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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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Affinity-Purified Anti-PATMK Antipeptide Antibodies Block Erythrophagocytosis by E. histolytica(A) Phagocytosis of calcium-treated or healthy erythrocytes by amebae was assayed in the presence of PBS (black), 50 μg/ml anti-Gal/GalNAc lectin light subunit (Lgl) (white), pre-immune (gray), 10 μg/ml anti-PATMK (horizontal hatch), 50 μg/ml anti-PATMK (vertical hatch), and 50 μg/ml anti-PATMK serum pre-absorbed with 25 μM of peptide (diagonal hatch). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to controls (*, p < 0.003 compared with pre-immune in M199s; #, p < 0.046 compared to pre-immune in 50 mM D-galactose; †, p < 0.002 compared to pre-immune in M199s [healthy erythrocytes], n = 6).(B) E. histolytica trophozoites interacting with CFSE-labeled erythrocytes were stained with pre-immune or anti-PATMK serum, magnified 100×.
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ppat-0040008-g004: Affinity-Purified Anti-PATMK Antipeptide Antibodies Block Erythrophagocytosis by E. histolytica(A) Phagocytosis of calcium-treated or healthy erythrocytes by amebae was assayed in the presence of PBS (black), 50 μg/ml anti-Gal/GalNAc lectin light subunit (Lgl) (white), pre-immune (gray), 10 μg/ml anti-PATMK (horizontal hatch), 50 μg/ml anti-PATMK (vertical hatch), and 50 μg/ml anti-PATMK serum pre-absorbed with 25 μM of peptide (diagonal hatch). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to controls (*, p < 0.003 compared with pre-immune in M199s; #, p < 0.046 compared to pre-immune in 50 mM D-galactose; †, p < 0.002 compared to pre-immune in M199s [healthy erythrocytes], n = 6).(B) E. histolytica trophozoites interacting with CFSE-labeled erythrocytes were stained with pre-immune or anti-PATMK serum, magnified 100×.

Mentions: Amebae were tested for their ability to ingest healthy or calcium-treated (apoptotic) erythrocytes following a 20 minute pre-incubation on ice in medium containing 50 μg/ml of anti-PATMK antibodies (Figure 4A). Anti-PATMK antibodies reduced the ingestion of calcium-treated (15.0% ± 4.0% vs. 30.7 ± 1.5%, p ≤ 0.003), as well as healthy erythrocytes (5.3% ± 2.5% vs. 22.0% ± 3.0%, p ≤ 0.002). Inhibition was also observed for the ingestion of calcium treated erythrocytes in the presence of 55 mM galactose (2.7% ± 2.1% vs. 12.7 ± 5.7, p ≤ 0.046). The effect of anti-PATMK antibodies could be reversed by pre-absorbing the antibodies against the antigen peptide at a concentration of 25 μM (Figure 4A). As expected, anti-Lgl antibodies (used as a control) had no affect on ingestion of either healthy or calcium-treated erythrocytes. We concluded that the ability of the anti-PATMK antibodies to block phagocytosis of not only healthy, but also calcium-treated, erythrocytes indicated a role for PATMK in erythrophagocytosis at a step after apoptotic killing of the red cell by the amebae.


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)

Affinity-Purified Anti-PATMK Antipeptide Antibodies Block Erythrophagocytosis by E. histolytica(A) Phagocytosis of calcium-treated or healthy erythrocytes by amebae was assayed in the presence of PBS (black), 50 μg/ml anti-Gal/GalNAc lectin light subunit (Lgl) (white), pre-immune (gray), 10 μg/ml anti-PATMK (horizontal hatch), 50 μg/ml anti-PATMK (vertical hatch), and 50 μg/ml anti-PATMK serum pre-absorbed with 25 μM of peptide (diagonal hatch). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to controls (*, p < 0.003 compared with pre-immune in M199s; #, p < 0.046 compared to pre-immune in 50 mM D-galactose; †, p < 0.002 compared to pre-immune in M199s [healthy erythrocytes], n = 6).(B) E. histolytica trophozoites interacting with CFSE-labeled erythrocytes were stained with pre-immune or anti-PATMK serum, magnified 100×.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211552&req=5

ppat-0040008-g004: Affinity-Purified Anti-PATMK Antipeptide Antibodies Block Erythrophagocytosis by E. histolytica(A) Phagocytosis of calcium-treated or healthy erythrocytes by amebae was assayed in the presence of PBS (black), 50 μg/ml anti-Gal/GalNAc lectin light subunit (Lgl) (white), pre-immune (gray), 10 μg/ml anti-PATMK (horizontal hatch), 50 μg/ml anti-PATMK (vertical hatch), and 50 μg/ml anti-PATMK serum pre-absorbed with 25 μM of peptide (diagonal hatch). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to controls (*, p < 0.003 compared with pre-immune in M199s; #, p < 0.046 compared to pre-immune in 50 mM D-galactose; †, p < 0.002 compared to pre-immune in M199s [healthy erythrocytes], n = 6).(B) E. histolytica trophozoites interacting with CFSE-labeled erythrocytes were stained with pre-immune or anti-PATMK serum, magnified 100×.
Mentions: Amebae were tested for their ability to ingest healthy or calcium-treated (apoptotic) erythrocytes following a 20 minute pre-incubation on ice in medium containing 50 μg/ml of anti-PATMK antibodies (Figure 4A). Anti-PATMK antibodies reduced the ingestion of calcium-treated (15.0% ± 4.0% vs. 30.7 ± 1.5%, p ≤ 0.003), as well as healthy erythrocytes (5.3% ± 2.5% vs. 22.0% ± 3.0%, p ≤ 0.002). Inhibition was also observed for the ingestion of calcium treated erythrocytes in the presence of 55 mM galactose (2.7% ± 2.1% vs. 12.7 ± 5.7, p ≤ 0.046). The effect of anti-PATMK antibodies could be reversed by pre-absorbing the antibodies against the antigen peptide at a concentration of 25 μM (Figure 4A). As expected, anti-Lgl antibodies (used as a control) had no affect on ingestion of either healthy or calcium-treated erythrocytes. We concluded that the ability of the anti-PATMK antibodies to block phagocytosis of not only healthy, but also calcium-treated, erythrocytes indicated a role for PATMK in erythrophagocytosis at a step after apoptotic killing of the red cell by the amebae.

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