Fig 1.
Deletion of the 3’ UTR of ama1 has no phenotypical impact in P. berghei.
A-B. Strategy to generate ama1Δutr (A) and ama1Con (B) parasites by modification of the wild type ama1 locus in PbDiCre parasites. C-D. Blood stage growth of untreated and rapamycin-treated ama1Δutr (C) or ama1Con (D) parasites. Rapamycin was administered at day 2. The graphs represent the parasitaemia (mean +/- SEM) in groups of 3 mice. E. Immunofluorescence staining of rapamycin-treated ama1Con and ama1Δutr blood stage schizonts with anti-AMA1 antibodies (blue). The right panels show mCherry (red), GFP (green) and AMA1 (blue) merged images. Scale bar = 10 μm. F. Immunofluorescence images of rapamycin-treated ama1Con and ama1Δutr sporozoites after staining with anti-AMA1 antibodies (magenta). The right panels show Hoechst (blue) and AMA1 (magenta) merged images. Scale bar = 5 μm.
Fig 2.
AMA1 is required during P. berghei invasion of mosquito salivary glands.
A. Strategy to generate ama1cKO parasites by modification of the ama1 locus in rapamycin-treated ama1Con parasites. B. Blood stage growth of rapamycin-treated and untreated ama1cKO parasites. The graph represents the parasitaemia (mean +/- SEM) in groups of 3 mice. Rapamycin was administered at day 2. **, p < 0.01; ****, p < 0.0001 (Two-way ANOVA). C-E. Quantification of midgut sporozoites (MG-SPZ, C), salivary gland sporozoites (SG-SPZ, D) or haemolymph sporozoites (HL-SPZ, E) isolated from mosquitoes infected with untreated or rapamycin-treated ama1Con and ama1cKO parasites. The graphs show the number of sporozoites per female mosquito (mean +/- SEM). Each dot represents the mean value obtained in independent experiments after dissection of 30–50 mosquitoes (MG, HL) or 50–70 mosquitoes (SG), respectively. Ns, non-significant; ****, p < 0.0001 (One-way ANOVA followed by Tukey’s multiple comparisons test). F-H. Quantification of excised (mCherry+/GFP-, red) and non-excised (mCherry+/GFP+, green) midgut sporozoites (MG-SPZ, F), salivary gland sporozoites (SG-SPZ, G) or haemolymph sporozoites (HL-SPZ, H) isolated from mosquitoes infected with untreated or rapamycin-treated ama1Con and ama1cKO parasites. I. Immunofluorescence imaging of untreated and rapamycin-treated ama1cKO salivary gland sporozoites after staining with anti-AMA1 antibodies (magenta). The right panels show Hoechst (blue) and AMA1 (magenta) merged images. Scale bar = 5 μm. J. Quantification of AMA1-positive and AMA1-negative sporozoites among untreated or rapamycin-exposed ama1Con, ama1Δutr and ama1cKO sporozoites, as assessed by microscopy.
Fig 3.
Sporozoite AMA1 is required for efficient infection of mammalian cells.
A. Quantification of sporozoite cell traversal activity (% of dextran-positive cells) in rapamycin-treated ama1Con and ama1cKO parasites. The values for rapamycin-treated ama1cKO parasites are represented as percentage of the rapamycin-treated ama1Con parasites (mean +/- SEM of three independent experiments). Each data point is the mean of five technical replicates. Ns, non-significant (Two-tailed ratio paired t test). B. Quantification of EEFs development in vitro, done by flow cytometry or microscopy analysis of HepG2 cells infected with sporozoites isolated from either untreated or rapamycin-treated ama1Con and ama1cKO infected mosquitoes. The data for rapamycin-treated ama1Con and ama1cKO parasites are represented as percentage of the respective untreated parasites (mean +/- SEM). Each data point is the mean of three technical replicates in one experiment. Ns, non-significant; *, p < 0.05 (Two-tailed ratio paired t test). C. Quantification of excised (mCherry+/GFP-, red) and non-excised (mCherry+/GFP+, green) EEF populations for untreated and treated ama1Con and ama1cKO parasites. D. Fluorescence microscopy of EEF development (24h p.i.) in vitro, in HepG2 cells infected with salivary gland sporozoites from untreated or rapamycin-treated ama1Con and ama1cKO parasites. The right panels show Hoechst (blue), mCherry (red) and GFP (green) merged images. Scale bar = 10 μm. E. Immunofluorescence imaging of mCherry+/GFP- (excised) rapamycin-treated ama1Con and ama1cKO EEFs after staining with anti-UIS4 antibodies (green). The right panels show Hoechst (blue), mCherry (red) and UIS4 (green) merged images. Scale bar = 10 μm.
Fig 4.
RON2 and RON4 are required for sporozoite invasion in the mosquito and mammalian hosts.
A. Strategy to generate ron2cKO and ron4cKO parasites in the PbDiCre line. B-C. Blood stage growth of rapamycin-treated and untreated ron2cKO (B) and ron4cKO (C) parasites. The graph represents the parasitaemia (mean +/- SEM) in groups of 5 mice. Rapamycin was administered at day 1. **, p < 0.01; ****, p < 0.0001 (Two-way ANOVA). D-F. Quantification of midgut sporozoites (MG-SPZ, D), haemolymph sporozoites (HL-SPZ, E) or salivary gland sporozoites (SG-SPZ, F) isolated from mosquitoes infected with untreated or rapamycin treated ron2cKO or ron4cKO parasites. The graphs show the number of sporozoites per infected female mosquito (mean +/- SEM). Each dot represents the mean value obtained in independent experiments after dissection of 30–50 mosquitoes (MG, HL) or 50–70 mosquitoes (SG), respectively. Ns, non-significant; *, p < 0.05; **, p < 0.01 (Two-tailed ratio paired t test). G-I. Quantification of excised (mCherry+/GFP-, red) and non-excised (mCherry+/GFP+, green) midgut sporozoites (MG-SPZ, G), haemolymph sporozoites (HL-SPZ, H) or salivary gland sporozoites (SG-SPZ, I) isolated from mosquitoes infected with untreated or rapamycin-treated ron2cKO and ron4cKO parasites. J. Quantification of EEFs development in vitro, done by microscopy analysis of HepG2 cells infected with sporozoites isolated from either untreated or rapamycin-treated ron2cKO and ron4cKO infected mosquitoes. The data for rapamycin-treated parasites are represented as percentage of the respective untreated parasites (mean +/- SEM). Each data point is the mean of five technical replicates in one experiment. Ns, non-significant; *, p < 0.05 (Two-tailed ratio paired t test). K. Quantification of sporozoite cell traversal activity (% of dextran-positive cells) in untreated and rapamycin-treated ron2cKO and ron4cKO parasites. The data for rapamycin-treated parasites are represented as percentage of the respective untreated parasites (mean +/- SEM). Each data point is the mean of five technical replicates from one experiment.
Fig 5.
Capturing sporozoite entry into salivary glands with serial block face-scanning electron microscopy (SBF-SEM).
A-F. SBF-SEM images showing an untreated ama1cKO sporozoite (noted as wt) penetrating into a mosquito salivary gland cell. Panels A and B show the same parasite in two different sections. In A, the sporozoite is cut twice (black arrows), with one part located outside the cell, underneath the basal lamina (BL, white arrow), and the other one inside the cell, within a vacuole surrounded by a membrane (white arrowhead). In B, a tight vacuole can be seen surrounding the intracellular portion of the invading sporozoite (arrowhead), as well as a full rhoptry (white arrow). The volume segmentation in C shows full rhoptries (blue) and empty vesicles (green) in the apical portion of the parasite. In D, the extracellular and intracellular parts of the sporozoite are colored in purple and pink, respectively, while the cell appears in yellow. The volume image in E shows the host cell surface (yellow), revealing a deep imprint of the extracellular parasite segment (black arrow) and the circular aperture at the point of entry (black arrowhead). In F, the entry site is shown at higher magnification. An overview of the segmentation process corresponding to panels A-F is shown in S4 Movie. Segmentation of the rhoptries is shown in S5 Movie. G-K. SBF-SEM images showing a rapamycin-treated ron2cKO sporozoite penetrating into a mosquito salivary gland cell. In G, the sporozoite is caught in the process of entry through an elevated host cell structure (arrow) associated with a tight constriction of the parasite body. The intracellular portion of the parasite is surrounded by a vacuole (white arrowhead). A volume segmentation of the sporozoite is shown in H, superimposed on the same section as in G. In the volume representations in I and J, the extracellular and intracellular parts of the sporozoite are colored in purple and pink, respectively, while the cell appears in yellow. The entry site is marked with an arrowhead, and shown at higher magnification in K. An overview of the segmentation process corresponding to panels G-K is shown in S6 Movie. Scale bars, 2 μm.
Fig 6.
Invasion by AMA1- and RON2-deficient sporozoites is associated with a loss of integrity of the mosquito salivary gland epithelium.
A-B. SBF-SEM sections of salivary glands infected with WT (A) or rapamycin-treated ama1cKO parasites (B), day 21 post-infection. The ama1cKO-infected gland shows signs of cellular damage (black arrows) despite low parasite density. A single intracellular sporozoite is indicated by a white arrow. Scale bars, 10 μm. C-E. SBF-SEM sections of salivary glands infected with rapamycin-treated ama1cKO parasites, day 15 post-infection. Disruption of the basal lamina is indicated by an arrow. In D, a large vacuole is visible around an intracellular sporozoite and is indicated by an asterisk. In E, both the basal lamina and the cell plasma membrane are ruptured (arrow), resulting in a large cellular vacuole that communicates with the outside (asterisk). Scale bars, 2 μm. F. SBF-SEM sections of salivary glands infected with rapamycin-treated ron2cKO parasites, day 15 post-infection. A large vacuole surrounding an intracellular sporozoite is indicated by an arrow. Scale bar, 2 μm. G. Fluorescence microscopy images of salivary glands infected with untreated (UT) or rapamycin-treated (+Rapa) ama1cKO or ron2cKO parasites, day 16 post-infection. Samples were stained with Hoechst 77742 (Blue). The panels show mCherry (red), GFP (green) and Hoechst (blue) and transmitted light merge images. Zones of retraction of the acinar epithelial cells are visible in the lobes infected with AMA1- and RON2-deficient sporozoites (arrows). Scale bars, 50 μm. H. Quantification of salivary gland lobes showing retracted epithelium after infection with untreated or rapamycin-treated ama1cKO and ron2cKO parasites. The data shown are from two independent experiments (Fisher’s exact test, P = 0.0286 for ama1cKO and P <0.0001 for ron2cKO).
Fig 7.
Model of AMA1-RON function in Plasmodium sporozoites.
AMA1 and RON proteins drive two distinct sporozoite invasion events in the mosquito and mammalian hosts. After egress from oocysts, sporozoites first rely on AMA1 and RONs to enter the mosquito salivary glands inside a transient vacuole, without causing epithelium damage, to eventually accumulate in the secretory cavities after crossing the acinar cells. Then, following parasite transmission to a mammalian host, AMA1 and RONs are required for efficient productive invasion of hepatocytes inside a parasitophorous vacuole. Both events supposedly involve rhoptry secretion and the formation of a junction, which however is uncoupled from the formation of a canonical parasitophorous vacuole during colonization of the insect salivary glands.