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CD36 selection of 3D7 Plasmodium falciparum associated with severe childhood malaria results in reduced VAR4 expression.

Magistrado PA, Staalsoe T, Theander TG, Hviid L, Jensen AT - Malar. J. (2008)

Bottom Line: Also, unlike var genes from groups B and C, those from group A do not have sequences consistent with CD36 binding--a major cytoadhesion phenotype of P. falciparum isolates.A selection-induced increased adhesion of 3D7(SM) iRBC to CD36 resulted in a reduced var4 transcription and VAR4 surface expression.The current findings are consistent with the notion that CD36 adhesion is not associated with particular virulent parasite phenotypes, such as those believed to be exhibited by VAR4 expressing parasites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Medical Parasitology at the Institute of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. pam@immi.ku.dk

ABSTRACT

Background: A subset of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1(SM)) is involved in the cytoadherence of P. falciparum-infected red blood cells (iRBC) contributing to the pathogenesis of severe disease among young children in malaria endemic areas. The PfEMP1(SM) are encoded by group A var genes that are composed of a more constrained range of amino acid sequences than groups B and C var genes encoding PfEMP1(UM) associated with uncomplicated malaria. Also, unlike var genes from groups B and C, those from group A do not have sequences consistent with CD36 binding--a major cytoadhesion phenotype of P. falciparum isolates.

Methods: A 3D7 PfEMP1(SM) sub-line (3D7(SM)) expressing VAR4 (PFD1235w/MAL8P1.207) was selected for binding to CD36. The protein expression of this parasite line was monitored by surface staining of iRBC using VAR4-specific antibodies. The serological phenotype of the 3D7(SM) parasites was determined by flow cytometry using malaria semi-immune and immune plasma and transcription of the 59 var genes in 3D7 were analysed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using var-specific primers.

Results: A selection-induced increased adhesion of 3D7(SM) iRBC to CD36 resulted in a reduced var4 transcription and VAR4 surface expression.

Conclusion: VAR4 is not involved in CD36 adhesion. The current findings are consistent with the notion that CD36 adhesion is not associated with particular virulent parasite phenotypes, such as those believed to be exhibited by VAR4 expressing parasites.

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Changes in VAR4 surface expression and transcription of the 3D7SM subline left to drift (3D7SM-drift). Left Y-axis, percentage of infected blood cells positive for VAR4 using flow cytometry and VAR4-specific CIDR1α (closed circle) and DBL5δ (open triangle) rabbit antibodies. Right Y-axis, percentage var4 gene transcription in ring stage (closed square) parasites calculated as: (var4 absolute copy number/∑copy number of all 3D7 var genes) × 100%. Fluorescence of 3D7SM-drift stained with pre-immune serum was used to separate positive cells from cells with background fluorescence.
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Figure 3: Changes in VAR4 surface expression and transcription of the 3D7SM subline left to drift (3D7SM-drift). Left Y-axis, percentage of infected blood cells positive for VAR4 using flow cytometry and VAR4-specific CIDR1α (closed circle) and DBL5δ (open triangle) rabbit antibodies. Right Y-axis, percentage var4 gene transcription in ring stage (closed square) parasites calculated as: (var4 absolute copy number/∑copy number of all 3D7 var genes) × 100%. Fluorescence of 3D7SM-drift stained with pre-immune serum was used to separate positive cells from cells with background fluorescence.

Mentions: In the absence of CD36 selection VAR4 is stably expressed on the surface of 3D7SM-drift for 44 generations – equivalent to five rounds of gelatine flotation (Figure 1iii-A). To analyse this further 3D7UM and 3D7SM were cultured without gelatine flotation or CD36 selection and monitored VAR4 surface expression and var4 transcription at specified time points for 94 generations. To avoid assay-to-assay variation, RBC infected with the two parasite sub-lines were frozen down at each sampling point. Frozen iRBC were thawed and cultured for another six generations and then assayed on the same day. Almost no VAR4 surface expression and var4 gene transcription was seen in the parental 3D7UM, whereas 3D7SM-iRBC showed high and stable surface expression of VAR4 as well as gene transcription for at least 94 generations (Figure 3). Using the percentageVAR4 positive iRBC and the ring-stage var4 transcription level at generation six (first sampling point) and 94 (last sampling point) the switch off-rate was calculated to be less than 0.005% (Figure 3). Alternatively, with a switch off-rate that is virtually 0, the minor differences observed at the transcription level may simply be due to experimental errors. Parallel to this, the two 3D7 sub-lines without prior freezing and thawing were assayed at every ~9th generation and similarly found VAR4 expression and transcription to be stable for over six months in continuous in vitro culture.


CD36 selection of 3D7 Plasmodium falciparum associated with severe childhood malaria results in reduced VAR4 expression.

Magistrado PA, Staalsoe T, Theander TG, Hviid L, Jensen AT - Malar. J. (2008)

Changes in VAR4 surface expression and transcription of the 3D7SM subline left to drift (3D7SM-drift). Left Y-axis, percentage of infected blood cells positive for VAR4 using flow cytometry and VAR4-specific CIDR1α (closed circle) and DBL5δ (open triangle) rabbit antibodies. Right Y-axis, percentage var4 gene transcription in ring stage (closed square) parasites calculated as: (var4 absolute copy number/∑copy number of all 3D7 var genes) × 100%. Fluorescence of 3D7SM-drift stained with pre-immune serum was used to separate positive cells from cells with background fluorescence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572619&req=5

Figure 3: Changes in VAR4 surface expression and transcription of the 3D7SM subline left to drift (3D7SM-drift). Left Y-axis, percentage of infected blood cells positive for VAR4 using flow cytometry and VAR4-specific CIDR1α (closed circle) and DBL5δ (open triangle) rabbit antibodies. Right Y-axis, percentage var4 gene transcription in ring stage (closed square) parasites calculated as: (var4 absolute copy number/∑copy number of all 3D7 var genes) × 100%. Fluorescence of 3D7SM-drift stained with pre-immune serum was used to separate positive cells from cells with background fluorescence.
Mentions: In the absence of CD36 selection VAR4 is stably expressed on the surface of 3D7SM-drift for 44 generations – equivalent to five rounds of gelatine flotation (Figure 1iii-A). To analyse this further 3D7UM and 3D7SM were cultured without gelatine flotation or CD36 selection and monitored VAR4 surface expression and var4 transcription at specified time points for 94 generations. To avoid assay-to-assay variation, RBC infected with the two parasite sub-lines were frozen down at each sampling point. Frozen iRBC were thawed and cultured for another six generations and then assayed on the same day. Almost no VAR4 surface expression and var4 gene transcription was seen in the parental 3D7UM, whereas 3D7SM-iRBC showed high and stable surface expression of VAR4 as well as gene transcription for at least 94 generations (Figure 3). Using the percentageVAR4 positive iRBC and the ring-stage var4 transcription level at generation six (first sampling point) and 94 (last sampling point) the switch off-rate was calculated to be less than 0.005% (Figure 3). Alternatively, with a switch off-rate that is virtually 0, the minor differences observed at the transcription level may simply be due to experimental errors. Parallel to this, the two 3D7 sub-lines without prior freezing and thawing were assayed at every ~9th generation and similarly found VAR4 expression and transcription to be stable for over six months in continuous in vitro culture.

Bottom Line: Also, unlike var genes from groups B and C, those from group A do not have sequences consistent with CD36 binding--a major cytoadhesion phenotype of P. falciparum isolates.A selection-induced increased adhesion of 3D7(SM) iRBC to CD36 resulted in a reduced var4 transcription and VAR4 surface expression.The current findings are consistent with the notion that CD36 adhesion is not associated with particular virulent parasite phenotypes, such as those believed to be exhibited by VAR4 expressing parasites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Medical Parasitology at the Institute of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. pam@immi.ku.dk

ABSTRACT

Background: A subset of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1(SM)) is involved in the cytoadherence of P. falciparum-infected red blood cells (iRBC) contributing to the pathogenesis of severe disease among young children in malaria endemic areas. The PfEMP1(SM) are encoded by group A var genes that are composed of a more constrained range of amino acid sequences than groups B and C var genes encoding PfEMP1(UM) associated with uncomplicated malaria. Also, unlike var genes from groups B and C, those from group A do not have sequences consistent with CD36 binding--a major cytoadhesion phenotype of P. falciparum isolates.

Methods: A 3D7 PfEMP1(SM) sub-line (3D7(SM)) expressing VAR4 (PFD1235w/MAL8P1.207) was selected for binding to CD36. The protein expression of this parasite line was monitored by surface staining of iRBC using VAR4-specific antibodies. The serological phenotype of the 3D7(SM) parasites was determined by flow cytometry using malaria semi-immune and immune plasma and transcription of the 59 var genes in 3D7 were analysed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using var-specific primers.

Results: A selection-induced increased adhesion of 3D7(SM) iRBC to CD36 resulted in a reduced var4 transcription and VAR4 surface expression.

Conclusion: VAR4 is not involved in CD36 adhesion. The current findings are consistent with the notion that CD36 adhesion is not associated with particular virulent parasite phenotypes, such as those believed to be exhibited by VAR4 expressing parasites.

Show MeSH
Related in: MedlinePlus