Fig 1.
Generation of P. berghei clampcKO parasites.
A. Strategy to modify the clamp locus in the PbDiCre line. A first transfection in PbDiCre parasites with the P1 construct led to the insertion of a GFP-hDHFR expression cassette flanked by two LoxN site upstream of clamp gene. Rapamycin-induced excision of the cassette caused retention of one single LoxN site upstream of clamp in the treated clamp-P1 parasites. In a second step, rapamycin-treated excised clamp-P1 parasites were transfected with the P2 construct to generate a clampcKO parasite line expressing CLAMP fused to a 3xFlag epitope tag in addition to GFP and hDHFR, and carrying two LoxN sites flanking clamp gene. Rapamycin-induced activation of DiCre leads to excision of the clamp gene together with the GFP-hDHFR cassette. B. Flow cytometry analysis of blood-stage parasites at different steps after initial transfection of the PbDiCre parental line to generate clampcKO parasites. NI, non-infected red blood cells. C. Blood stage growth of rapamycin-treated and untreated clampcKO parasites. Rapamycin was administered at day 1. The graph shows the parasitaemia (mean +/- SEM) in groups of 5 mice, as quantified by flow cytometry based on mCherry detection. **, p < 0.01; ****, p < 0.0001 (Two-way ANOVA). The experiment shown in panel C was only performed once.
Fig 2.
Deletion of clamp impacts invasion of mosquito salivary glands by sporozoites.
A-B. Fluorescence microscopy imaging of midguts (A) and salivary glands (B) from rapamycin-exposed and untreated clampcKO-infected mosquitoes, dissected at day 16 or 21 post-feeding, respectively. Exposure time and contrast were adjusted at the same level for each channel in both conditions. Scale bar, 200 μm. C-E. Comparison of sporozoite numbers collected from midguts (C), salivary glands (D) and haemolymph (E) of female mosquitoes infected with rapamycin-exposed and untreated clampcKO parasites. All results shown in C-E are mean +/- SEM of at least four independent experiments. Ns, non-significant; *, p < 0.05; ****, p<0.0001 (Two-tailed ratio paired t test).
Fig 3.
Conditional gene deletion abrogates CLAMP protein expression in sporozoites.
A. Immunofluorescence analysis of rapamycin-exposed and untreated clampcKO sporozoites labelled with anti-Flag antibodies (magenta). Both rapamycin-exposed and untreated clampcKO parasites express mCherry (red), while only untreated clampcKO parasites express GFP (green). Nuclei were stained with Hoechst 77742 (blue). Scale bar, 5 μm. B. Western bot analysis of haemolymph sporozoite lysates from untreated or rapamycin-treated parasites, using anti-Flag antibodies to detect CLAMP. CSP was used as a loading control.
Fig 4.
CLAMP-deficient sporozoites are impaired in transcellular migration, host cell infection and motility.
A. Quantification of traversed (dextran-positive) HepG2 cells by FACS after incubation for 3h with rapamycin-exposed and untreated clampcKO salivary gland sporozoites in the presence of fluorescein-labelled dextran. Results shown are mean +/- SEM of three independent experiments, each performed with 5 technical replicates. B. Quantification of UIS4-labelled exo-erythrocytic forms (EEFs) in HepG2 cells as determined by fluorescence microscopy 24h post-invasion with rapamycin-exposed and untreated clampcKO salivary gland sporozoites. Results shown are mean +/- SEM of four independent experiments with 5 technical replicates for each. ***, p<0.001; ****, p<0.0001 (Two-tailed ratio paired t test). C. Maximum intensity projection of video-microscopy images of untreated clampcKO (mCherry+/GFP+, yellow) and rapamycin-exposed clampcKO (mCherry+/GFP-, red) salivary gland sporozoites, recorded for 3 min. Sporozoites were mixed and activated at 37°C in the presence of albumin before imaging. Their movement patterns were classified in four categories: circular gliding, waving, floating and fixed (shown in magnified images). Scale bars, 50 μm for projection and 10 μm for magnified images. D. Quantification of motility patterns in untreated (n = 1496) and rapamycin-exposed (n = 864) clampcKO sporozoites. Sporozoites were classified in three groups based on their motility pattern. ****, p<0.0001 (Chi-square).
Fig 5.
CLAMP is essential for TRAP shedding.
A. P. berghei proteins identified by mass spectrometry after anti-Flag immunoprecipitation from two independent CLAMP-flag sporozoite lysates, and not from control samples. B. Additional lysates were prepared from CLAMP-flag and parental P. berghei salivary gland sporozoites, and used for anti-Flag IP followed by western blotting using anti-TRAP antibodies. TRAP was detected in total extracts (input) from both CLAMP-flag (C) and parental (P) samples, but only in CLAMP-Flag immunoprecipitate (IP). C. TRAP secretion assay using haemolymph sporozoites (5 x 104 or equivalent per lane) from untreated or rapamycin-treated clampcKO parasites. Microneme secretion was stimulated by incubation for 15 min at 37°C in the presence of BSA and ethanol. After stimulation of microneme secretion, samples were fractionated by centrifugation in sporozoite pellets (spz) and supernatants containing secreted proteins (SN), and analyzed by western blot using anti-TRAP antibodies. TRAP protein was detected as a ~100 kDa major band (black arrow) and two minor bands (grey arrows) in sporozoite lysates, and as a single ~75 kDa in supernatants (red arrow). D. Quantification of signal intensity of TRAP western blot bands in sporozoite pellets (main band only) and supernatants. The data show TRAP levels in rapamycin-treated parasites relative to untreated parasites, both in pellets and supernatants.
Fig 6.
Expansion microscopy reveals a unique distribution of CLAMP in sporozoites.
A. Salivary gland sporozoites expressing Flag-tagged CLAMP (untreated clampcKO parasites) were examined by expansion microscopy using antibodies against Flag (green) and TRAP (red). Specific accumulation of CLAMP is clearly visible at the apical tip of sporozoites, as indicated by arrows in the upper panels. The lower i and ii magnified panels illustrate the distinct distribution of CLAMP (green arrowheads) and TRAP (red arrowheads), and the colocalization of both proteins in a subset of micronemes (yellow arrowheads). Scale bars, 10 μm (upper left panel) or 2.5 μm (other panels). B. Sporozoites labeled with antibodies against Flag (green) and AMA1 (red) were examined by expansion microscopy. Apical accumulation of CLAMP is indicated by arrows in the upper panels. The lower i and ii magnified panels illustrate the distinct distribution of CLAMP (green arrowheads) and AMA1 (red arrowhead). Scale bars, 10 μm (upper left panel) or 2.5 μm (other panels).