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Research Article
Pawan Malhotra
International Centre for Genetic Engineering and Biotechnology
Corresponding Author
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The human malaria parasite, Plasmodium falciparum possess a unique mechanism of gliding motility guided by glideosome that powers its entry into insect and vertebrate hosts to facilitate its invasion and internalization within the targeted host cell. Photosensitized INA-labelled protein 1 (PhIL1) forms a novel protein complex that is associated with glideosome motor complex in the inner membrane complex of invasive merozoite. To establish the role of PfPhIL1 associated novel complex at asexual blood stages, we characterized 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). GFP targeting and co-immunoprecipitation analysis confirmed that these proteins are part of a PhIL1 associated novel complex, which co-exists with the glideosomal complex. To know the functional significance of PhIL1 associated complex, transgenic parasites were generated for glmS mediated conditional knock-down of each of the three proteins. Parasites lacking PfPhIP or PfGAPM2 were unable to invade the RBCs. PfPhIP deficient parasites also showed defects in merozoite segmentation. PfPhIP and PfGAPM2 depleted parasites revealed abrogation of reorientation/gliding, although initial attachment with human RBCs was not affected in these knock-down parasites. Together, the data presented here shows that proteins of the PhIL1 associated complex play an important role in orientation of P. falciparum merozoites post initial attachment, which is crucial for formation of tight junction and hence invasion of host erythrocytes.
Keywords
Malaria, P. falciparum, Inner Membrane Complex, Merozoite invasion, Glideosome
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- FigureS1.TIF
Figure S1: 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.
- FigureS2.TIF
Figure S2: 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. M denotes known molecular weight marker.
- FigureS3.TIF
Figure S3: 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 arrowhead. (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.
- FigureS4.TIF
Figure S4: 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 smears showing agglomerates and arrested merozoites following PfPhIP knockdown.
- FigureS5.tif
Figure S5: 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 (B) and PfAMA1. Arrowheads show agglomerates showing loss of signal for AMA1 and MSP1 in unsegmented nuclei. Scale bar = 5 μm.
- FigureS6.tif
Figure S6: 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, anti-GAP50, anti-EBA175 antibody (green) and anti-AMA1 antibody (red) (A) PfPhIP deficient merozoites (B) PfGAPM2 deficient merozoites. Scale bar = 5 μm.
- TableS1.TIF
Table S1: Oligonucleotides used in this study
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research Article
Pawan Malhotra
International Centre for Genetic Engineering and Biotechnology
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
The most recent version of this article is available here.
No community comments so far
Version 1
Posted 06 Oct, 2020
Community comments: 2
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Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Declarations:
Declarations
- The author has confirmed that a statement listing potential conflicts of interest or lack thereof is included in the text.
The most recent version of this article is available here.
The human malaria parasite, Plasmodium falciparum possess a unique mechanism of gliding motility guided by glideosome that powers its entry into insect and vertebrate hosts to facilitate its invasion and internalization within the targeted host cell. Photosensitized INA-labelled protein 1 (PhIL1) forms a novel protein complex that is associated with glideosome motor complex in the inner membrane complex of invasive merozoite. To establish the role of PfPhIL1 associated novel complex at asexual blood stages, we characterized 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). GFP targeting and co-immunoprecipitation analysis confirmed that these proteins are part of a PhIL1 associated novel complex, which co-exists with the glideosomal complex. To know the functional significance of PhIL1 associated complex, transgenic parasites were generated for glmS mediated conditional knock-down of each of the three proteins. Parasites lacking PfPhIP or PfGAPM2 were unable to invade the RBCs. PfPhIP deficient parasites also showed defects in merozoite segmentation. PfPhIP and PfGAPM2 depleted parasites revealed abrogation of reorientation/gliding, although initial attachment with human RBCs was not affected in these knock-down parasites. Together, the data presented here shows that proteins of the PhIL1 associated complex play an important role in orientation of P. falciparum merozoites post initial attachment, which is crucial for formation of tight junction and hence invasion of host erythrocytes.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- FigureS1.TIF
Figure S1: 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.
- FigureS2.TIF
Figure S2: 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. M denotes known molecular weight marker.
- FigureS3.TIF
Figure S3: 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 arrowhead. (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.
- FigureS4.TIF
Figure S4: 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 smears showing agglomerates and arrested merozoites following PfPhIP knockdown.
- FigureS5.tif
Figure S5: 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 (B) and PfAMA1. Arrowheads show agglomerates showing loss of signal for AMA1 and MSP1 in unsegmented nuclei. Scale bar = 5 μm.
- FigureS6.tif
Figure S6: 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, anti-GAP50, anti-EBA175 antibody (green) and anti-AMA1 antibody (red) (A) PfPhIP deficient merozoites (B) PfGAPM2 deficient merozoites. Scale bar = 5 μm.
- TableS1.TIF
Table S1: Oligonucleotides used in this study
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