Fig 1.
PhIL1 colocalize with ALV5, PhIP, and GAPM2 in the P. falciparum IMC.
(A) ALV5-GFP expression pattern in asexual blood stages (merozoite, and schizont) of P. falciparum. (B) PhIP-GFP expression pattern in asexual blood stages showed typical IMC localization. (C) GAPM2-GFP expression pattern in asexual blood stages. (D) Co-staining of PhIL1 with ALV5, PhIP, and GAPM2 in P. falciparum blood-stage schizont. These proteins co-localized with PhIL1 in the IMC at the schizont stage of the parasite with a Pearson’s colocalization coefficient of more than 0.7. Scale bar = 5 μm. (E) List of proteins pulled down by GFP-trap beads from lysates obtained from parasites expressing GFP-tagged ALV5, PhIP, or GAPM2 respectively. n = 3 experiments.
Fig 2.
PhIL1-associated novel complex overlaps with components of the linear motor in the IMC but is a separate complex.
(A) Native PAGE analysis of schizont stage parasite lysate shows co-existence of PhIL1-associated complex with the glideosomal complex via. some overlapping components. A complex of ∼800 kDa was detected containing components of PfPhIL1-associated complex and GAP50. Furthermore, a smaller complex of ∼250 kDa was also detected that included PfPhIL1 and PfGAPM2. These results indicate a possible association of PhIL1-associated complex and glideosomal complex. n = 2 experiments. (B) PhIL1-associated complex co-exists with the glideosomal complex as seen by Glycerol gradient co-sedimentation analysis. Glycerol gradient fractionation of P. falciparum schizont extract using 5 to 45% Glycerol gradient followed by immunoblotting of the fractions using specific antibodies showed the co-sedimentation of PfGAP50, PfPhIL1, PfALV5, PfPhIP, and PfGAPM2 together in fractions 5 to 11, particularly, in fraction 9 corresponding to ∼250 kDa molecular mass. n = 2 experiments. (C) Recombinant GAP50 and BSA were subjected to SDS-PAGE followed by (D) far western analysis showing the interaction of recombinant PfGAP50 protein with PfPhIL1. GAP50 was denatured and renatured on the membrane followed by incubation with recombinant PhIL1 protein and probed with α-PhIL1 antisera which recognized a band of ~43kDa corresponding to the size of GAP50. BSA used as control protein did not show any interaction with PfPhIL1. (E) Model showing molecular motor and partially overlying proposed PhIL1-associated complex (dotted box) in the parasite IMC. Photosensitized 5-[125I] Iodonaphthalene-1-azide Labelled Protein-1 (PhIL1); PhIL1 Interacting Protein (PhIP); Glideosome-Associated Protein 40, 45 and 50 (GAP40, 45 and 50); Glideosome-associated protein with multiple membrane spans (GAPM); myosin-A (MyoA); Myosin-A Tail domain Interacting Protein (MTIP); Connector Protein between actin and protein of the merozoite surface (CP); Protein of the Merozoite surface (MzP); Parasite Plasma Membrane (PPM).
Fig 3.
Functional characterization of P. falciparum ALV5, PhIP, and GAPM2 by inducible regulation of endogenous protein levels and their effect on parasite growth.
(A) Western blot analysis of lysate from PfALV5-pHA-glmS line with α-HA rat serum showing conditional knock-down on glucosamine treatment. (B) Effect of conditional knockdown of PfALV5 on parasite invasion of the host RBC. Glucosamine-treated (+GlcN) and untreated (-GlcN) cultures were incubated till the formation of new rings and the parasitemia was estimated by flow cytometry. (C) Representative parasites from the Giemsa-stained smears showing morphology following ALV5 knockdown. (D) Western blot analysis of lysate from PfPhIP-HA-glmS line with α-HA rat antibody to check for conditional knock-down shows a robust knockdown of PhIP-HA protein. (E) Effect of conditional knockdown of PfPhIP on parasite invasion. (F) Representative parasites from the Giemsa-stained smears showing morphology following PhIP knockdown. Percentage of different phenotypes was calculated from Giemsa smears of glucosamine treated PhIP-HA-glmS parasites, highlighting the arrest of growth in segmented schizont stage, and altered efficacy of released merozoites to invade following PhIP knockdown. See also S4 Fig. (G) Western blot analysis of lysate from PfGAPM2-HA-glmS line with α-HA rat serum showing efficient knockdown of the GAPM2-HA protein when compared to the PfBiP loading control. (H) Effect of conditional knockdown of PfGAPM2 in parasite showing up to 85% invasion inhibition. (I) Representative Giemsa-stained smears showing the arrest in development following GAPM2 knockdown due to inefficiency of released merozoites to invade the host RBC. Zoomed Giemsa smear show phenotype for PhIP- and GAPM2-HA-glmS parasites in presence of glucosamine, highlighting the arrest in development following knockdown. Data represent mean ± SD. n = 3 experiments. PfBiP was used as a loading control for western blot analysis.
Fig 4.
PfPhIP deficient parasites show defects in the segmentation of daughter cells.
PfPhIP-HA-glmS parasites maintained with and without GlcN were E64-treated (10 μM) and probed with the anti-HA antibody (green) and counterstained with DAPI. (A) Apparent knockdown of PhIP expression leads to the formation of agglomerates of unsegmented daughter nuclei. (B) Anti-GAP50 antibody (IMC marker) (red) showed defects in IMC formation in agglomerates. Merozoite plasma membrane and micronemes were stained with antibodies against P. falciparum (C) merozoite surface protein 1 (PfMSP1) (red) and (D) PfAMA1 (red), respectively, in E64-treated schizonts cultivated with and without GlcN. PfMSP1 was visible in schizonts but forms relatively larger rings that surround multiple nuclei in the agglomerates. PfAMA1 showed a loss of signal in agglomerates. Scale bar = 5 μm. Based upon the staining pattern parasites were grouped and the percentage parasites in each group are presented in the bar graphs. (E) 3D reconstruction of the PfPhIP-HA-glmS schizonts with and without GlcN is presented using Imaris 7.6.1. Arrowheads indicate agglomerates of unsegmented daughter nuclei. See also S5 and S6 Figs.
Fig 5.
PfGAPM2 and PfPhIP function is essential for invasion of the host cell by the merozoites.
Merozoites released from GlcN treated (A) PfPhIP-HA-glmS and (B) PfGAPM2-HA-glmS parasites were stained using antibodies against PfGAP50 (green, top panel), PfEBA175 (green, middle panel), PfRON2 (green) and PfAMA1 (red) (lower panel). Staining for these marker proteins showed normal micronemal and rhoptry organelles, however, it was observed that the discharge of contents from these apical organelles was affected. Also, the apical end of the merozoites, attached to the host was not aligned towards the erythrocyte membrane. Scale bar = 5 μm. (C) 3D reconstruction of the merozoites from PfPhIP or PfGAPM2 deficient parasites arrested on the RBC surface using Imaris 9. See also S7 Fig. (D) Percentage distribution of PhIP- and GAPM2-iKD parasites in the presence of glucosamine w.r.t to PfRON2 was quantified for staining pattern which is either distant from or in proximity to the erythrocyte surface. (E) Schematic showing the effect of PhIP and GAPM2 depletion on the secretion of invasion ligands from the apical complex of merozoite compared to a healthy, invasive merozoite.
Fig 6.
Function of PhIL1-associated complex is crucial for reorientation mediated by gliding motor complex and thus host-cell invasion by Plasmodium falciparum merozoites.
The schematic illustrates GAPM2 and PhIP are essential for blood-stage infection and their genetic attenuation arrests merozoite invasion by impeding the function of glideosomal motor machinery resulting in failure of merozoite to reorient its apical end towards the host RBC. Depletion of PhIP also leads to the formation of agglomerates of unsegmented schizonts suggesting its probable role in IMC-mediated parasite cell division. In the left panel, the level of PfPhIP or GAPM2 protein is maintained by the absence of GlcN. The middle panel shows consequences of PhIP deficiency, wherein a portion of daughter cells get segmented while others remain trapped as an agglomerate under a common plasma membrane. In the agglomerate, IMC fails to form, parasite plasma membrane surrounds multinucleated unsegmented daughter cell, and microneme secretion is affected. Few segmented merozoites which egressed were seen arrested at the erythrocyte surface and are unable to invade. The right panel summarizes the effect of GAPM2 deficiency in the parasite where the merozoites fail to invade the erythrocytes due to the disability of these merozoites to align their apical end towards the host cell surface. Apical reorientation of merozoites is imperative for invasion so that the apical organelles are aligned to the erythrocyte membrane which leads to the discharge of apical organellar proteins followed by invasion. Created with BioRender.com.