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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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Related in: MedlinePlus

PATMK Is Expressed on the Surface of Trophozoites as a Type 1 Integral Membrane Protein(A) Western blot with affinity-purified anti-PATMK peptide antibodies was performed on amebic lysates in the absence (lane 1) or presence of 50-μM (lane 2) and 5-nM (lane 3) competing peptide, or pre-immune sera (lane 4).(B) Confocal microscopy of E. histolytica trophozoites stained with pre-immune or affinity purified anti-PATMK antibodies with or without cell permeabilization with 0.2% Triton X-100. Magnified 40×.
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ppat-0040008-g002: PATMK Is Expressed on the Surface of Trophozoites as a Type 1 Integral Membrane Protein(A) Western blot with affinity-purified anti-PATMK peptide antibodies was performed on amebic lysates in the absence (lane 1) or presence of 50-μM (lane 2) and 5-nM (lane 3) competing peptide, or pre-immune sera (lane 4).(B) Confocal microscopy of E. histolytica trophozoites stained with pre-immune or affinity purified anti-PATMK antibodies with or without cell permeabilization with 0.2% Triton X-100. Magnified 40×.

Mentions: Rabbit anti-serum against a peptide specific for the ectodomain of PATMK (EIQKQNPISTSLKISKISSD) (underlined in Figure 1A) revealed a single band at ∼140 kDa. This band disappeared when antibody was pre-absorbed against 50 μM but not 5 nM of the antigen peptide (Figure 2A). Affinity-purified anti-PATMK antibodies stained the surface of both permeabilized and non-permeabilized trophozoites, whereas pre-immune serum yielded little staining (Figure 2B). We concluded that PATMK was expressed in E. histolytica trophozoites as a plasma membrane protein of the expected mass and with the amino-terminus extra-cellular.


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 Is Expressed on the Surface of Trophozoites as a Type 1 Integral Membrane Protein(A) Western blot with affinity-purified anti-PATMK peptide antibodies was performed on amebic lysates in the absence (lane 1) or presence of 50-μM (lane 2) and 5-nM (lane 3) competing peptide, or pre-immune sera (lane 4).(B) Confocal microscopy of E. histolytica trophozoites stained with pre-immune or affinity purified anti-PATMK antibodies with or without cell permeabilization with 0.2% Triton X-100. Magnified 40×.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040008-g002: PATMK Is Expressed on the Surface of Trophozoites as a Type 1 Integral Membrane Protein(A) Western blot with affinity-purified anti-PATMK peptide antibodies was performed on amebic lysates in the absence (lane 1) or presence of 50-μM (lane 2) and 5-nM (lane 3) competing peptide, or pre-immune sera (lane 4).(B) Confocal microscopy of E. histolytica trophozoites stained with pre-immune or affinity purified anti-PATMK antibodies with or without cell permeabilization with 0.2% Triton X-100. Magnified 40×.
Mentions: Rabbit anti-serum against a peptide specific for the ectodomain of PATMK (EIQKQNPISTSLKISKISSD) (underlined in Figure 1A) revealed a single band at ∼140 kDa. This band disappeared when antibody was pre-absorbed against 50 μM but not 5 nM of the antigen peptide (Figure 2A). Affinity-purified anti-PATMK antibodies stained the surface of both permeabilized and non-permeabilized trophozoites, whereas pre-immune serum yielded little staining (Figure 2B). We concluded that PATMK was expressed in E. histolytica trophozoites as a plasma membrane protein of the expected mass and with the amino-terminus extra-cellular.

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