larger and rounder than those maintained in axenic conditions. Similarly, parasites that did not invade cells but instead developed extracellularly, between hepatic cells for 12-h underwent early metamorphosis but exhibited a delayed conversion as they displayed morphological features closer to those of intracellular 4-h forms (data not shown). These results underline the substantial contribution of host cell factors to optimal sporozoite conversion.

4. At the completion of metamorphosis, the parasites only retain organelles involved in biosyntheses

During the process of conversion, the PV membrane also underwent dynamic transformations to adapt to the shape changes of the sporozoite (Fig. 5H). Astonishingly, host ER-PV interaction was constantly maintained over the course of metamorphosis, indicative of strong association between the vacuolar membrane and the host ER network (Figs 5 and 6).

By 24-h p.i., the parasites increased in volume by 4 to 5-times compared to sporozoites and intrahepatic 12-h forms, which both have comparable cell volumes (~35 μm³). The 24-forms still retained some portion of the IMC (Fig. 6A-D). 3-D visualization of MTIP-labeled membranes at this stage using spinning disk confocal microscopy illustrated the persistence of residual IMC, as a retracting network within the parasite, confined in part to the parasite periphery (Fig. 6E; see movie S1). By contrast, the anterior ring and secretory organelles involved in invasion were absent in all the sections viewed. The cytoplasm, which had a high electron-density in sporozoites due to abundant ribosomes, now appeared less electron-dense with scattered ribosomes. The nucleus became enlarged and lobed, which is indicative of mitotic events occurring within the intact nuclear envelope (Fig. 6A). At this stage of development, the 24-h forms - or trophozoites - showed no resemblance to sporozoites. The nucleoplasm exhibited the same texture as the cytoplasm (Fig. 6A-D). The general appearance of the ER and mitochondrion was also noticeably different in trophozoites (Fig. 6F-G) compared to sporozoites (Fig. 6H). The ER expanded markedly and reorganized into several local dense networks with shorter sheets and dilated cisternae, resembling mitotic ER in mammalian cells [24]. The single mitochondrion of the sporozoite developed into a major network with numerous small branches, suggesting major restructuring of this organelle as visualized by staining with mito-Tracker (Fig. 6I). The parasite also retained the apicoplast, a relict plastid that contains essential anabolic pathways [25], as shown by immunofluorescence using antibodies against the apicoplast resident acyl-carrier binding protein (ACP). Like the mitochondrion, the apicoplast develops from a simple structure to a highly branched continuous network during schizogony [26].

It has been previously observed that sporozoites occasionally penetrate into the host nucleus but these intranuclear forms display a limited degree of maturation [27]. We also detected P. yoelii in the host nucleoplasm. These few intranuclear parasites were never surrounded by any membrane (Fig. S6). Within 24-h, only a small number of these parasites completed metamorphosis unlike their intravacuolar counterparts. Additionally, the intranuclear parasites never underwent endomitoses. A decrease in parasite viability was similarly observed for sporozoites cultivated under axenic conditions for 24-h as no nuclear divisions were observed in extracellular trophozoites but instead parasites became progressively vacuolized (data not shown). This indicates that the PV represents an environment that is favorable for optimal sporozoite conversion and essential for schizogony.

5. Micronemes are compartmentalized in the cytoplasm prior to their discharge in the parasitophorous vacuole

Sporozoite micronemes are small, oval structures with a diameter of ~50 nm. These organelles are abundantly distributed throughout the cytoplasm of sporozoites as shown by the diffuse pattern observed by fluorescence microscopy on parasites labeled with anti-TRAP antibodies (Fig. 7) [28, 29]. ImmunoEM staining of sporozoites with the same antibodies confirmed the abundance of micronemes dispersed in the cytoplasm (see Fig. 9A). We monitored the localization of TRAP by fluorescence microscopy to follow the kinetics of microneme removal over the time. Surprisingly, during the process of parasite transformation, large and well-defined punctate structures containing TRAP were visible. The diameter of the TRAP-containing organelles varied from 0.2 to 0.4 μm, and likely reflected either an aggregation of micronemes in the cytoplasm or their compartmentalization within a membrane-bound structure. Eight- and 24-h p.i., the TRAP-containing organelles were distributed randomly in the cytoplasm (Fig. 7). By 31-h p.i., these compartments became noticeably smaller and were more abundantly concentrated at the periphery of the cytoplasm (Fig. 8A). On late schizonts, the fluorescence staining was predominantly at the outer surface of the parasite, likely in the vacuolar space, suggesting a discharge of TRAP into the PV or the expulsion of intact TRAP-containing organelles by the parasite. Double immunostaining of parasites 36-h p.i. with anti-TRAP and anti-CSP antibodies confirmed that TRAP staining was outside of the parasite plasma membrane. The majority of the 55-h forms did not show any TRAP labeling in their PV, implying mechanisms of organelle degradation operational in the vacuolar space.

To further characterize these large TRAP-positive structures, we scrutinized the parasites at the ulltrastructural level. EM studies on parasites 8-h post-conversion revealed the presence of ~0.25 μm organelles delineated by a single membrane and containing intraluminal vesicles having the same size and electron-density as sporozoite micronemes (Fig. 9B, compared with Fig. 8A panel a). ImmunoEM labeling demonstrated the presence of TRAP on vesicles sequestered within single membrane-bound compartments (Fig. 9B, panel b), which confirms that these structures are indeed micronemes. Interestingly, some microneme-containing compartments were docked onto the plasma membrane at sites devoid of IMC. This suggests the occurrence of fusion events permitting the exocytosis of micronemes into the PV (see serial sections a-a’ in Fig. 9B).

Reagents and antibodies

All chemicals were obtained from Sigma Chem. Co. (St. Louis, MO) unless indicated otherwise. Mito-Tracker Green FM and ER-Tracker Red (Bodipy-TR glibenclamide) were obtained from Molecular Probes (Eugene, OR). Antibodies used for immunofluorescence assays included: rabbit anti-MTIP (diluted at 1:500) from Dr L. Bergman (Drexel University), rabbit anti-TRAP (diluted at 1:300) from Dr U. Frevert (New York University), mouse anti-Hsp70 (diluted at 1:400), mouse anti-CSP (diluted at 1:1000) from Dr. F. Zavala (Johns Hopkins University), rabbit anti-ACP (diluted at 1:100) from Dr. S. Prigge (Johns Hopkins University) and rabbit anti-UIS4 (diluted at 1:250) from S. Kappe (Seattle Biomedical Research Institute).

Mammalian cell lines and parasite strains

The commercial mammalian cell lines used in this study are the mouse Hepa1-6 cells (ATCC CRL-1830) and the human HepG2-A16 cells (ATCC HB 8065). The HepG2 cell line stably transfected with CD81 (HepG2-CD81) was kindly provided by Dr. D. Mazier (Université Pierre et Marie Curie, France) [27]. All cell lines were grown as monolayers at 37°C in an atmosphere of 5% CO2 in α-minimum essential medium (Gibco BRL, Gaithersburg, MD), supplemented with 10% (v/v) of fetal bovine serum (FBS), 2 mM L-glutamine and penicillin/streptomycin (100 units/ml per 100 μg/ml). The P. berghei ANKA line expressing GFP [57] was grown in Anopheles stephensi mosquitoes blood-fed on infected Swiss CD-1/ICR mice as described [12]. P. yoelii strain 17 XNL was similarly grown in A. stephensi mosquitoes blood-fed on infected Swiss Webster mice.

Plasmodium cultivation and infection in vitro

P. berghei sporozoites were isolated from disrupted salivary glands of infected A. stephensi mosquitoes and further purified as described [12] before incubation in the presence of different mammalian cells for various times. Alternatively, sets of experiments were carried out on sporozoites maintained as axenic cultures for various times at 37°C in DMEM containing 4.5 g/liter glucose and supplemented with L-glutamine to a final concentration of 2 mM, FBS at a final concentration of 10%, penicillin-streptomycin at 500 U/ml and 500 μg/ml respectively, and Amphotericin B at 25μg/ml.

Light microscopy

Immunofluorescence assays on intracellular parasites were performed as previously described [15]. Infected cells were incubated with the organelle-Trackers according to the manufacturer’s instructions. Images were acquired on a Nikon Eclipse E800 microscope equipped with a Spot RT CCD Camera and processed using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) and Adobe Photoshop software (Adobe Systems Inc.). To visualize the IMC in 3-D, intracellular P. berghei were labeled with anti-MTIP antibodies and fluorescence pattern was analyzed by a spinning disk confocal microscope (Nikon TE2000E) using a Plan-Apochromat 63x/1.4 NA, and processed using the Volocity software (Improvison, Waltham, MA).

Electron microscopy

For thin-section transmission EM, pellets of P. berghei sporozoites, axenic parasites (P. berghei or P. yoelii) or P. yoelii-infected HepG2-CD81 were fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences; EMS, Hatfield, PA) in 0.1 M sodium cacodylate buffer (pH 7.4) for 1-h at room temperature, and processed as described [58] before examination with a Philips CM120 Electron Microscope (Eindhoven, the Netherlands) under 80-kV. For scanning EM imaging, preparations of axenic P. yoelii were washed in PBS, allowed to adhere on to poly(L-lysine)-coated glass coverslips and subsequently fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2). After fixation, samples were rinsed in 0.1 M cacodylate buffer for 30 min at 4°C. They were then post-fixed for 1-h in 2% osmium tetroxide at 4°C. After a brief rinse in distilled water, samples were en-bloc stained in 2% aqueous uranyl acetate for 1-h at room temperature in the dark. Following a graded ethanol dehydration, cells were critical point dried with liquid CO₂, mounted onto SEM stubs with double stick carbon tape, and sputter coated with 10 nm gold palladium. Samples were viewed and photographed with a LEO FESEM 1530 operating at 1 kV. For immunoelectron microscopy, axenic P. yoelii were fixed in 4% paraformaldehyde (EMS) in 0.25 M HEPES (ph7.4) for 1 h at room temperature, then in 8% paraformaldehyde in the same buffer overnight at 4°C. They were infiltrated, frozen and sectioned as previously described [58]. The sections were immunolabeled with anti-TRAP antibodies (1:50) or anti-MTIP antibodies (1:100), then with mouse anti-rabbit IgG antibodies, followed directly by 10 nm protein A-gold particles (Department of Cell Biology, Medical School, Utrecht University, the Netherlands) .