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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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Analysis of EBA-140 in 3D7-A and 3D7-B(A) Western blot analysis of schizonts extracts (Sch.) or culture supernatants (S.n.) probed with rat anti-EBA-140 antibodies. The same membrane was re-probed with rabbit anti-EBA-175 antibodies to control for the amount of stage-specific material. The position of SeeBlue Plus2 pre-stained standards (Invitrogen) is shown (kDa).(B) Immunoprecipitation of NP-40-extracted schizonts and culture supernatants with anti-EBA-140 antibodies. The position of Precision Plus All Blue pre-stained standards (Bio-Rad, http://www.bio-rad.com/) is shown.(C) Erythrocyte binding assay with radiolabelled supernatants from 3D7-A and 3D7-B. The lane IP corresponds to 3D7-B supernatant immunoprecipitated with anti-EBA-140 antibodies.(D) IFA of 3D7-A and 3D7-B schizonts with rabbit anti-EBA-140 and mouse anti-EBA-175 antibodies. Middle and lower panels are representative of 93% and 7% of EBA-175-positive 3D7-A schizonts, respectively. Scale bar = 5 μm.
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ppat-0030107-g002: Analysis of EBA-140 in 3D7-A and 3D7-B(A) Western blot analysis of schizonts extracts (Sch.) or culture supernatants (S.n.) probed with rat anti-EBA-140 antibodies. The same membrane was re-probed with rabbit anti-EBA-175 antibodies to control for the amount of stage-specific material. The position of SeeBlue Plus2 pre-stained standards (Invitrogen) is shown (kDa).(B) Immunoprecipitation of NP-40-extracted schizonts and culture supernatants with anti-EBA-140 antibodies. The position of Precision Plus All Blue pre-stained standards (Bio-Rad, http://www.bio-rad.com/) is shown.(C) Erythrocyte binding assay with radiolabelled supernatants from 3D7-A and 3D7-B. The lane IP corresponds to 3D7-B supernatant immunoprecipitated with anti-EBA-140 antibodies.(D) IFA of 3D7-A and 3D7-B schizonts with rabbit anti-EBA-140 and mouse anti-EBA-175 antibodies. Middle and lower panels are representative of 93% and 7% of EBA-175-positive 3D7-A schizonts, respectively. Scale bar = 5 μm.

Mentions: eba-140 is the only gene among those expressed at very different levels between the two parasite lines that is known to participate in erythrocyte invasion [10], whereas pfg27/25 is known to have an important role in gametocyte development [11], and the function of acbp-14 is not known. We first aimed to determine whether lower eba-140 transcript abundance in 3D7-A compared to that of 3D7-B resulted in lower abundance of EBA-140 protein. Western blot and immunoprecipitation experiments revealed that EBA-140 was abundant both in schizont extracts and in culture supernatants of 3D7-B, but only present at very low levels in 3D7-A (Figure 2A and 2B). The multiple EBA-140-specific bands in Figure 2A and 2B correspond to proteolytic processing products. Furthermore, erythrocyte binding assays revealed a very different composition of proteins that bind to erythrocytes in supernatants from 3D7-A or 3D7-B (Figure 2C). A band of approximately 175 kDa with an identical mobility to EBA-175 (as determined by western blot on samples run side by side, unpublished data) and a band of approximately 152 kDa were observed for both 3D7-A and 3D7-B, but two strong bands with a mobility identical to two of the EBA-140-immunoprecipitated bands were observed only in 3D7-B.


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)

Analysis of EBA-140 in 3D7-A and 3D7-B(A) Western blot analysis of schizonts extracts (Sch.) or culture supernatants (S.n.) probed with rat anti-EBA-140 antibodies. The same membrane was re-probed with rabbit anti-EBA-175 antibodies to control for the amount of stage-specific material. The position of SeeBlue Plus2 pre-stained standards (Invitrogen) is shown (kDa).(B) Immunoprecipitation of NP-40-extracted schizonts and culture supernatants with anti-EBA-140 antibodies. The position of Precision Plus All Blue pre-stained standards (Bio-Rad, http://www.bio-rad.com/) is shown.(C) Erythrocyte binding assay with radiolabelled supernatants from 3D7-A and 3D7-B. The lane IP corresponds to 3D7-B supernatant immunoprecipitated with anti-EBA-140 antibodies.(D) IFA of 3D7-A and 3D7-B schizonts with rabbit anti-EBA-140 and mouse anti-EBA-175 antibodies. Middle and lower panels are representative of 93% and 7% of EBA-175-positive 3D7-A schizonts, respectively. Scale bar = 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1937010&req=5

ppat-0030107-g002: Analysis of EBA-140 in 3D7-A and 3D7-B(A) Western blot analysis of schizonts extracts (Sch.) or culture supernatants (S.n.) probed with rat anti-EBA-140 antibodies. The same membrane was re-probed with rabbit anti-EBA-175 antibodies to control for the amount of stage-specific material. The position of SeeBlue Plus2 pre-stained standards (Invitrogen) is shown (kDa).(B) Immunoprecipitation of NP-40-extracted schizonts and culture supernatants with anti-EBA-140 antibodies. The position of Precision Plus All Blue pre-stained standards (Bio-Rad, http://www.bio-rad.com/) is shown.(C) Erythrocyte binding assay with radiolabelled supernatants from 3D7-A and 3D7-B. The lane IP corresponds to 3D7-B supernatant immunoprecipitated with anti-EBA-140 antibodies.(D) IFA of 3D7-A and 3D7-B schizonts with rabbit anti-EBA-140 and mouse anti-EBA-175 antibodies. Middle and lower panels are representative of 93% and 7% of EBA-175-positive 3D7-A schizonts, respectively. Scale bar = 5 μm.
Mentions: eba-140 is the only gene among those expressed at very different levels between the two parasite lines that is known to participate in erythrocyte invasion [10], whereas pfg27/25 is known to have an important role in gametocyte development [11], and the function of acbp-14 is not known. We first aimed to determine whether lower eba-140 transcript abundance in 3D7-A compared to that of 3D7-B resulted in lower abundance of EBA-140 protein. Western blot and immunoprecipitation experiments revealed that EBA-140 was abundant both in schizont extracts and in culture supernatants of 3D7-B, but only present at very low levels in 3D7-A (Figure 2A and 2B). The multiple EBA-140-specific bands in Figure 2A and 2B correspond to proteolytic processing products. Furthermore, erythrocyte binding assays revealed a very different composition of proteins that bind to erythrocytes in supernatants from 3D7-A or 3D7-B (Figure 2C). A band of approximately 175 kDa with an identical mobility to EBA-175 (as determined by western blot on samples run side by side, unpublished data) and a band of approximately 152 kDa were observed for both 3D7-A and 3D7-B, but two strong bands with a mobility identical to two of the EBA-140-immunoprecipitated bands were observed only in 3D7-B.

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