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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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Comparison of Proteins Secreted to the Culture Supernatant between 3D7-A and 3D7-B(A) Radiolabelled culture supernatants run on 20-cm-long 6% SDS-PAGE. The arrows indicate bands present in supernatants from 3D7-A but not from 3D7-B. The arrowhead indicates the band that forms a doublet with the 148-kDa band present only in 3D7-A.(B) Western blot of two lanes identical to those in (A) and run contiguously, probed with rabbit anti-PfRh2b antibodies.(C) Culture supernatants run side by side with identical supernatants immunoprecipitated with the monoclonal antibody 61.3 against RhopH2 (lanes IP). The position of the three members of the RhopH complex is indicated.(D) RT-PCR analysis of clag3.1 and clag3.2 in RNAs from 3D7-A and 3D7-B schizonts.
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ppat-0030107-g003: Comparison of Proteins Secreted to the Culture Supernatant between 3D7-A and 3D7-B(A) Radiolabelled culture supernatants run on 20-cm-long 6% SDS-PAGE. The arrows indicate bands present in supernatants from 3D7-A but not from 3D7-B. The arrowhead indicates the band that forms a doublet with the 148-kDa band present only in 3D7-A.(B) Western blot of two lanes identical to those in (A) and run contiguously, probed with rabbit anti-PfRh2b antibodies.(C) Culture supernatants run side by side with identical supernatants immunoprecipitated with the monoclonal antibody 61.3 against RhopH2 (lanes IP). The position of the three members of the RhopH complex is indicated.(D) RT-PCR analysis of clag3.1 and clag3.2 in RNAs from 3D7-A and 3D7-B schizonts.

Mentions: To detect differences in the expression of invasion-related proteins that might have escaped microarray analysis, we compared radiolabelled culture supernatants from 3D7-A and 3D7-B by SDS-PAGE. Two abundant bands were present in 3D7-A but not in 3D7-B (Figure 3A). A very high molecular mass band corresponds to a form of PfRh2b with an insertion that is present only in 3D7-A [12], as demonstrated by western blot with anti-PfRh2b antibodies on samples run side by side (Figure 3B). The other band has an electrophoretic mobility of approximately 148 kDa and forms a doublet with a band of slightly higher mobility. Because the size of these bands is similar to the size of RhopH1/Clag, we immunoprecipitated supernatants of the two lines with an anti-RhopH2 monoclonal antibody that immunoprecipitates the whole RhopH complex. The immunoprecipitated complex contained an additional band in 3D7-A with an identical mobility to the band present in supernatants of 3D7-A but not of 3D7-B, indicating that the protein present only in 3D7-A supernatants is part of the RhopH complex (Figure 3C). Mass spectrometry analysis revealed the identity of this polypeptide as Clag3.2 (PFC0110w) (25% coverage), whereas the lower band of the doublet (also present in 3D7-B, arrowhead in Figure 3A) was identified as Clag3.1 (PFC0120w) in both parasite lines (33% coverage). Despite the 95% identity between the two proteins, the identification was unambiguous because it was based on three peptides that were specific for one or the other protein (Table 1). As expected from these results, reverse transcriptase (RT)-PCR analysis revealed that clag3.1 transcripts are present at similar levels in 3D7-A and 3D7-B schizonts, but clag3.2 transcripts are almost absent in 3D7-B (Figure 3D).


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)

Comparison of Proteins Secreted to the Culture Supernatant between 3D7-A and 3D7-B(A) Radiolabelled culture supernatants run on 20-cm-long 6% SDS-PAGE. The arrows indicate bands present in supernatants from 3D7-A but not from 3D7-B. The arrowhead indicates the band that forms a doublet with the 148-kDa band present only in 3D7-A.(B) Western blot of two lanes identical to those in (A) and run contiguously, probed with rabbit anti-PfRh2b antibodies.(C) Culture supernatants run side by side with identical supernatants immunoprecipitated with the monoclonal antibody 61.3 against RhopH2 (lanes IP). The position of the three members of the RhopH complex is indicated.(D) RT-PCR analysis of clag3.1 and clag3.2 in RNAs from 3D7-A and 3D7-B schizonts.
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Related In: Results  -  Collection

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ppat-0030107-g003: Comparison of Proteins Secreted to the Culture Supernatant between 3D7-A and 3D7-B(A) Radiolabelled culture supernatants run on 20-cm-long 6% SDS-PAGE. The arrows indicate bands present in supernatants from 3D7-A but not from 3D7-B. The arrowhead indicates the band that forms a doublet with the 148-kDa band present only in 3D7-A.(B) Western blot of two lanes identical to those in (A) and run contiguously, probed with rabbit anti-PfRh2b antibodies.(C) Culture supernatants run side by side with identical supernatants immunoprecipitated with the monoclonal antibody 61.3 against RhopH2 (lanes IP). The position of the three members of the RhopH complex is indicated.(D) RT-PCR analysis of clag3.1 and clag3.2 in RNAs from 3D7-A and 3D7-B schizonts.
Mentions: To detect differences in the expression of invasion-related proteins that might have escaped microarray analysis, we compared radiolabelled culture supernatants from 3D7-A and 3D7-B by SDS-PAGE. Two abundant bands were present in 3D7-A but not in 3D7-B (Figure 3A). A very high molecular mass band corresponds to a form of PfRh2b with an insertion that is present only in 3D7-A [12], as demonstrated by western blot with anti-PfRh2b antibodies on samples run side by side (Figure 3B). The other band has an electrophoretic mobility of approximately 148 kDa and forms a doublet with a band of slightly higher mobility. Because the size of these bands is similar to the size of RhopH1/Clag, we immunoprecipitated supernatants of the two lines with an anti-RhopH2 monoclonal antibody that immunoprecipitates the whole RhopH complex. The immunoprecipitated complex contained an additional band in 3D7-A with an identical mobility to the band present in supernatants of 3D7-A but not of 3D7-B, indicating that the protein present only in 3D7-A supernatants is part of the RhopH complex (Figure 3C). Mass spectrometry analysis revealed the identity of this polypeptide as Clag3.2 (PFC0110w) (25% coverage), whereas the lower band of the doublet (also present in 3D7-B, arrowhead in Figure 3A) was identified as Clag3.1 (PFC0120w) in both parasite lines (33% coverage). Despite the 95% identity between the two proteins, the identification was unambiguous because it was based on three peptides that were specific for one or the other protein (Table 1). As expected from these results, reverse transcriptase (RT)-PCR analysis revealed that clag3.1 transcripts are present at similar levels in 3D7-A and 3D7-B schizonts, but clag3.2 transcripts are almost absent in 3D7-B (Figure 3D).

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