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Epigenetic silencing of Plasmodium falciparum genes linked to erythrocyte invasion.

Cortés A, Carret C, Kaneko O, Yim Lim BY, Ivens A, Holder AA - PLoS Pathog. (2007)

Bottom Line: Silencing was clonally transmitted and occurred in the absence of detectable DNA alterations, suggesting that it is epigenetic.This was demonstrated for eba-140.Clonal variant expression of invasion-related ligands increases the flexibility of the parasite to adapt to its human host.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, Medical Research Council National Institute for Medical Research (NIMR), London, United Kingdom. acortes@pcb.ub.es

ABSTRACT
The process of erythrocyte invasion by merozoites of Plasmodium falciparum involves multiple steps, including the formation of a moving junction between parasite and host cell, and it is characterised by the redundancy of many of the receptor-ligand interactions involved. Several parasite proteins that interact with erythrocyte receptors or participate in other steps of invasion are encoded by small subtelomerically located gene families of four to seven members. We report here that members of the eba, rhoph1/clag, acbp, and pfRh multigene families exist in either an active or a silenced state. In the case of two members of the rhoph1/clag family, clag3.1 and clag3.2, expression was mutually exclusive. Silencing was clonally transmitted and occurred in the absence of detectable DNA alterations, suggesting that it is epigenetic. This was demonstrated for eba-140. Our data demonstrate that variant or mutually exclusive expression and epigenetic silencing in Plasmodium are not unique to genes such as var, which encode proteins that are exported to the surface of the erythrocyte, but also occur for genes involved in host cell invasion. Clonal variant expression of invasion-related ligands increases the flexibility of the parasite to adapt to its human host.

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Related in: MedlinePlus

Expression of Invasion-Related Genes in Subclones of 3D7-A(A) Analysis of expression of the rhoph1/clag family. Top, silver staining of RhopH components in concentrated, Albumax-free culture supernatants from 3D7-A, 3D7-B, and 11 subclones of 3D7-A resolved in 20 cm 6% SDS-PAGE. Middle, RT-PCR analysis from RNA of tightly synchronised schizonts. cDNA samples were the same across the four panels. The single copy rhoph2 gene was used to control the amount of stage-specific cDNA. The intensity of bands in left and right columns cannot be directly compared (in any of the panels) because they correspond to separate experiments. Bottom, same samples as in the top panel analysed by western blot with anti-Clag2 and anti-Clag3.2 antibodies.(B) Analysis of expression of members of the eba family. Top panels, RT-PCR analysis. The single-copy ama1 gene was used to control the amount of stage-specific cDNA. Lower panels, western blot analysis of schizont extracts with rat anti-EBA-140 antibodies. The same membranes were probed with rabbit anti-EBA-175 and mouse anti-AMA1 antibodies.(C) RT-PCR analysis of expression of genes of the pfRh family. The timing of expression of these genes is similar to that of ama1; thus, RT-PCR analysis of this gene in (B) controls the amount of stage-specific cDNA.(D) RT-PCR analysis of genes expressed at very different levels between 3D7-A and 3D7-B, and of other members of the acbp gene family: acbp gene in Chromosomes 8 (PF08_0099), 10 (PF10_0015 and PF10_0016), and 14 (PF14_0749). See Text S1 for the control of stage-specific cDNA.
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ppat-0030107-g004: Expression of Invasion-Related Genes in Subclones of 3D7-A(A) Analysis of expression of the rhoph1/clag family. Top, silver staining of RhopH components in concentrated, Albumax-free culture supernatants from 3D7-A, 3D7-B, and 11 subclones of 3D7-A resolved in 20 cm 6% SDS-PAGE. Middle, RT-PCR analysis from RNA of tightly synchronised schizonts. cDNA samples were the same across the four panels. The single copy rhoph2 gene was used to control the amount of stage-specific cDNA. The intensity of bands in left and right columns cannot be directly compared (in any of the panels) because they correspond to separate experiments. Bottom, same samples as in the top panel analysed by western blot with anti-Clag2 and anti-Clag3.2 antibodies.(B) Analysis of expression of members of the eba family. Top panels, RT-PCR analysis. The single-copy ama1 gene was used to control the amount of stage-specific cDNA. Lower panels, western blot analysis of schizont extracts with rat anti-EBA-140 antibodies. The same membranes were probed with rabbit anti-EBA-175 and mouse anti-AMA1 antibodies.(C) RT-PCR analysis of expression of genes of the pfRh family. The timing of expression of these genes is similar to that of ama1; thus, RT-PCR analysis of this gene in (B) controls the amount of stage-specific cDNA.(D) RT-PCR analysis of genes expressed at very different levels between 3D7-A and 3D7-B, and of other members of the acbp gene family: acbp gene in Chromosomes 8 (PF08_0099), 10 (PF10_0015 and PF10_0016), and 14 (PF14_0749). See Text S1 for the control of stage-specific cDNA.

Mentions: To determine whether further heterogeneity in the expression of invasion-related genes occurs in the cloned parasite line 3D7, we analysed expression of these genes in 11 subclones of 3D7-A (described in Materials and Methods). Silver-stained SDS-PAGE analysis of culture supernatants from these 11 subclones revealed that all of them expressed either Clag3.1 or Clag3.2, but none of them expressed both (Figure 4A, top panels). Each subclone had gone through at least 11 cycles of replication from a single parasite to harvesting for analysis. The subclones reflect the clonally transmitted expression pattern of the individual parasites from which they originated. This result indicates that 3D7-A is a mixture of parasites expressing one or the other protein. Mutually exclusive expression of the two genes was confirmed by semi-quantitative RT-PCR (Figure 4A, middle panels). All clones that expressed clag3.1 at high levels had only low-level expression of clag3.2 and vice versa. Furthermore, another member of the clag family, clag2, also showed clonal variant expression between the subclones, whereas clag8 and clag9 were expressed at very similar levels in all subclones (Figure 4A). Western blot analysis with antibodies specific for Clag2 and Clag3.2 confirmed that low levels of transcripts resulted in low abundance or absence of the corresponding proteins in culture supernatants (Figure 4A, bottom panels). Expression patterns remained stable over continuous culture for at least one additional month (Figure S1A). Interestingly, a stock of the cloned line HB3 at Ehime University (Japan) derived from HB3B, which had been passed through chimpanzees [13], expressed only clag3.1, but a stock at the same university derived from HB3A (prior to chimpanzee passage) expressed only clag3.2, supporting the idea of mutually exclusive expression and switching between the two genes (Figure S2). HB3 at the National Institute for Medical Research (United Kingdom) and W2mef lines only expressed clag3.1 at detectable levels (unpublished data).


Epigenetic silencing of Plasmodium falciparum genes linked to erythrocyte invasion.

Cortés A, Carret C, Kaneko O, Yim Lim BY, Ivens A, Holder AA - PLoS Pathog. (2007)

Expression of Invasion-Related Genes in Subclones of 3D7-A(A) Analysis of expression of the rhoph1/clag family. Top, silver staining of RhopH components in concentrated, Albumax-free culture supernatants from 3D7-A, 3D7-B, and 11 subclones of 3D7-A resolved in 20 cm 6% SDS-PAGE. Middle, RT-PCR analysis from RNA of tightly synchronised schizonts. cDNA samples were the same across the four panels. The single copy rhoph2 gene was used to control the amount of stage-specific cDNA. The intensity of bands in left and right columns cannot be directly compared (in any of the panels) because they correspond to separate experiments. Bottom, same samples as in the top panel analysed by western blot with anti-Clag2 and anti-Clag3.2 antibodies.(B) Analysis of expression of members of the eba family. Top panels, RT-PCR analysis. The single-copy ama1 gene was used to control the amount of stage-specific cDNA. Lower panels, western blot analysis of schizont extracts with rat anti-EBA-140 antibodies. The same membranes were probed with rabbit anti-EBA-175 and mouse anti-AMA1 antibodies.(C) RT-PCR analysis of expression of genes of the pfRh family. The timing of expression of these genes is similar to that of ama1; thus, RT-PCR analysis of this gene in (B) controls the amount of stage-specific cDNA.(D) RT-PCR analysis of genes expressed at very different levels between 3D7-A and 3D7-B, and of other members of the acbp gene family: acbp gene in Chromosomes 8 (PF08_0099), 10 (PF10_0015 and PF10_0016), and 14 (PF14_0749). See Text S1 for the control of stage-specific cDNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1937010&req=5

ppat-0030107-g004: Expression of Invasion-Related Genes in Subclones of 3D7-A(A) Analysis of expression of the rhoph1/clag family. Top, silver staining of RhopH components in concentrated, Albumax-free culture supernatants from 3D7-A, 3D7-B, and 11 subclones of 3D7-A resolved in 20 cm 6% SDS-PAGE. Middle, RT-PCR analysis from RNA of tightly synchronised schizonts. cDNA samples were the same across the four panels. The single copy rhoph2 gene was used to control the amount of stage-specific cDNA. The intensity of bands in left and right columns cannot be directly compared (in any of the panels) because they correspond to separate experiments. Bottom, same samples as in the top panel analysed by western blot with anti-Clag2 and anti-Clag3.2 antibodies.(B) Analysis of expression of members of the eba family. Top panels, RT-PCR analysis. The single-copy ama1 gene was used to control the amount of stage-specific cDNA. Lower panels, western blot analysis of schizont extracts with rat anti-EBA-140 antibodies. The same membranes were probed with rabbit anti-EBA-175 and mouse anti-AMA1 antibodies.(C) RT-PCR analysis of expression of genes of the pfRh family. The timing of expression of these genes is similar to that of ama1; thus, RT-PCR analysis of this gene in (B) controls the amount of stage-specific cDNA.(D) RT-PCR analysis of genes expressed at very different levels between 3D7-A and 3D7-B, and of other members of the acbp gene family: acbp gene in Chromosomes 8 (PF08_0099), 10 (PF10_0015 and PF10_0016), and 14 (PF14_0749). See Text S1 for the control of stage-specific cDNA.
Mentions: To determine whether further heterogeneity in the expression of invasion-related genes occurs in the cloned parasite line 3D7, we analysed expression of these genes in 11 subclones of 3D7-A (described in Materials and Methods). Silver-stained SDS-PAGE analysis of culture supernatants from these 11 subclones revealed that all of them expressed either Clag3.1 or Clag3.2, but none of them expressed both (Figure 4A, top panels). Each subclone had gone through at least 11 cycles of replication from a single parasite to harvesting for analysis. The subclones reflect the clonally transmitted expression pattern of the individual parasites from which they originated. This result indicates that 3D7-A is a mixture of parasites expressing one or the other protein. Mutually exclusive expression of the two genes was confirmed by semi-quantitative RT-PCR (Figure 4A, middle panels). All clones that expressed clag3.1 at high levels had only low-level expression of clag3.2 and vice versa. Furthermore, another member of the clag family, clag2, also showed clonal variant expression between the subclones, whereas clag8 and clag9 were expressed at very similar levels in all subclones (Figure 4A). Western blot analysis with antibodies specific for Clag2 and Clag3.2 confirmed that low levels of transcripts resulted in low abundance or absence of the corresponding proteins in culture supernatants (Figure 4A, bottom panels). Expression patterns remained stable over continuous culture for at least one additional month (Figure S1A). Interestingly, a stock of the cloned line HB3 at Ehime University (Japan) derived from HB3B, which had been passed through chimpanzees [13], expressed only clag3.1, but a stock at the same university derived from HB3A (prior to chimpanzee passage) expressed only clag3.2, supporting the idea of mutually exclusive expression and switching between the two genes (Figure S2). HB3 at the National Institute for Medical Research (United Kingdom) and W2mef lines only expressed clag3.1 at detectable levels (unpublished data).

Bottom Line: Silencing was clonally transmitted and occurred in the absence of detectable DNA alterations, suggesting that it is epigenetic.This was demonstrated for eba-140.Clonal variant expression of invasion-related ligands increases the flexibility of the parasite to adapt to its human host.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, Medical Research Council National Institute for Medical Research (NIMR), London, United Kingdom. acortes@pcb.ub.es

ABSTRACT
The process of erythrocyte invasion by merozoites of Plasmodium falciparum involves multiple steps, including the formation of a moving junction between parasite and host cell, and it is characterised by the redundancy of many of the receptor-ligand interactions involved. Several parasite proteins that interact with erythrocyte receptors or participate in other steps of invasion are encoded by small subtelomerically located gene families of four to seven members. We report here that members of the eba, rhoph1/clag, acbp, and pfRh multigene families exist in either an active or a silenced state. In the case of two members of the rhoph1/clag family, clag3.1 and clag3.2, expression was mutually exclusive. Silencing was clonally transmitted and occurred in the absence of detectable DNA alterations, suggesting that it is epigenetic. This was demonstrated for eba-140. Our data demonstrate that variant or mutually exclusive expression and epigenetic silencing in Plasmodium are not unique to genes such as var, which encode proteins that are exported to the surface of the erythrocyte, but also occur for genes involved in host cell invasion. Clonal variant expression of invasion-related ligands increases the flexibility of the parasite to adapt to its human host.

Show MeSH
Related in: MedlinePlus