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. 2003 Apr 21;197(8):1045-50.
doi: 10.1084/jem.20022100.

Transformation of sporozoites into early exoerythrocytic malaria parasites does not require host cells

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Transformation of sporozoites into early exoerythrocytic malaria parasites does not require host cells

Karine Kaiser et al. J Exp Med. .

Abstract

Malaria parasite species that infect mammals, including humans, must first take up residence in hepatic host cells as exoerythrocytic forms (EEF) before initiating infection of red blood cells that leads to malaria disease. Despite the importance of hepatic stages for immunity against malaria, little is known about their biology and antigenic composition. Here, we show that sporozoites, the parasites' transmission stage that resides in the mosquito vector salivary glands, can transform into early EEF without intracellular residence in host hepatocytes. The morphological sequence of transformation and the expression of proteins in the EEF appear indistinguishable from parasites that develop within host cells. Transformation depends on temperature elevation to 37 degrees C and serum. Our findings demonstrate that residence in a host hepatocyte or specific host cell-derived factors are not necessary to bring about the profound morphological and biochemical changes of the parasite that occur after its transmission from vector to mammalian host.

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Figures

Figure 1.
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 cells transform through the classic sequence of morphological changes into EE trophozoites. (A) After 4 h of culture, many parasites exhibit the transformation bulb (arrows), which is the first morphological indication of transformation. (B) After 10 h of culture, parasites have progressed further in transformation, as indicated by enlargement of the transformation bulb and concurrent retraction of the armlike sporozoite remnants (arrows). The insets show transforming parasites at higher magnification. After 24 h, (C–E) spherical trophozoites have developed that are indistinguishable from (F–H) trophozoites that developed within host cells. Micrographs A–D, F, and G are GFP fluorescence images; E and H are differential interference contrast images (DIC) showing the same trophozoites as in D and G, respectively. Bars, 10 µm.
Figure 2.
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) Spherical 18-h EE trophozoite showing intense HSP70 expression. (C) Large 48-h EEF showing compartmentalization of HSP70 staining. Micrographs on the right show the corresponding DIC images. (D) Western blot analysis of HSP70 expression. Sporozoites (Spz) express HSP70 at barely detectable levels. Host cell-free EEF, cultured for 24 h, show a dramatic increase in HSP70 expression. (E) Spherical 24-h EE trophozoite showing surface expression of circumsporozoite (CS) protein. (F) Spherical 24-h EE trophozoites do not express TRAP. Nuclei in E and F are visualized with DAPI. (G) EEF grown for 24 h without host cells express transcripts encoding MSP-1 (left) and HEP17 (right). RT-PCR with gene-specific oligonucleotide primers amplified specific fragments of 120 base pairs (bp). Controls are PCR reactions without prior RT. Bars, 5 µm.
Figure 3.
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 indicating little IMC loss. The trophozoite has a single elongate nucleus. (B) An extracellular EEF showing partial IMC loss. Two closely associated nuclei are visible showing that this parasite has undergone a first round of nuclear division. (C) An extracellular EEF showing further IMC loss to ∼30% of its original size. Two separated nuclei are visible. (D) An EEF developing within a HepG2 cell 24 h after infection. Loss of the IMC occurs similar to the loss observed in host cell-free development. Nuclei in A–D are visualized with DAPI. Micrographs on the right show the corresponding DIC images. (E) A culture of EE forms expressing GFP grown at 22°C for 24 h. Initiation of transformation has occurred as indicated by the transformation bulbs (arrows). The EEF are arrested at this stage and do not develop into spherical trophozoites. Bars, 10 µm. (F) rRNA type switch in EEF grown for 24 h without host cells. RT-PCR with type-specific oligonucleotide primers detects expression of A-type rRNA in cultures grown at 22°C and 37°C. Expression of S-type rRNA is not detectable in 37°C cultures but still detectable in 22°C cultures. Controls are PCR reactions without prior RT for 37°C/A-type and 22°C/S-type.

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