Fig 1.
PfGNA1 is essential for parasite growth during asexual development.
A) Diagram illustrating the enzymatic reactions involved in UDP-GlcNAc biosynthesis through the Hexosamine Biosynthetic Pathway. Glucosamine-phosphate N-acetyltransferase (GNA1) converts glucosamine-6-phosphate (GlcN6P) to N-acetylglucosamine-6-phosphate (GlcN6P). The chemical structures of the pathway intermediates are shown. B) Scheme outlining the timing of rapamycin (or mock, DMSO) treatment, administered at cycle 0 after tight synchronization of parasites within a 5-hour window. The times at which samples were collected for gene excision (white arrowheads) and protein expression (black arrowhead) analyses are also shown. C) PCR analysis of gna1 was conducted at different times post-DMSO (D) or rapamycin (R) treatment, using primers P5 and P6 specified S2A Fig and S2 Table. D) Parasite growth across cycles 1 and 2 following PfGNA1 disruption assessed by flow cytometry. E) Invasion rates for parasites treated with either DMSO or rapamycin were measured during the transition between developmental cycle 1 and 2. F) Western blot (left) with anti-HA antibody and Coomassie-stained gel (right) as loading control showing protein from trophozoites at cycle 1, treated with DMSO (D) or rapamycin (R) during cycle 0. The arrowhead indicates a band of approximately 30-35 kDa, efficiently depleted upon rapamycin addition. In panels D and E the graph shows mean ± SD values of three independent biological replicates. Statistical analyses were performed using unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Abbreviations: G6PI: Glucose-6-phosphate isomerase; GFPT: Glucosamine-fructose-6-phosphate aminotransferase; GNA1: Glucosamine-phosphate N-acetyltransferase; PAGM: Phosphoacetylglucosamine mutase; UAP: UDP-N-acetylglucosamine pyrophosphorylase.
Fig 2.
PfGNA1-disrupted parasites show depletion of HBP intermediates and UDP-GlcNAc.
A) Volcano plot showing global metabolomic changes between PfGNA1-disrupted and DMSO-treated parasites. Detailed analyses of GlcNAc-1/6-P (B), UDP-GlcNAc (C) and GDP-Man (D) levels in PfGNA1-disrupted parasites (treated with rapamycin), non-disrupted parasites (DMSO-treated) and the parental cell line. Data represent the mean and standard deviation from four independent replicates. p-values from two-sided Student’s t-tests are shown in B, C and D, comparing the specified conditions. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Abbreviations: GlcNAc-1/6-P, N-acetylglucosamine-1/6-phosphate; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine; GDP-Man, Guanosine diphosphate mannose.
Fig 3.
GPI synthesis is altered in PfGNA1-deficient parasites, disrupting the localization of the GPI-anchored protein MSP1.
A) Quantification of GPI molecules per cell in DMSO- and rapamycin-treated segmented schizonts from cycle 1. B) Immunofluorescence microscopy showing MSP1 distribution in segmented schizonts. MSP1 was labelled with a mouse anti-MSP1 antibody (green), and nuclei were stained with Hoechst 33342 dye (blue). Scale bar is 5 µm. C) Subcellular fractionation of schizonts treated with DMSO or rapamycin was performed sequentially using saponin, followed by Triton X-100 extraction. Both fractions were analysed by SDS-PAGE and Western blotting, using anti-MSP1 (left), anti-AMA1 (middle) and anti-BiP (right) antibodies, respectively. Bands migrating at varying heights, likely reflecting initial MSP1 and AMA1 processing, are observed and indicated in the Triton lanes (left panel). FL: Full-length. Panels B and C display representative images from four independent biological replicates.
Fig 4.
Disruption of PfGNA1 hints at segmentation defects and prevents parasite egress.
A) Histograms showing a population shift from ring stages to trophozoite/schizont stages. For flow cytometry analysis, the DNA of parasite cultures was stained with SYTO 11, and RBCs were gated using bivariate plots (SSC-H vs. FSC-H), recording 50,000 events within this region. The recorded events were then plotted as a function of fluorescence intensity detected through Filter 1, where subpopulations are distinguished according to their DNA content. B) Microscopy images of Giemsa-stained smears from tightly synchronized (5-hour window) PfGNA1 conditional knockout parasites treated with DMSO (control) or rapamycin. The images show time points during cycle 1 and the transition to cycle 2. Scale bar is 5 µm. C) Egress of synchronized (5-hour window) PfGNA1 conditional knockout parasites treated with DMSO (control) and rapamycin. Total parasitemia and the levels of young forms (rings) and mature forms (trophozoites-schizonts) were measured every two hours by flow cytometry during the transition between cycles 1 and 2, as described in A. The percentage of mature forms at each time point was normalized to the percentage observed at 91 hpp, when the trophozoite-schizont peak was reached. The ring levels are represented as an absolute number (hpp: hours post-Percoll). D) PfGNA1 conditional knockout parasite, treated with either rapamycin or DMSO, were enriched using Percoll, and merozoites were mechanically released by filtration. The number of merozoites post-filtration was measured by flow cytometry and normalized to the percentage of schizonts in culture before filtration. Panels A and B shows a representative image from four biological replicates. Panels C and D are based on three independent biological replicates, each with technical replicates. The graphs depict the means and standard deviation derived from one representative biological replicate, averaged over technical triplicates. The statistical analysis of panels C and D were performed using unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Fig 5.
PfGNA1 disrupted parasites show a severe segmentation defect.
Transmission electron microscopy showing the ultrastructure of PfGNA1 conditional-knockout schizonts treated with DMSO (control) or rapamycin, with or without E64 exposure. A) DMSO-treated parasites show multiple merozoites (M) along with distinct structures, including nuclei (N), apical organelles (AO), and food vacuoles (FV) containing hemozoin crystals (H). B) Rapamycin-treated parasites exhibited multinucleated, fused merozoites enclosed within the parasitophorous membrane (PVM). C) Zoom from B) showing the proper formation of apical organelles. D) and E) Detail of a PfGNA1-disrupted parasite surrounded by multiple layers of stacked membranes (indicated by arrowheads). F) An E64-treated, DMSO-control parasite, showing isolated merozoites with their respective organelles. Membrane vesicle whorls, marked by an asterisk are also visible. G) An E64-exposed, rapamycin-treated parasite with ribosomes (R) dispersed irregularly throughout the cytoplasm. Multiple nuclei display distinct chromatin condensation. H) Zoom of a FV in a PfGNA1-disrupted parasite as in G), containing internal membranous structures. A square highlights a ruptured FV with liberated hemozoin crystals. I) Detail of a nucleus of an E64-exposed, rapamycin-treated parasite showing chromatin condensation. J) Numerous stacked membrane layers are also observed.
Fig 6.
Diagram depicting impaired parasite egress following disruptions in HBP and GPI biosynthesis.
Rapamycin-treated PfGNA1-disrupted parasites show a marked reduction in GPI molecule levels, compromising MSP1 anchoring to the merozoite membrane and causing it to diffuse away. These parasites also display severe abnormalities during schizont development, characterized by aberrant morphology and the inability to form fully segmented, multinucleated schizonts with distinct nuclei. Furthermore, they fail to fully rupture the PVM, which prevents later breakdown of the red blood cell host membrane and ultimately blocks egress.