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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

Sequence and Domain Structure of PATMK (NCBI ID: XP_655593)(A) Amino acid sequence with the sequence used to produce anti-peptide antibodies underlined, and the boxed sequence showing the peptide found in 5- and 10-minute phagosome preparations.(B) Domains of PATMK. The transmembrane domain begins at amino acid 841.(C) Alignment of PATMK with Hank's consensus of conserved residues for serine/threonine or tyrosine kinase. Upper cased residues are conserved, and positions requiring any amino acid are denotated by “X,” whereas positions requiring hydrophobic residues are denoted by “O”.
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ppat-0040008-g001: Sequence and Domain Structure of PATMK (NCBI ID: XP_655593)(A) Amino acid sequence with the sequence used to produce anti-peptide antibodies underlined, and the boxed sequence showing the peptide found in 5- and 10-minute phagosome preparations.(B) Domains of PATMK. The transmembrane domain begins at amino acid 841.(C) Alignment of PATMK with Hank's consensus of conserved residues for serine/threonine or tyrosine kinase. Upper cased residues are conserved, and positions requiring any amino acid are denotated by “X,” whereas positions requiring hydrophobic residues are denoted by “O”.

Mentions: The amino acid sequence of PATMK (Figure 1A) predicted a 146 kDa protein, containing a 21 amino acid signal peptide sequence, an ectodomain containing 25 CXXC repeats, a 22 amino acid membrane spanning domain and an intracellular domain with the catalytic residues of a kinase (Figure 1B and 1C) [29]. The kinase specificity for PATMK could not be predicted given that its sequence contained the necessary residues for both serine/threonine and tyrosine kinase family members. Attempts to biochemically define the in vitro kinase activity by expressing the kinase domain in E. coli or by immunoprecipitating PATMK from trophozoites were unsuccessful (data not shown). Whether PATMK is a pseudokinase, as the lack of a conserved ATP-orienting glycine-rich motif suggests (domain I, Figure 1C), will require additional studies. We concluded that the presence of PATMK in the early phagosome proteome was consistent with it having a role in phagocytosis of the apoptotic corpse.


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)

Sequence and Domain Structure of PATMK (NCBI ID: XP_655593)(A) Amino acid sequence with the sequence used to produce anti-peptide antibodies underlined, and the boxed sequence showing the peptide found in 5- and 10-minute phagosome preparations.(B) Domains of PATMK. The transmembrane domain begins at amino acid 841.(C) Alignment of PATMK with Hank's consensus of conserved residues for serine/threonine or tyrosine kinase. Upper cased residues are conserved, and positions requiring any amino acid are denotated by “X,” whereas positions requiring hydrophobic residues are denoted by “O”.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040008-g001: Sequence and Domain Structure of PATMK (NCBI ID: XP_655593)(A) Amino acid sequence with the sequence used to produce anti-peptide antibodies underlined, and the boxed sequence showing the peptide found in 5- and 10-minute phagosome preparations.(B) Domains of PATMK. The transmembrane domain begins at amino acid 841.(C) Alignment of PATMK with Hank's consensus of conserved residues for serine/threonine or tyrosine kinase. Upper cased residues are conserved, and positions requiring any amino acid are denotated by “X,” whereas positions requiring hydrophobic residues are denoted by “O”.
Mentions: The amino acid sequence of PATMK (Figure 1A) predicted a 146 kDa protein, containing a 21 amino acid signal peptide sequence, an ectodomain containing 25 CXXC repeats, a 22 amino acid membrane spanning domain and an intracellular domain with the catalytic residues of a kinase (Figure 1B and 1C) [29]. The kinase specificity for PATMK could not be predicted given that its sequence contained the necessary residues for both serine/threonine and tyrosine kinase family members. Attempts to biochemically define the in vitro kinase activity by expressing the kinase domain in E. coli or by immunoprecipitating PATMK from trophozoites were unsuccessful (data not shown). Whether PATMK is a pseudokinase, as the lack of a conserved ATP-orienting glycine-rich motif suggests (domain I, Figure 1C), will require additional studies. We concluded that the presence of PATMK in the early phagosome proteome was consistent with it having a role in phagocytosis of the apoptotic corpse.

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