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A malaria membrane skeletal protein is essential for normal morphogenesis, motility, and infectivity of sporozoites.

Khater EI, Sinden RE, Dessens JT - J. Cell Biol. (2004)

Bottom Line: Knockout of PbIMC1a protein expression reduces, but does not abolish, sporozoite gliding locomotion.We identify a family of proteins related to PbIMC1a in Plasmodium and other apicomplexan parasites.These results provide new functional insight in the role of membrane skeletons in apicomplexan parasite biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Imperail College London, London SW7 2AZ, England, UK.

ABSTRACT
Membrane skeletons are structural elements that provide mechanical support to the plasma membrane and define cell shape. Here, we identify and characterize a putative protein component of the membrane skeleton of the malaria parasite. The protein, named PbIMC1a, is the structural orthologue of the Toxoplasma gondii inner membrane complex protein 1 (TgIMC1), a component of the membrane skeleton in tachyzoites. Using targeted gene disruption in the rodent malaria species Plasmodium berghei, we show that PbIMC1a is involved in sporozoite development, is necessary for providing normal sporozoite cell shape and mechanical stability, and is essential for sporozoite infectivity in insect and vertebrate hosts. Knockout of PbIMC1a protein expression reduces, but does not abolish, sporozoite gliding locomotion. We identify a family of proteins related to PbIMC1a in Plasmodium and other apicomplexan parasites. These results provide new functional insight in the role of membrane skeletons in apicomplexan parasite biology.

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Targeted disruption of PbIMC1a. (A) Diagram of the recombination strategy. Indicated are sequences specific for PbIMC1a (dark gray); PbDHFR (light gray); TgDHFR/TS (white); positions of SphI restriction sites (s); and probes used in Southern blot. (B) Southern analysis of SphI-digested genomic DNA (gDNA) from WT and PbIMC1a-KO parasite clones 104 and 204. (C) RT-PCR analysis of blood stages total RNA for mRNA of PbIMC1a and the reference gene tubulin-1.
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fig3: Targeted disruption of PbIMC1a. (A) Diagram of the recombination strategy. Indicated are sequences specific for PbIMC1a (dark gray); PbDHFR (light gray); TgDHFR/TS (white); positions of SphI restriction sites (s); and probes used in Southern blot. (B) Southern analysis of SphI-digested genomic DNA (gDNA) from WT and PbIMC1a-KO parasite clones 104 and 204. (C) RT-PCR analysis of blood stages total RNA for mRNA of PbIMC1a and the reference gene tubulin-1.

Mentions: To study the role of PbIMC1a we generated PbIMC1a gene-disrupted parasites. A modified T. gondii dihydrofolate reductase/thymidylate synthase (DHFR/TS) gene cassette (conferring resistance to pyrimethamine) was inserted into the PbIMC1a gene by double homologous recombination replacing nucleotides 550–2,404 of the PbIMC1a gene (Fig. 3 A). Southern analyses of SphI-digested genomic DNA from the resulting parasite (named PbIMC1a-KO) confirmed correct integration into the target locus: A probe corresponding to the central PbIMC1a region gave rise to a single band in parental wild-type (WT), but no signal in PbIMC1a-KO parasites. Conversely, a probe corresponding to the DHFR/TS gene gave rise to three DHFR/TS-specific bands in PbIMC1a-KO, but no signal in WT parasites (Fig. 3 B). In addition, PbIMC1a-specific mRNA was readily amplified by RT-PCR from total RNA samples of WT, but not from PbIMC1a-KO blood stage parasites, confirming disruption of the PbIMC1a gene (Fig. 3 C).


A malaria membrane skeletal protein is essential for normal morphogenesis, motility, and infectivity of sporozoites.

Khater EI, Sinden RE, Dessens JT - J. Cell Biol. (2004)

Targeted disruption of PbIMC1a. (A) Diagram of the recombination strategy. Indicated are sequences specific for PbIMC1a (dark gray); PbDHFR (light gray); TgDHFR/TS (white); positions of SphI restriction sites (s); and probes used in Southern blot. (B) Southern analysis of SphI-digested genomic DNA (gDNA) from WT and PbIMC1a-KO parasite clones 104 and 204. (C) RT-PCR analysis of blood stages total RNA for mRNA of PbIMC1a and the reference gene tubulin-1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172497&req=5

fig3: Targeted disruption of PbIMC1a. (A) Diagram of the recombination strategy. Indicated are sequences specific for PbIMC1a (dark gray); PbDHFR (light gray); TgDHFR/TS (white); positions of SphI restriction sites (s); and probes used in Southern blot. (B) Southern analysis of SphI-digested genomic DNA (gDNA) from WT and PbIMC1a-KO parasite clones 104 and 204. (C) RT-PCR analysis of blood stages total RNA for mRNA of PbIMC1a and the reference gene tubulin-1.
Mentions: To study the role of PbIMC1a we generated PbIMC1a gene-disrupted parasites. A modified T. gondii dihydrofolate reductase/thymidylate synthase (DHFR/TS) gene cassette (conferring resistance to pyrimethamine) was inserted into the PbIMC1a gene by double homologous recombination replacing nucleotides 550–2,404 of the PbIMC1a gene (Fig. 3 A). Southern analyses of SphI-digested genomic DNA from the resulting parasite (named PbIMC1a-KO) confirmed correct integration into the target locus: A probe corresponding to the central PbIMC1a region gave rise to a single band in parental wild-type (WT), but no signal in PbIMC1a-KO parasites. Conversely, a probe corresponding to the DHFR/TS gene gave rise to three DHFR/TS-specific bands in PbIMC1a-KO, but no signal in WT parasites (Fig. 3 B). In addition, PbIMC1a-specific mRNA was readily amplified by RT-PCR from total RNA samples of WT, but not from PbIMC1a-KO blood stage parasites, confirming disruption of the PbIMC1a gene (Fig. 3 C).

Bottom Line: Knockout of PbIMC1a protein expression reduces, but does not abolish, sporozoite gliding locomotion.We identify a family of proteins related to PbIMC1a in Plasmodium and other apicomplexan parasites.These results provide new functional insight in the role of membrane skeletons in apicomplexan parasite biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Imperail College London, London SW7 2AZ, England, UK.

ABSTRACT
Membrane skeletons are structural elements that provide mechanical support to the plasma membrane and define cell shape. Here, we identify and characterize a putative protein component of the membrane skeleton of the malaria parasite. The protein, named PbIMC1a, is the structural orthologue of the Toxoplasma gondii inner membrane complex protein 1 (TgIMC1), a component of the membrane skeleton in tachyzoites. Using targeted gene disruption in the rodent malaria species Plasmodium berghei, we show that PbIMC1a is involved in sporozoite development, is necessary for providing normal sporozoite cell shape and mechanical stability, and is essential for sporozoite infectivity in insect and vertebrate hosts. Knockout of PbIMC1a protein expression reduces, but does not abolish, sporozoite gliding locomotion. We identify a family of proteins related to PbIMC1a in Plasmodium and other apicomplexan parasites. These results provide new functional insight in the role of membrane skeletons in apicomplexan parasite biology.

Show MeSH
Related in: MedlinePlus