Figure 1.
puf mRNA expression and immunolocalization in P. berghei RFP+ salivary gland sporozoites.
A, Relative and stage-specific transcription levels of puf1 and puf2 in wild type parasites. puf1 and puf2 steady state mRNA levels were analyzed by RT-PCR using cDNA from highly purified gametocytes and day 18 salivary gland (SG) sporozoites. Genomic DNA amplification is also provided, together with a map showing the localisation of primers used for amplifying puf1 and puf2 and the different sizes to be expected in cDNA and genomic DNA. See also Fig. S3C for Northern analysis of puf2 throughout parasite life cycle. B, Sporozoites tagged with red fluorescent protein (line 733cl1) were stained with anti-Puf2 peptide antibody 904 (top panel) and 905 (lower panel) revealing distinct cytoplasmic protein speckles. Scale bars = 10 µm.
Figure 2.
puf2- parasites transform into early liver stage EEF's in mosquito salivary glands.
A, maximum projection of a z-series scan of infected mosquito salivary glands on day 30 of mosquito infection. B, Development of salivary gland-resident parasites from day 18 to day 30 after mosquito infection; puf2- undergo exo-erythrocytic liver stage metamorphosis with bulging visible at day 22 and complete transformation at day 30. C, Proportion of sporozoites versus EEF-like's of wild type, puf1- and puf2- parasites in mosquito SG's during 13 days of salivary gland infection. Scale bars = 10 µm.
Figure 3.
Functionality and liver stage infectivity of wild type and mutant sporozoites.
A, 3×104 sporozoites were allowed to glide on cover slips pre-coated with anti-circumsporozoite protein (CSP) antibody for 40 minutes at 37°C. The number of parasites associated with CSP trails is a measure of gliding motility ability. B, Dextran tetramethylrhodamine was added to Huh7 cells before sporozoite addition. Two hours p.i. the number of traversed cells (Dextran+) was quantified by fluorescence-activated cell sorting. C, Huh7 cells fixed and double-stained with anti-CSP antibody to distinguish extracellular from intracellular sporozoites after a 2-hour incubation with 3×104 sporozoites. D, EEF development 48 h after addition of sporozoites to Huh7 cells. EEF areas were quantified with Image J of fluorescence microscopy images. For Figure 3A-D, n = 3. E, Plasmodium liver load 44 h after intravenous injection of 1×104 sporozoites; parasite load measured by qRT-PCR of P. berghei 18S rRNA normalized to hypoxanthine-guanine phosphoribosyltransferase. Five C57Bl/6 mice per group. F, Appearance of blood parasitaemia following infection of C57Bl/6 with 1×104 intravenously injected sporozoites (wild type n = 10 mice; puf1- n = 10; puf2- n = 25). G, Appearance of blood parasitaemia after mosquito bite (wild type n = 4 mice; puf1- n = 8; puf2- n = 10). All experiments used sporozoites 18 days after mosquito infection. T-test * p<0.05; ** p<0.01. All data show mean±SD.
Figure 4.
Transcriptional changes in puf2-.
A, Quantitative RT-PCR analysis was done on cDNA from wild type and puf2- salivary gland sporozoites (SGS) at day 18 after mosquito infection. B, Heatmap of expression changes measured by microarray analysis for >300 genes in wild type and puf2- at days 18 and 27 after mosquito infection. Expression values are scaled up to the rows and range from -3 (blue) to +3 (red). C, Correlation analysis of quantitative RT-PCR and microarray results.
Figure 5.
Ultrastructural evidence of puf2- parasites transforming into early liver stage EEFs in A. stephensi mosquito salivary glands.
Transmission electron microsocopy of 29-day salivary gland parasites showing the presence of slender-shaped, wild type and puf1- sporozoites with outer plasma membrane (PM) and entirely intact, inner membrane complex (IMC). puf2- salivary gland parasites developing into early EEFs show clear IMC disruption in the bulging region (arrows). Left pictures: longitudinal sections; right picture: transversal sections. Scale bars = 1 µm.
Figure 6.
Plasmodium sporozoite transformation into EEF-like form is protein translation dependent.
A, Wild type 18 day SGS were incubated at 37°C or room temperature (RT) for 2 h and 4 h and with or without the presence of cycloheximide (CHD, 100 µg/ml) as indicated. Transformation of WT SGS into EEF-like in vitro is more pronounced at 37°C for 4 h and relies on protein translation. T-test ** p<0.01. All data show mean±SD. B, WT and puf2- 18 day SGS were incubated at 37°C for 4 h with and without the presence of cycloheximide (CHD, 100 µg/ml) as indicated, showing that transformation of puf2- sporozoites does not rely on protein translation. C, Protein expression in puf2- and wild type SGS, 18 days after infection.
Figure 7.
Proposed model for Puf2-mediated regulation of Plasmodium developmental progression during transmission from the mosquito vector to the mammalian host.
In the mosquito Puf2 controls the translation of key protein(s). In its absence, this(ese) proteins are translated and sporozoites initiate transformation into the round EEF, which only occurs in the mammalian host in wild-type parasites.