The human malaria parasite, Plasmodium falciparum possess a unique gliding machinery referred as glideosome that powers its entry into the insect and vertebrate hosts. A number of parasite proteins including Photosensitized INA-labelled protein 1 (PhIL1) have been shown to associate with glideosome machinery. Here we describe a novel PhIL1 associated protein complex that co-exists with glideosome motor complex in the inner membrane complex of the merozoite. Furthermore, using experimental genetics approach we characterized the role(s) of three proteins associated with PhIL1: a glideosome associated protein- PfGAPM2, an IMC structural protein- PfALV5 and a previously uncharacterised protein - referred here as PfPhIP (PhIL1 Interacting Protein). Parasites lacking PfPhIP or PfGAPM2 were unable to invade the host RBCs. Additionally, the down regulation of PfPhIP resulted in significant defects in merozoite segmentation. Furthermore, the PfPhIP and PfGAPM2 depleted parasites revealed abrogation of reorientation/gliding, however initial attachment with host RBCs was not affected in these parasites. Together, the data presented here shows that proteins of the PhIL1 associated complex plays an important role in orientation of P. falciparum merozoites following initial attachment, which is crucial for formation of tight junction and hence invasion of host erythrocytes. The identification and characterization of PhIL1 associated complex opens new avenues for future anti-malarial drug development.
Research Article
Plasmodium falciparum PhIL1 associated complex play an essential role in merozoite reorientation and invasion of host erythrocytes
https://doi.org/10.21203/rs.3.rs-87875/v2
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Malaria
P. falciparum
Inner Membrane Complex
Merozoite invasion
Glideosome
Merozoite Reorientation
Table 1 List of proteins pulled down by GFP-Trap beads from lysates of ALV5, PhIP and GAPM2-GFP parasites, respectively. n = 3 experiments.
- FigS1.tif
Generation and conformation of PfALV5-, PfPhIP- and PfGAPM2-GFP fusion parasite lines: (A) Schematic showing vector map of pSSPF2 construct, indicating different vector cassettes used for generation of GFP protein in fusion with PfALV5, PfPhIP or PfGAPM2. Western blot analysis of lysate from (B) PfALV5-GFP, (C) PfPhIP-GFP, and (D) PfGAPM2-GFP tag line, with α-GFP rabbit serum. M denotes known molecular weight marker.
- FigS2.tif
Glycerol gradient fractionation for cosedimentation of PhIL1 associated complex along with PfGAP50: Western blot analysis following glycerol gradient centrifugation in P. falciparum blood-stage schizonts using protein specific antibodies for: (A) PfGAP50 (B) PfPhIL1 (C) PfALV5 (D) PfPhIP and (E) PfGAPM2. n = 2 experiments. (F) Protein-protein interaction between PfPhIL1 and PfGAP50 was further confirmed using Far western blotting. Recombinant GAP50 protein was subjected to SDS-PAGE (lane 1, 2 and 3 having 0.7, 2.8 and 7 µg of purified protein respectively) and transferred to PVDF membrane. Proteins were denatured on the membrane followed by renaturation and subsequently incubated with 14 µg rPhIL1 protein. Blot was probed with α-PhIL1 antibody (lane 5, 6, 7, and 8). BSA (lane 4 and 8; 10 µg) was used as a control. M denotes known molecular weight marker.
- FigS3.tif
Generation of PfALV5, PfPhIP and PfGAPM2 inducible knockdown parasite lines: (A) Schematic of the glmS ribozyme reverse genetic tool: The ribozyme is inserted in the 3′-UTR after the coding region so that it is present in the expressed mRNA. Following addition of the inducer, glucosamine, which binds to the ribozyme, the mRNA self-cleaves resulting in degradation of the mRNA and knock down of protein expression. Integration into the parasite genome was confirmed by PCR using different primer sets: cloned C-terminus region (a/b), upstream of cloned region (c) and from the glmS ribozyme sequence, 1236A (d). Position of primers are marked by arrowheads. (B) PfALV5-pHA_glmS integrants in parasite genome were selected by PCR analysis using primer sets: 1003600_FglmS-HA (a) / 1003600_RglmS-HA (b) and 1003600_Int. (c) /1236A (d). (C) PCR was set up for confirmation of successful integration of PfPhIP-pHA_glmS construct in parasite genome using different set of primers: 1310700_FglmS-HA (a) /1310700_RglmS-HA (b) and 1310700_Int (c) /1236A (d). (D) Successful integration of PfGAPM2-pHA_glmS construct in parasite genome was confirmed using primer set: 0423500_FglmS-HA (a) / 0423500_RglmS-HA (b) and 0423500_Int. (c) / 1236A (d). M denotes known molecular weight marker.
- FigS4.tif
The two distinct phenotypes observed in PhIP knock-down parasites demonstrate the divergent functions of the components of IMC during asexual lifecycle of the parasite. (A) Number of parasites showing the arrest in development following knockdown due to two different phenotypes was calculated from Giemsa-stained smears of PhIP-HA-glmS parasites after 42 h of glucosamine treatment (1.25 mM). (B) Representative parasites from the Giemsa-stained smears showing agglomerates and arrested merozoites following PfPhIP knockdown.
- FigS5.tif
PfPhIP deficiency leads to incomplete formation of the IMC. Representative images of E64-treated schizont stage in [-]/ [+] GlcN PfPhIP-HA-glmS parasites using antibodies against (A) PfMSP1 and (B) PfAMA1. Arrowheads show agglomerates showing loss of signal for AMA1 and MSP1 in unsegmented nuclei. Scale bar = 5 μm.
- FigS6.tif
Released merozoites from PfPhIP- and PfGAPM2-deficient schizonts fail to invade erythrocytes. Merozoites appeared to be arrested at RBC surface due to failure to align its apical end towards the host cell were probed with DAPI; anti-RON2 (green), anti-GAP50 (green), anti-EBA175 antibody (green) and anti-AMA1 antibody (red) in (A) PfPhIP deficient merozoites (B) PfGAPM2 deficient merozoites. Scale bar = 5 μm.
- FigS7.tif
Oligonucleotides used in this study