Immunofluorescence assay and expansion microscopy (PS-ExM) reveals liver stage parasite subpellicular microtubule.

(A), and (B), show maximum projection of z-stacks confocal images of infected HeLa cells and (C) sporozoites. Host and parasite microtubules were stained with anti-α-tubulin (magenta), and nuclei with DAPI (blue). The parasitophorous membrane vacuole (PVM) was stained with anti-UIS4 (green). (A) HeLa cells were infected with Plasmodium salivary glands sporozoites and methanol-fixed at 24, 30, 48, and 56 hpi. IFA was performed. (B) Fixed infected HeLa cells were expanded using the PS-ExM protocol. Inlets show LSPMB structures at 6, 30, and 48 hpi. Scale bar: 10 µm. (C) To determine whether the LSPMB originates from the sporozoite stage, PS-ExM was performed on salivary gland sporozoites, and parasite liver stage at 2 and 6 hpi. (D) LSPMB volume was quantified from PS-ExM z-stacks using 3D and 4D analysis in IMARIS software. Normalised mean volumes were: 1294.12 µm³ in sporozoites, 628.34 µm³ at 2 hpi, 414.84 µm³ at 24 hpi, 231.4 µm³ at 48 hpi, to 5.6 µm³ at 54 hpi. (E) Live cell confocal imaging of infected HeLa cells incubated with tubulin tracker SPY650. Images show microtubules at 10, 30, 36, and 48 hpi. Host and parasite microtubules are shown in magenta; the parasite expresses cytosolic GFP (green). Scale bar: 5 µm.

Microtubules are essential for Plasmodium liver stage development.

(A and B) show the effect of oryzalin on parasite microtubules during liver stage development. Sporozoites isolated from mosquito salivary glands were incubated in medium containing either 10 or 20 µM of oryzalin. HeLa cells were infected and fixed at 48 hpi with cold methanol prior to PS-ExM. (A) confocal z-stack projections of control cells (DMSO) and cells treated with 10 and 20 µM of Oryzalin. (B) confocal z-stack projections of infected cells where oryzalin was washed out at 24 hpi. Microtubule was stained with anti-α-tubulin (magenta), the PVM with anti UIS4 (green), and nuclei with DAPI (blue). Scale bar: 10 µm. ‘’+’’ indicates cells treated with Oryzalin; ‘’ - ‘’ indicates cells from which the drug has been removed at 24 hpi. (C) Normalized total parasite DNA volume at 48 hpi. Mean DNA volume was 1952.02 µm³ in the DMSO control, 827.26 µm³ in 10 µM oryzalin-treated cells, and 1411.73 µm³ after drug removal. (D) Normalized total hemi-spindle volume. The mean volume was 539.3 µm ³ in the control, 0 µm³ with oryzalin treatment, and 89.08 µm³ after drug removal. (E) Normalized LSPMB volume at 48 hpi. Mean volume was 5.92 µm³ in the control, 2.3 µm³ with oryzalin, and 1.65 µm³ after drug removal. (F, G and H) Parasite size measurement at 6, 24, and 48 hpi, respectively. At 24 and 48 hpi, mean parasite size was 145.9 µm² in the control, 71.75 µm² with oryzalin, and 115.3 µm² after drug removal. (I) Parasite survival rate from 6 to 48 hpi. A slight increase in parasite death was observed in oryzalin-treated cells compared to the DMSO control. The red line on the graphs represents the mean and standard deviation. (C, D, E, F, and G), and (H): each dot represents an individual quantified parasite. (J) Confocal z-stack projection of IFA images at 56 hpi in cells treated with 10 µM oryzalin and DMSO control. Cells were fixed with cold methanol and stained for MSP1 (magenta), α-tubulin (grey) and nuclei (DAPI, blue). Scale bar: 5 µm. Statistical comparisons were performed using one-way ANOVA with Dunnett’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

LSPMB is polyglutamylate.

(A and B) show maximum projection of confocal z-stacks form sporozoites and infected primary hepatocytes at different time points during liver stage development. Primary hepatocytes were isolated from mice and infected with sporozoites. Cells were fixed and stained with antibodies against (A) polyglutamylated tubulin (anti-IN105), (B) tyrosinated tubulin (anti-Ty), and α-tubulin (anti-α-tubulin). Nuclei were stained with DAPI (blue), and the PVM with anti-UIS4 (grey). ROIs in (A) show the LSPMB at 6 and 36 hpi and polyglutamylated hemi-spindle poles at 48 hpi. ROIs in (B) display LSPMB and α-tubulin evolution across liver stages. (C) Normalized LSPMB volume and the hemi-spindle pole counts from 6 hpi to 56 hpi, quantified using IMARIS 3D/4D analysis. LSPMB volume decreased from 1038.9 µm³ at 6 hpi to 0 µm³ at 56 hpi, while hemi-spindle poles count increased from 0 at 6 hpi to a median of 928 at 56 hpi, showing a perfect Spearman anti-correlation coefficient of -1. n = 15 per time point. (D) Single-plane PS-ExM confocal image of infected HeLa cells at 36 hpi with parasites expressing PMP1::GFP. Cells were PFA-fixed and stained with anti-GFP (magenta), anti-IN105 (green), and anti-UIS4 (PVM). The yellow arrow indicates the direction of Cell RGB Profiler analysis from outside the PVM to inside the parasite. (E) Pixel intensity profiles (arbitrary units) for PVM (grey), LSPMB (green), and parasite plasma membrane (magenta) in expanded (PS-ExM) cells (E) and non-expanded (NE) cells. Overlap was observed in NE cells, while PS-ExM resolved LSPMB localization between the PVM and the parasite plasma membrane. n=10, in triplicate. Scare bar: 10 µm. (F) Single-plane confocal image of infected primary hepatocytes fixed with cold methanol. Cells were stained with anti-IN105 (green), α-tubulin (magenta), and DAPI (blue). The ROI shows a LSPMB tubule in direct contact with a parasite hemi-spindle pole. MZP: Maximum Z projection. Scale bars: 10 µm.

Plasmodium α1-tubulin C-terminal post-translational modification is important for the mosquito stage development.

(A) Schematic of the double homologous recombination (DHR) construct targeting the Plasmodium α1-tubulin gene (PBANKA_0417700) for C-terminal mutagenesis. The cassette (blue box) includes the 3’UTR P. berghei dihydrofolate reductase (PbDHFR), elongation factor-1 alpha (ef1α) promoter, human dihydrofolate reductase (hdhfr), yeast FCU (yfcu), 3’UTR PbDHFR, Hsp70 promoter, mCherry and the 3’ UTR Hsp70, with the 3’UTR region of the PBANKA_0417700 gene. This cassette was flanked by a 5′ homologous region (5′HR) containing the α1-tubulin coding region including the stop codon (with introduced mutations in the C-terminal region, cyan box) and a part of the 3′UTR of the gene (3’HR). The construct was transfected into the PbWT (P. berghei ANKA line cl15cy1). Black arrows indicate PCR primer binding sites and expected product sizes. Primer sequences are listed in Table 2 and 3. (B) Integration PCR results for the mutant parasite lines, showing expected band sizes on a 1% agarose gel: 5’HR = 4.4 kb, 3’HR = 2.7 kb, whole locus (WL) = 8.8 kb. Ladder: 1 kb DNA marker. Genotypes: WT, (wild-type); PsΔ (glutamine-to-alanine substitution); TyΔ (deletion the C-terminal tyrosination/detyrosination residues); CtΔ (complete C-terminal deletion), and NoΔ (construct control with no mutation). To ensure that all samples contained the same amount of DNA, a control gene representing a portion of tubulin (Ctrl tub about 470 bp) was used for integration PCR. (C) Representative confocal images of live oocysts at 11 days post infection (dpi). Nuclei were stained with Hoechst 33342 (hot cyan) and cytosolic mCherry (red) was expressed under the HSp70 promoter. BF, brightfield. (D) Normalized salivary sporozoite counts at 18 dpi. The control (NoΔ) averaged 14,033 sporozoites count per mosquito (C./mosq.), compared to 7,300 (PsΔ), 8,066 (TyΔ), and 200 (CtΔ). Experiment performed in triplicate; n = 20 per replicate. (E and F) Wide-field microscope motility assay images of salivary gland sporozoites showing the typical circular movement (maximum z projection in Fiji) of the control, PsΔ, and TyΔ salivary gland (SG) sporozoites (E) and the control and CtΔ hemolymph (H) sporozoites (F). For CtΔ, hemolymph sporozoites were used due to the absence of salivary gland sporozoites; these lacked typical motility. (G)) Quantification of sporozoite gliding velocity for PsΔ, and TyΔ salivary gland sporozoites. Mean velocities: NoΔ = 1.3 µm/s, PsΔ = 1.38 µm/s, TyΔ = 1.9 µm/s, CtΔ = 0.5 µm/s. Statistical analysis: one-way ANOVA with Dunnett’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (H) Quantification of sporozoite gliding velocity of CtΔ haemolymph sporozoites. The average gliding speed for control hemolymph sporozoites was 1.4 µm/s, whereas CtΔ sporozoites exhibited a slower speed of 0.5 µm/s. Statistical comparison tests were performed using two tailed unpaired t tests (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Liver stage development of Plasmodium α1-tubulin mutant parasites.

(A, B and C) Maximum projections of PS-ExM-acquired z-stack confocal images of infected HeLa cells at 6 and 36 hpi, showing the control (NoΔ), PsΔ, and CtΔ mutant parasites. (D and E) Maximum projections of PS-ExM-acquired z-stack confocal images of control and TyΔ salivary gland sporozoites, as well as CtΔ hemolymph sporozoites (E). In (A, B and C), cells were stained with antibodies against polyglutamylated tubulin (anti-IN105), while (D and E) antibodies against tyrosinated tubulin (anti-Ty) were used. In all panels, α-tubulin was labeled with anti-α-tubulin, nuclei with DAPI (blue), and the parasitophorous vacuole membrane (PVM) with anti-UIS4 (grey). (F and G) represent the fluorescence intensity (arbitrary units, a.u.) of anti-Ty in sporozoites (F) and anti-IN105 at 36 hpi in the liver stage (G). In (F), the mean fluorescence intensity of CtΔ (22 a.u.) was significantly reduced compared to TyΔ (33.4 a.u.) and NoΔ (49.6 a.u.), while in (G), PsΔ (79.9 a.u.) showed a significant reduction compared to NoΔ (121 a.u.). (H and L) showed infection rates of mutant parasites in HeLa cells at 6 hpi, determined by pre-counted sporozoites and quantitative microscopy. PsΔ (23.7%) and TyΔ (27.3%) exhibited typical infection rates compared to control (NoΔ) (16%) and WT (14%) (H), whereas CtΔ hemolymph sporozoites had a drastically reduced infection rate (2%) relative to controls hemolymph sporozoites (7.4%) (L). (K and O) Parasite survival from 6 to 48 hpi, normalized to 100% at 6 hpi. At 48 hpi, survival rates were 63% (NoΔ), 60% (WT), 50% (PsΔ), and 47% (TyΔ) (K). In contrast, CtΔ hemolymph sporozoites showed a significantly lower survival rate (2%) compared to controls (52%) (O), indicating impaired viability. (I and J, M and N) Parasite size measurements at 6 and 48 hpi for control, PsΔ, and TyΔ salivary gland sporozoites (I and J), as well as control and CtΔ hemolymph mutants (M and N). At 48 hpi, PsΔ (208.16 µm²) and TyΔ (203.7 µm²) were significantly smaller than NoΔ (237.9 µm²) and WT (235.7 µm²) (J). The CtΔ hemolymph mutants failed to establish liver stage infection, with an average size (88.1 µm²) markedly smaller than controls (187.4 µm²) (N). Each dot represents an individual parasite. PsΔ: Glutamine-to-alanine substitution at the polyglutamylation site; TyΔ: Deletion of C-terminal tyrosination/detyrosination residues; CtΔ: Complete deletion of PBANKA_0417700 α-tubulin C-terminal PTM residues and NoΔ: Control construct with no mutation. WT: wild type parasite. The experiments were conducted using, (F, H, I, J, K): One-way ANOVA with Dunnett’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (G, L, M, N, O): Two-tailed unpaired t-tests (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). All experiments were performed in triplicate.

Model of the tubulin behavior during Plasmodium berghei salivary gland sporozoite and exo-erythrocytic stage.

The graphical abstract summarizes the key finding of this study. It illustrates the development of Plasmodium parasites within primary hepatocytes, from invasion to the cytomere stage. α-tubulin was stained with anti-α-tubulin (magenta), nuclei with DAPI (blue), and the parasitophorous vacuole membrane (PVM) with anti-UIS4 (grey). The model highlights the evolution of sporozoite subpellicular microtubules (SSPM) into liver stage subpellicular microtubules (LSPMB), along with their associated tubulin post-translational modifications (PTMs). We propose that polyglutamylated LSPMB originates from the SSPM and is recycled during liver stage development to support hemi-spindle pole formation, membrane biogenesis, and potentially other functions. Upon hepatocyte invasion, the SSPM loses tyrosination, a modification that initially confers stability and supports sporozoite motility. This detyrosination likely facilitates parasite shape transformation, increases microtubule dynamics, and enables interactions with ER structures and MAPs. Tyrosination reappears in late stages, possibly stabilizing merozoite microtubules for erythrocyte invasion. Microtubule-stabilizing drugs did not affect the SSPM but inhibited liver stage microtubule polymerization, confirming their dynamic nature. Inhibition of microtubule dynamics also prevented merozoite formation. Finally, C-terminal tubulin PTM residues were shown to be essential for salivary gland sporozoite development and successful liver stage infection.

Plasmodium berghei eB1egfp parasites integration PCR primer

Plasmodium berghei α1-tubulin mutant parasites integration PCR primer

Plasmodium berghei α1-tubulin mutagenesis QuickChangeII primers sequences

Effect of Plasmodium-specific microtubule inhibitors on HeLa cell.

HeLa cells were seeded in 6-well plates and treated the following day with various concentrations of microtubule-targeting drugs. Media were replaced daily with fresh drug-supplemented medium. After five days, surviving cells were detached and counted. (A) Effect of amiprophos-methyl (AMP) on HeLa cell viability. A significant reduction in cell count was observed only at 50 µM compared to the DMSO control. (B) effect of the oryzalin on HeLa cells viability. No significant differences in cell count were observed at any tested concentrations tested relative to DMSO. (C) represent the parasite size at 48 hpi in infected HeLa cells treated with oryzalin and AMP at 50 µM. oryzalin showed a significant inhibitory effect on parasite growth compared to AMP and DMSO control. Statistical comparison tests were performed using a one-way ANOVA test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (D and E) Maximum projection confocal IFA z-stacks of HeLa cells treated with amiprophos-methyl (D) and oryzalin (E) tested at different concentrations, respectively. Cells were fixed with cold and stained with anti-α-tubulin (magenta) and DAPI (blue). (F) Confocal IFA z-projection of HeLa cells treated with low-dose oryzalin (0.5 µM), showing unusually elongated microtubule structures. (G) PS-ExM confocal z-stack of oryzalin-treated cells showing the very long LSPMB and hemi-spindles. (H) Quantification of LSPMB and hemi-spindle lengths. Oryzalin-treated microtubules were on average four times longer than those in DMSO-treated controls. Each dot represents a measurement from a single cell. LMT, Long microtubule; NMT, normal microtubule. Statistical comparison tests were performed using two tailed unpaired t tests (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Plasmodium end-binding protein 1 (eB1) is expressed in mosquito and liver stag.

(A) Pearson correlation coefficients between polyglutamylated LSPMB and α-tubulin LSPMB at 6, 36, and 48 hpi during liver stage development. On average, 92% of polyglutamylated tubulin colocalized with the LSPMB. (B) Maximum projection of PS-ExM confocal z-stacks of infected HeLa cells fixed at 30 and 48 hpi. Cells were stained with anti-γ-tubulin (green), anti-α-tubulin (magenta), anti-UIS4 (grey, PVM), and DAPI (blue, nuclei). White arrows indicate host centrosomes; yellow arrows highlight the LSPMB. Parasite hemi-spindle poles were also detected with γ-tubulin. (C) Schematic of the double homologous recombination construct used to tag Plasmodium eB1 (PBANKA_0405600) with eGFP at the C-terminus. The expression cassette (blue box) includes 3′UTR PbDHFR, EF1α promoter, hDHFR, yeast fcu, and the 3′UTR of PBANKA_0405600, flanked by the 5′HR (eB1 coding region) and 3′UTR. The construct was transfected into the PbmCherry 1868 mother line. (D) Integration PCR confirming correct insertion of the eGFP-tagged eB1 cassette. PCR product sizes: 5′HR = 1.5 kb, 3′HR = 1.3 kb, whole locus (WL) = 6 kb. Primer sequences listed in Table 1. Lane labels: Tr (transgenic), WT (wild type), ctrl (control). Ladder: 1 kb DNA marker. (E) Flow cytometry profile of erythrocytes infected with eB1-GFP-expressing parasites. Gate P1 includes all analyzed cells; P11 identifies mCherry-positive (PE-Texas Red-H) infected erythrocytes; R10 identifies GFP+/mCherry+ gametocytes. (F) Summary table showing parasitemia percentages across gates defined in (E). (G) Representative confocal live imaging of P. berghei eB1-GFP oocysts at 7 and 12 days post-infection (dpi), showing eB1-GFP (green), parasite mCherry (red), and Hoechst-stained nuclei (blue). Scale bar: 5 µm. (H) Confocal IFA image of P. berghei eB1-GFP sporozoites stained with anti-GFP (green), anti-α-tubulin (magenta), and anti-circumsporozoite protein (CSP, grey). (I) Maximum projection of PS-ExM confocal z-stacks of HeLa cells infected with P. berghei eB1-GFP parasites at 36 and 48 hpi. Cells were stained with anti-GFP (green), anti-α-tubulin (magenta), and anti-UIS4 (grey). Scale bar: 10 µm.

Plasmodium α-tubulin proteins and mutant parasite characterization.

(A) Comparison of Plasmodium α1- and α2-tubulin protein sequences highlighting predicted post-translational modification (PTM) sites. Polyglutamylation site E445 is conserved in both isoforms (green). α1-tubulin contains a predicted tyrosination site at Y453 (magenta), while α2-tubulin has a predicted detyrosination site at E450 (magenta). Neither isoform has the conserved acetylation site K40 (yellow). C-terminal PTM regions are underlined in red. (B) Sequence alignment of PBANKA_0417700 gene showing mutations introduced in the tubulin mutant parasites. Red highlights indicate substitutions or deletions. Dashes (—) indicate deleted nucleotides. Ps″: glutamine (E) substituted with alanine (A) at the polyglutamylation site; Ty″: deletion of the C-terminal tyrosination/detyrosination motif; Ct″: deletion of the entire C-terminal PTM region; No″: unmodified construct control. (C) Structural comparison of α1-tubulin mutant proteins using AlphaFold 3 predictions, aligned with PyMOL (Version 2.6.0). Green: wild type (No″); cyan: Ps″ mutant; magenta: Ty″ mutant; and yellow: Ct″ mutant. All predicted structures retained overall folding. (D) Maximum projection of confocal z-stacks of HeLa cells infected with mutant parasites (adapted from Freedy et al.). Microtubules were stained with anti-α-tubulin (magenta) and polyglutamylated tubulin with anti-IN105 (green). Nuclei were stained with DAPI (blue); the parasitophorous vacuole membrane (PVM) was stained with anti-UIS4 (grey). IntronΔ: line with deleted introns in the Plasmodium α1-tubulin gene; C-termΔ: line with deletion of C-terminal residues 540ADY. (E) In vivo infection kinetics in mice infected with mutant parasites. Parasitemia was measured daily from day 1 to day 5 post-infection. No significant differences were observed between mutant lines (intronΔ and C-termΔ) and wild type (WT). ExonΔ: mutant line in which α1-tubulin exons were replaced with α2-tubulin introns. This line failed to produce salivary gland sporozoites. Liver stage development in this line was delayed by 4 days due to hemolymph sporozoites used. Experiments were performed in triplicate.

Plasmodium α-tubulin mutant parasite oocyst formation and sporozoite infectivity.

(A) Comparison of oocyst area at day 7 and 11 post-infection. No significant difference was observed between the WT (NoΔ) and the PsΔ or TyΔ mutants at day 7 post-infection. However, by day 11, the CtΔ mutant exhibited a significantly reduced oocyst size compared to the WT. (B) Normalized midgut sporozoites count per mosquito (C./mosq.) at day 14 post-feeding. No significant difference in sporozoite numbers was observed between NoΔ and PsΔ, whereas CtΔ showed a drastic reduction. In contrast, TyΔ displayed both a significantly larger oocyst size at day 11 and a higher sporozoite count at day 14 compared to the WT. (C) Blood parasitemia levels in mice infected with mutant parasites, measured from day 0 to day 7 post-infection. All mutants (PsΔ and TyΔ) established infections and progressed similarly to the WT (NoΔ). PsΔ: glutamine-to-alanine substitution at polyglutamylation site; TyΔ: deletion of C-terminal tyrosination/detyrosination residues; CtΔ: complete deletion of PBANKA_0417700 α-tubulin C-terminal PTM residues; NoΔ: control construct with no mutation. The experiments were conducted in triplicate. Statistical multiple comparisons were performed using one-way ANOVA with Dunnett’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).