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Malaria IMC1 membrane skeleton proteins operate autonomously and participate in motility independently of cell shape.

Tremp AZ, Dessens JT - J. Biol. Chem. (2010)

Bottom Line: Because of the coincident effects of previous imc1 gene disruptions on both zoite shape and locomotion, it has been impossible to ascribe a direct involvement in motility to these proteins.By generating double mutant parasites for IMC1h and IMC1b, we tested this hypothesis: double knock-out exacerbated the phenotypes of the single knock-outs in terms of ookinete strength, motility, and infectivity but did not further affect ookinete morphology.These findings provide the first genetic evidence that IMC1 proteins can function independently of each other and contribute to gliding motility independently of cell shape.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.

ABSTRACT
Plasmodium IMC1 (inner membrane complex 1) proteins comprise components of the subpellicular network, a lattice of intermediate filaments that form a structural part of the pellicle in the zoite stages of malaria parasites. Family members IMC1a and IMC1b are differentially expressed in sporozoites and ookinetes, respectively, but have functionally equivalent roles affecting cell morphology, strength, motility, and infectivity. Because of the coincident effects of previous imc1 gene disruptions on both zoite shape and locomotion, it has been impossible to ascribe a direct involvement in motility to these proteins. We show here that a third family member, IMC1h, has a distinct differential expression pattern and localizes to the pellicle of both ookinetes and sporozoites. Knock-out of IMC1h mimics the loss-of-function phenotypes of IMC1a and IMC1b in their respective life stages, indicating that IMC1 proteins could be operating co-dependently. By generating double mutant parasites for IMC1h and IMC1b, we tested this hypothesis: double knock-out exacerbated the phenotypes of the single knock-outs in terms of ookinete strength, motility, and infectivity but did not further affect ookinete morphology. These findings provide the first genetic evidence that IMC1 proteins can function independently of each other and contribute to gliding motility independently of cell shape.

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Southern analysis of genetically modified parasite lines IMC1h/GFP and IMC1h-KO. A, schematic diagram of WT and genetically modified imc1h loci on genomic DNA. Indicated are coding sequences (bars), positions of the HindIII restriction sites (H), and expected HindIII restriction fragments (horizontal arrows) with sizes shown in kb. Sequences corresponding to probes used are indicated by thick lines. B, Southern blot of HindIII-digested parasite genomic DNA using probes specific to imc1h and hDHFR sequences.
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Figure 2: Southern analysis of genetically modified parasite lines IMC1h/GFP and IMC1h-KO. A, schematic diagram of WT and genetically modified imc1h loci on genomic DNA. Indicated are coding sequences (bars), positions of the HindIII restriction sites (H), and expected HindIII restriction fragments (horizontal arrows) with sizes shown in kb. Sequences corresponding to probes used are indicated by thick lines. B, Southern blot of HindIII-digested parasite genomic DNA using probes specific to imc1h and hDHFR sequences.

Mentions: To study the expression and localization of IMC1h, a genetically modified P. berghei parasite line stably expressing full-length IMC1h fused to a C-terminal enhanced GFP tag was generated. This was achieved by replacing, via double crossover homologous recombination, the native imc1h allele with a recombinant allele linked in-frame to the enhanced gfp coding sequence. Concomitantly, a human dihydrofolate reductase (hdhfr) gene cassette was introduced, which confers resistance to the antimalarial drugs pyrimethamine and WR99210. To study the function of IMC1h and its contribution to parasite development, a genetically modified P. berghei parasite line was generated in which all of the imc1h coding sequence was removed except for the first 25 residues. In this parasite line, GFP acts as a reporter gene under the control of the native imc1h promoter. After transfection of purified schizont preparations, pyrimethamine-resistant parasites were selected and cloned. Diagnostic PCR across the predicted integration sites showed correct integration of the hDHFR gene cassette into the imc1h locus as well as the absence of the unmodified imc1h alleles (data not shown). This was confirmed by assessing the integrity of the genetically modified parasite lines (named IMC1h/GFP and IMC1h-KO, respectively) by Southern blot analysis of HindIII-digested genomic DNA. Two DNA probes were used: one specific to the imc1h coding sequence and one specific to the hDHFR gene (Fig. 2A). The imc1h probe gave rise to a 4.1-kb band in the parental WT parasites, a 1.8-kb band in IMC1h/GFP parasites, and no signal in IMC1h-KO parasites, as predicted (Fig. 2B). The hDHFR probe gave rise to a hDHFR-specific signal of ∼7.9 kb in IMC1h/GFP and IMC1h-KO parasites, but not in WT parasites, as expected (Fig. 2B). These combined results confirmed the correct integration of the recombinant imc1h and hDHFR alleles into the imc1h locus.


Malaria IMC1 membrane skeleton proteins operate autonomously and participate in motility independently of cell shape.

Tremp AZ, Dessens JT - J. Biol. Chem. (2010)

Southern analysis of genetically modified parasite lines IMC1h/GFP and IMC1h-KO. A, schematic diagram of WT and genetically modified imc1h loci on genomic DNA. Indicated are coding sequences (bars), positions of the HindIII restriction sites (H), and expected HindIII restriction fragments (horizontal arrows) with sizes shown in kb. Sequences corresponding to probes used are indicated by thick lines. B, Southern blot of HindIII-digested parasite genomic DNA using probes specific to imc1h and hDHFR sequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037651&req=5

Figure 2: Southern analysis of genetically modified parasite lines IMC1h/GFP and IMC1h-KO. A, schematic diagram of WT and genetically modified imc1h loci on genomic DNA. Indicated are coding sequences (bars), positions of the HindIII restriction sites (H), and expected HindIII restriction fragments (horizontal arrows) with sizes shown in kb. Sequences corresponding to probes used are indicated by thick lines. B, Southern blot of HindIII-digested parasite genomic DNA using probes specific to imc1h and hDHFR sequences.
Mentions: To study the expression and localization of IMC1h, a genetically modified P. berghei parasite line stably expressing full-length IMC1h fused to a C-terminal enhanced GFP tag was generated. This was achieved by replacing, via double crossover homologous recombination, the native imc1h allele with a recombinant allele linked in-frame to the enhanced gfp coding sequence. Concomitantly, a human dihydrofolate reductase (hdhfr) gene cassette was introduced, which confers resistance to the antimalarial drugs pyrimethamine and WR99210. To study the function of IMC1h and its contribution to parasite development, a genetically modified P. berghei parasite line was generated in which all of the imc1h coding sequence was removed except for the first 25 residues. In this parasite line, GFP acts as a reporter gene under the control of the native imc1h promoter. After transfection of purified schizont preparations, pyrimethamine-resistant parasites were selected and cloned. Diagnostic PCR across the predicted integration sites showed correct integration of the hDHFR gene cassette into the imc1h locus as well as the absence of the unmodified imc1h alleles (data not shown). This was confirmed by assessing the integrity of the genetically modified parasite lines (named IMC1h/GFP and IMC1h-KO, respectively) by Southern blot analysis of HindIII-digested genomic DNA. Two DNA probes were used: one specific to the imc1h coding sequence and one specific to the hDHFR gene (Fig. 2A). The imc1h probe gave rise to a 4.1-kb band in the parental WT parasites, a 1.8-kb band in IMC1h/GFP parasites, and no signal in IMC1h-KO parasites, as predicted (Fig. 2B). The hDHFR probe gave rise to a hDHFR-specific signal of ∼7.9 kb in IMC1h/GFP and IMC1h-KO parasites, but not in WT parasites, as expected (Fig. 2B). These combined results confirmed the correct integration of the recombinant imc1h and hDHFR alleles into the imc1h locus.

Bottom Line: Because of the coincident effects of previous imc1 gene disruptions on both zoite shape and locomotion, it has been impossible to ascribe a direct involvement in motility to these proteins.By generating double mutant parasites for IMC1h and IMC1b, we tested this hypothesis: double knock-out exacerbated the phenotypes of the single knock-outs in terms of ookinete strength, motility, and infectivity but did not further affect ookinete morphology.These findings provide the first genetic evidence that IMC1 proteins can function independently of each other and contribute to gliding motility independently of cell shape.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.

ABSTRACT
Plasmodium IMC1 (inner membrane complex 1) proteins comprise components of the subpellicular network, a lattice of intermediate filaments that form a structural part of the pellicle in the zoite stages of malaria parasites. Family members IMC1a and IMC1b are differentially expressed in sporozoites and ookinetes, respectively, but have functionally equivalent roles affecting cell morphology, strength, motility, and infectivity. Because of the coincident effects of previous imc1 gene disruptions on both zoite shape and locomotion, it has been impossible to ascribe a direct involvement in motility to these proteins. We show here that a third family member, IMC1h, has a distinct differential expression pattern and localizes to the pellicle of both ookinetes and sporozoites. Knock-out of IMC1h mimics the loss-of-function phenotypes of IMC1a and IMC1b in their respective life stages, indicating that IMC1 proteins could be operating co-dependently. By generating double mutant parasites for IMC1h and IMC1b, we tested this hypothesis: double knock-out exacerbated the phenotypes of the single knock-outs in terms of ookinete strength, motility, and infectivity but did not further affect ookinete morphology. These findings provide the first genetic evidence that IMC1 proteins can function independently of each other and contribute to gliding motility independently of cell shape.

Show MeSH
Related in: MedlinePlus