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Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family.

Boettner DR, Huston CD, Linford AS, Buss SN, Houpt E, Sherman NE, Petri WA - PLoS Pathog. (2008)

Bottom Line: Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact.The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMK(delta932)).In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK), was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMK(delta932)). Expression of the carboxy-truncation of PATMK(delta932) also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

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PATMK Co-Localizes with Carboxylate-Modified 2.0-mm Beads during Phagocytosis(A) Anti-PATMK did not directly bind to beads in the absence of amebae. 2 μm carboxylate-modified fluorescent beads were stained with anti-PATMK and RPE-conjugated goat-anti-rabbit antibodies as described and representative images are shown.(B) Ingested beads did not co-localize with anti-rabbit IgG:PE in a permeabilized cell. Ameba were allowed to ingest beads, fixed and stained with RPE-conjugated goat-anti rabbit antibodies.(C) PATMK co-localized with an ingested bead at the surface of an unpermeabilized cell.(D) PATMK co-localized with ingested beads in a permeabilized cell. Amebae were allowed to ingest beads, left unpermeabilized (C) or permeabilized (D) and stained as described with anti-PATMK and secondary antibody.
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ppat-0040008-g003: PATMK Co-Localizes with Carboxylate-Modified 2.0-mm Beads during Phagocytosis(A) Anti-PATMK did not directly bind to beads in the absence of amebae. 2 μm carboxylate-modified fluorescent beads were stained with anti-PATMK and RPE-conjugated goat-anti-rabbit antibodies as described and representative images are shown.(B) Ingested beads did not co-localize with anti-rabbit IgG:PE in a permeabilized cell. Ameba were allowed to ingest beads, fixed and stained with RPE-conjugated goat-anti rabbit antibodies.(C) PATMK co-localized with an ingested bead at the surface of an unpermeabilized cell.(D) PATMK co-localized with ingested beads in a permeabilized cell. Amebae were allowed to ingest beads, left unpermeabilized (C) or permeabilized (D) and stained as described with anti-PATMK and secondary antibody.

Mentions: In order to understand the role PATMK played in ingestion, E. histolytica trophozoites were stained with anti-PATMK after ingestion of 2 μm carboxylate-modified fluorescent beads. To ensure that the PATMK antibody did not directly bind beads, we used identical methods to stain beads and bead containing cells and imaged the cells using the Amnis Imagestream imaging cytometer (Amnis Corporation; Seattle, WA). There was no evidence of non-specific binding of anti-PATMK to the carboxylate-modified beads (Figure 3A). Additionally, no staining was observed with secondary antibody alone (Figure 3B). However, the location of PATMK aggregates in both permeabilized and non-permeabilized trophozoites did correlate with that of the ingested bead (Figure 3C and 3D), suggesting that PATMK may directly interact with cargo during ingestion.


Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family.

Boettner DR, Huston CD, Linford AS, Buss SN, Houpt E, Sherman NE, Petri WA - PLoS Pathog. (2008)

PATMK Co-Localizes with Carboxylate-Modified 2.0-mm Beads during Phagocytosis(A) Anti-PATMK did not directly bind to beads in the absence of amebae. 2 μm carboxylate-modified fluorescent beads were stained with anti-PATMK and RPE-conjugated goat-anti-rabbit antibodies as described and representative images are shown.(B) Ingested beads did not co-localize with anti-rabbit IgG:PE in a permeabilized cell. Ameba were allowed to ingest beads, fixed and stained with RPE-conjugated goat-anti rabbit antibodies.(C) PATMK co-localized with an ingested bead at the surface of an unpermeabilized cell.(D) PATMK co-localized with ingested beads in a permeabilized cell. Amebae were allowed to ingest beads, left unpermeabilized (C) or permeabilized (D) and stained as described with anti-PATMK and secondary antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211552&req=5

ppat-0040008-g003: PATMK Co-Localizes with Carboxylate-Modified 2.0-mm Beads during Phagocytosis(A) Anti-PATMK did not directly bind to beads in the absence of amebae. 2 μm carboxylate-modified fluorescent beads were stained with anti-PATMK and RPE-conjugated goat-anti-rabbit antibodies as described and representative images are shown.(B) Ingested beads did not co-localize with anti-rabbit IgG:PE in a permeabilized cell. Ameba were allowed to ingest beads, fixed and stained with RPE-conjugated goat-anti rabbit antibodies.(C) PATMK co-localized with an ingested bead at the surface of an unpermeabilized cell.(D) PATMK co-localized with ingested beads in a permeabilized cell. Amebae were allowed to ingest beads, left unpermeabilized (C) or permeabilized (D) and stained as described with anti-PATMK and secondary antibody.
Mentions: In order to understand the role PATMK played in ingestion, E. histolytica trophozoites were stained with anti-PATMK after ingestion of 2 μm carboxylate-modified fluorescent beads. To ensure that the PATMK antibody did not directly bind beads, we used identical methods to stain beads and bead containing cells and imaged the cells using the Amnis Imagestream imaging cytometer (Amnis Corporation; Seattle, WA). There was no evidence of non-specific binding of anti-PATMK to the carboxylate-modified beads (Figure 3A). Additionally, no staining was observed with secondary antibody alone (Figure 3B). However, the location of PATMK aggregates in both permeabilized and non-permeabilized trophozoites did correlate with that of the ingested bead (Figure 3C and 3D), suggesting that PATMK may directly interact with cargo during ingestion.

Bottom Line: Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact.The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMK(delta932)).In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK), was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMK(delta932)). Expression of the carboxy-truncation of PATMK(delta932) also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

Show MeSH
Related in: MedlinePlus