Malaria parasite LIMP protein regulates sporozoite gliding motility and infectivity in mosquito and mammalian hosts
- Faculdade de Medicina da Universidade de Lisboa, Edifício Egas Moniz, Av. Prof. Egas Moniz, Portugal
- University of Heidelberg Medical School, Germany
- Indian Institute of Technology Delhi, India
- Leiden University Medical Center, The Netherlands
Figures
Figure 1 with 3 supplements
Gene and protein structure of limp and its translational regulation during transmission.
(A) The Plasmodium berghei limp gene (1194 bp) is composed of six exons (shaded bars) and five introns (lines). (B) Protein alignment of LIMP orthologues from seven Plasmodium species. ‘+' signal …
Figure 1—figure supplement 1
Multiple sequence alignment of LIMP orthologues from related apicomplexan parasites.
EfaB: Eimeria falciformis; NCLIV: Neospora caninum; TGGT1: Toxoplasma gondii; HHA: Hammondia hammondi. All sequences from eupathdb.org.
Figure 1—figure supplement 2
Generation and genotyping of the limp::gfp parasite line.
(A) limp GFP tagging construct pLIS0079 (i) was obtained by cloning the last 1149 bp of the limp ORF excluding the stop codon upstream and in frame with the gfp gene. This construct includes Toxoplas…
Figure 1—figure supplement 3
limp::gfp parasites show no defects in liver- and blood-stage infections.
(A) Dextran assay of sporozoite hepatocyte traversal. Bars show means±SEM; p-value for Student´s t-test. WT and limp::gfp (one experiment; n = 3). (B) Exoerythrocytic form (EEF) numbers during in …
Figure 2 with 2 supplements
Δlimp parasites suffer cumulative population loss during mosquito and rodent passage.
(A) Ookinete (Ook) conversion rates of both Δa and Δb parasites (50–90% dashed lines). Bars show means, dots represent individual conversion rates. Δa and Δb (four independent experiments, n = 8). (B…
Figure 2—figure supplement 1
Generation and genotyping of Δlimp parasite lines.
(A) limp gene deletion construct pLIS0060 (i) was obtained by cloning limp 5' and 3' targeting regions (TR) upstream and downstream of the Toxoplasma gondii dhfr/ts selectable marker cassette, …
Figure 2—figure supplement 2
Δlimp parasites develop normal blood-stage parasitaemia and gametocytaemias over the course of infection.
(A) WT and Δlimp-a total parasitaemias over time. (B) WT and Δlimp-a male gametocytaemias over time. (C) WT and Δlimp-a female gametocytaemias over time. (A–C) Lines show means±SEM values from three …
Figure 3
Δlimp parasites are severely impaired in their gliding motility, adhesion and liver cell invasion capabilities.
(A) Dextran assay of sporozoite hepatocyte traversal. NISG: cells incubated with non-infected salivary gland material. WT (four independent experiments; n = 12); Δa (five independent experiments; n =…
Figure 4 with 3 supplements
LIMP localises to the parasite plasma membrane but its function cannot be targeted by blocking antibodies.
(A) Immunofluorescence assays of WT, limp::gfp, mcherry::limp and limp::myc salivary gland sporozoites. Sporozoites were stained with anti-GFP (α-GFP), α-mCherry or α-MYC antibodies at days 17–20 …
Figure 4—figure supplement 1
Phenotypic characterisation of tagged limp parasite lines.
Oocysts per mosquito midgut. All tagged lines show oocyst numbers similar to WT parasites indicating no defect in midgut colonisation. Lines show means±SEM from two independent experiments. WT (n = 2…
Figure 4—figure supplement 2
Generation and genotyping of the mcherry::limp parasite line.
(A) mCherry limp tagging construct pLIS0505 (i) was obtained by inserting the mcherry gene downstream and in frame of the signal peptide (SP) of limp. To ensure correct cleavage of the N-terminal …
Figure 4—figure supplement 3
Generation and genotyping of the limp::myc parasite line.
(A) limp MYC tagging construct pLIS0472 (i) was obtained analogously to limp GFP tagging construct by cloning the last 1149 bp of the limp ORF excluding the stop codon upstream and in frame with the …
Figure 5
limp::gfp salivary gland sporozoites glide with a ‘limp’.
(A) The percentage of circular moving limp::gfp salivary gland (SG) sporozoites is comparable to WT, mcherry::limp and limp::myc sporozoites. Four independent experiments were performed for each …
Figure 6 with 2 supplements
GFP tag occludes solvent accessible surface area of LIMP helices.
Modelled LIMP structures linked to different tags (GFP in green, MYC in orange and mCherry in red) extracted after 100 ns of all-atom molecular dynamics simulations. Solvent accessible surface area …
Figure 6—figure supplement 1
LIMP secondary structure prediction and destabilisation upon GFP tagging.
(A) Multiple sequence alignment of Plasmodium LIMP orthologues with secondary structure prediction shown for P. berghei LIMP. α: alpha helix; β: beta sheet. (B) All-atom molecular dynamics …
Figure 6—figure supplement 2
All-atom molecular dynamics simulations of LIMP tagged with GFP, MYC and mCherry.
Starting modelled structures (bottom), stable intermediate structures at 50 ns (middle) and structures after 100 ns (top) are shown. Linker residues between LIMP and tags are highlighted in cyan in …
Figure 7
Role of motility- and invasion-related Plasmodium factors during mosquito stage development and transmission of malaria parasites.
While previously published malaria proteins participate in just one or few motility and invasion steps of Plasmodium mosquito stages, LIMP plays a role in midgut invasion/oocyst formation, salivary …
Videos
Video 1
Live gliding motility of a representative wild-type sporozoite
https://doi.org/10.7554/eLife.24109.015
Video 2
Live gliding motility of a representative limp::gfp sporozoite
https://doi.org/10.7554/eLife.24109.016Additional files
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Supplementary file 1
Summary of Plasmodium surface-, microneme- or rhoptry-localised proteins with function in ookinete and sporozoite motility, adhesion, cell traversal and cell invasion.
- https://doi.org/10.7554/eLife.24109.021
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Supplementary file 2
Primers used in the present work.
(A) Primers used in life-cycle RT-PCRs and qPCR of RNA extracts from infected livers. (B) Primers used in the generation of gene deletion, GFP-tagging, mCherry-tagging and MYC-tagging constructs. (C) Primers used in genotyping and RT-PCR of mutant parasite lines.
- https://doi.org/10.7554/eLife.24109.022
