To test if sporozoites can transform in the absence of host cells, they were cultured without HepG2 cells in microchamber slides coated with basement membrane extract (Matrigel) and DMEM containing 10% FBS for 4, 10, and 24 h at 37°C. Microscopic examination showed that after 4 h, sporozoites frequently developed the transformation bulb ( A) that was also typically observed during early intracellular transformation (2
). At 10 h, the extracellular transformation had proceeded further to intermediate EE-like forms displaying a more extended bulb and further retraction of the armlike remnants of the sporozoite cell body ( B), closely resembling the progression of transformation observed during intracellular transformation (3
). At 24 h, ~13% of sporozoites had transformed into completely spherical EE-like forms (, and ). This rate of transformation exceeds the published rates of transformation achieved in HepG2 cells, which range from 3 to 8% (6
) and the rates of transformation in HepG2 cells observed by us (~5%, unpublished data). EE-like forms that were cultured without HepG2 cells were similar in size (6–10 µm) and morphology when compared with EEF that grew within HepG2 cells (). No sporozoites were detectable in 24-host cell–free cultures, indicating that untransformed sporozoites did not survive.
Figure 1. Transformation of Plasmodium sporozoites into EEF does not require host cells. P. berghei sporozoites and EEF express green fluorescent protein enabling the visualization of live parasites. (A–E) Sporozoites cultured at 37°C without HepG2 (more ...)
Host Cell–free Transformation into Hepatic Stages
We tested if expression of EEF proteins is comparable between EEF that undergo intracellular development and EE-like forms that develop without host cells, using antibodies against HSP70, CSP, TRAP, and MTIP. Although HSP70 of Plasmodium
is highly expressed in EEF and erythrocytic stages of the parasite, it is barely detectable in sporozoites (10
). Therefore, HSP70 expression can be used to follow the transformation of sporozoites into hepatic stages. After 6 h, early extracellular EE-like forms identified by the transformation bulb showed increasing HSP70 expression ( A). After 18–24 h, completely spherical EE-like forms had developed that showing intense HSP70 staining ( B). When cultures were incubated for 48 h, most of these early EE-like forms seemed not to develop further, but a few EE-like forms showed significant increase in size and compartmentalization of HSP70 staining ( C). This might correspond to the cytoplasmic compartmentalization observed in intracellular EEF at this time point (13
). HSP70 staining patterns closely resembled the staining patterns of intracellular EEF developing inside HepG2 cells. Up-regulation of HSP70 expression was confirmed by immunoblot analysis ( D). HSP70 expression was barely detectable in protein extracts of 800,000 salivary gland sporozoites. However, expression increased dramatically when the same number of parasites where cultured in the host cell–free system for 24 h (13% transformation rate). HSP70 expression might be induced by temperature elevation, the transformation event itself, or both. However, because parasites kept at 37°C for 24 h either transformed or died, it was not possible to investigate this further.
Figure 2. EEF cultured without host cells express key antigens. (A–C) Different stages of EEF development show increased expression of HSP70. (A) Early 6-h EEF showing the typical transformation bulb. HSP70 expression is mostly localized to the bulb. (B) (more ...)
CSP, the major sporozoite surface protein (14
), is involved in sporozoite host cell recognition. It is also expressed on the plasma membrane of early EEF (15
) and might fulfill additional functions during hepatic stage development. Robust CSP surface expression was detected on EE-like forms cultured in the absence of host cells for 24 h using a monoclonal antibody specific for CSP ( E; reference 17
). In contrast, TRAP, a sporozoite micronemal protein involved in host cell invasion (18
), was not detectable on spherical EE-like forms at this time ( F). The loss of TRAP expression coincides with the disappearance of micronemes after sporozoite transformation in vivo (3
). We also determined that P. berghei
EE-like forms cultured in the absence of host cells for 24 h expressed transcripts ( G) encoding MSP-1 (19
), and the hepatic and erythrocytic stage protein 17 (HEP17; reference 20
Apicomplexan zoites, including the Plasmodium
sporozoite, are delimited by a tri-laminar pellicle consisting of a plasma membrane and two closely aligned inner membranes that form the inner membrane complex (IMC). After hepatocyte invasion, the sporozoite plasma membrane becomes the EEF plasma membrane; however, the IMC is disassembled and is not detectable at ~30 h after invasion (3
). Dismantling the IMC might be essential for entry of the parasite into the trophic phase because its rigidity could interfere with growth. MTIP localizes to the IMC of sporozoites (21
) and can serve as a marker to follow the loss of the IMC during hepatic stage development. Dual fluorescence assays with antibodies against CSP and MTIP showed that the IMC was lost in EE-like forms grown without host cells () . IMC loss was sometimes simultaneous with a first round of nuclear division but these events did not occur in a synchronized fashion (). The IMC loss in host cell–free EEF closely resembled its loss in intracellular EEF ( D). Lactacystin (10 µM), a highly specific inhibitor of proteasome activity, inhibited host cell–free transformation by ~70% (unpublished data), indicating that EE-like transformation was a proteasome-dependent process. This was consistent with its previously observed inhibitory effect on sporozoite transformation into intracellular EEF (22
). Together, the data show that, in the absence of intact host cells or host cell–derived components, sporozoites transform into EEF that undergo nuclear division and express proteins similar to early EEF that develop within host cells.
Figure 3. (A–D) The IMC is lost during intracellular and host cell-free development of transformed EEF. EEF (24-h culture) stained with antibodies against CS and MTIP, an IMC marker. (A) An extracellular EEF that shows complete circumferential MTIP staining (more ...)
The host cell–free culture system provides a unique opportunity to study what environmental factors directly govern transformation. The stimulus for transformation might be a shift in temperature experienced by the parasite during transmission. To test this, we incubated sporozoites at either 22°C (temperature of mosquito vector) or 37°C (temperature of mammalian host) for 24 h. In contrast to sporozoites cultured at 37°C, sporozoites cultured at 22°C initiated transformation (revealed by the occurrence of transformation bulbs), but rarely developed into completely spherical EEF ( E and ). Therefore, a shift from low to high temperature is not necessary for initiation of the transformation process but high temperature is required for complete transformation of sporozoites into EEF. Interestingly, parasites kept at 22°C for 24 h could still develop into spherical EEF when the temperature was subsequently shifted to 37°C for 24 h (), and this occurred with higher efficiency of transformation (~19%) than in cultures directly incubated at 37°C. Thus, parasites kept at low temperature remained viable, experiencing a reversible arrest of transformation. In addition, we determined that the presence of serum was also critical for transformation (). Without serum, few parasites transformed into spherical EEF and most parasites were not detectable in 37°C cultures after 24 h.
Switching occurs between the expression of different types of ribosomal RNA (rRNA) at transition points in the Plasmodium
life cycle (23
). One such transition is the transformation of sporozoites into EEF when rRNA expression switches from S-type in sporozoites (S for sporozoite) to A-type in EEF and succeeding blood stages (A for Asexual; reference 24
). We determined rRNA expression in the host cell-free transformation system at 22°C and 37°C after 24 h of culture by reverse transcriptase-PCR using oligonucleotide primers specific for either A-type or S-type. Expression of A-type rRNA was detected in host cell-free EEF cultures at both temperatures ( F). Thus, expression of A-type rRNA was not dependent on temperature elevation, however, it seemed to increase with the elevated temperature. S-type rRNA was detectable at 22°C but was not detectable at 37°C, confirming the rRNA switch ( F). This indicated that either the elevated temperature repressed expression or that the S-type is under control of a cold-stimulated promoter. Thermoregulation of rRNA gene expression was described recently to also occur in the parasites' blood stages (25
A central tenet of Plasmodium
transmission is that the invasive sporozoite must penetrate a host hepatocyte and take up intracellular residence to initiate development into EEF. However, we have shown that in principle, sporozoites do not require an intact host cell or any specific host cell–derived factors to transform into EEF. Our results suggest that in vivo, sporozoites might initiate the transformation program before invasion as soon as they enter the mammalian bloodstream. After sporozoites enter the bloodstream, they rapidly sequester in the liver and invade hepatocytes within minutes (26
). This rapid homing ensures that transformation occurs only after invasion of a suitable host cell. Although sporozoites can transform into EEF without host cells, intracellular residence is likely to be essential for the parasites' trophic phase when host cell–derived factors are needed for further growth. Plasmodium
merozoites can transform into trophozoites and develop outside a red blood cell (27
) only in the presence of red cell extract, indicating that merozoite transformation and subsequent parasite development was dependent on some yet undefined host cell factors.
More than 50 yr after their discovery (1
), the biology and antigenic repertoire of EEF remains largely unstudied, mainly due to the small number that can be obtained in vivo and in vitro and the technical challenge of separating the intracellular EEF from uninfected hepatocytes and surrounding host hepatocytes. Sterile, protective immunity against malaria in humans and animal models has only been achieved by immunization with radiation-attenuated sporozoites (5
). Irradiated sporozoites invade hepatocytes and transform into early EEF, but remain arrested at this stage (30
). Thus, it is the antigens expressed in early EEF that confer the sterile, protective immunity (32
). Therefore, identification of these antigens must be regarded as the most important goal toward development of a preerythrocytic malaria vaccine (5
), and this might be facilitated by a host cell–free transformation system.