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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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Expression of Carboxy-Truncated PATMK Reduced Ingestion of Eythrocytes by E. histolytica(A) Two constructs were assembled by PCR and cloned behind the cysteine synthase promoter in the vector pEhEx and transfected into HM1:IMSS trophozoites: a full-length, carboxy Flag epitope tagged PATMK (PATMK1279), and a truncation at residue 932, with a carboxy Flag epitope tag (PATMK_932).(B) Amebic lysates (107 cells of PATMK1279, PATMK_932, or empty vector) were subjected to immunoprecipitation using anti-Flag resin. Proteins from the IP were separated on an 8% polyacrylamide gel, transferred to PVDF and blotted with anti-PATMK or pre-immune serum. (In every lane, the heavy chain from the immunoprecipitating antibody appears at ∼50 kDa).(C) Phagocytosis of calcium-treated erythrocytes by amebae expressing PATMK_932, PATMK1279, and empty vector controls were assayed in M199S (hatched bars) or M199S competed with 55 mM D-galactose (black bars). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to empty vector controls (*, p < 0.003, n = 6).(D) Amebic surface staining was performed on non-permeablized fixed E. histolytica trophozoites using pre-immune (bold line) or anti-Gal/GalNAc Hgl specific serum (thin line) and analyzed by flow cytometry.
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ppat-0040008-g006: Expression of Carboxy-Truncated PATMK Reduced Ingestion of Eythrocytes by E. histolytica(A) Two constructs were assembled by PCR and cloned behind the cysteine synthase promoter in the vector pEhEx and transfected into HM1:IMSS trophozoites: a full-length, carboxy Flag epitope tagged PATMK (PATMK1279), and a truncation at residue 932, with a carboxy Flag epitope tag (PATMK_932).(B) Amebic lysates (107 cells of PATMK1279, PATMK_932, or empty vector) were subjected to immunoprecipitation using anti-Flag resin. Proteins from the IP were separated on an 8% polyacrylamide gel, transferred to PVDF and blotted with anti-PATMK or pre-immune serum. (In every lane, the heavy chain from the immunoprecipitating antibody appears at ∼50 kDa).(C) Phagocytosis of calcium-treated erythrocytes by amebae expressing PATMK_932, PATMK1279, and empty vector controls were assayed in M199S (hatched bars) or M199S competed with 55 mM D-galactose (black bars). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to empty vector controls (*, p < 0.003, n = 6).(D) Amebic surface staining was performed on non-permeablized fixed E. histolytica trophozoites using pre-immune (bold line) or anti-Gal/GalNAc Hgl specific serum (thin line) and analyzed by flow cytometry.

Mentions: Because gene replacement is not currently possible in E. histolytica, we sought a fourth (in addition to shRNA knockdown, anti-PATMK antibodies and proteomics identification of PATMK in the early phagosome) independent approach to test the role of PATMK in erythrophagocytosis. PATMK1279 (full length carboxy-FLAG epitope-tagged PATMK), PATMKΔ932 (a truncated, carboxy-FLAG epitope-tagged PATMK), and empty vector control (Figure 6A) were stably transfected into trophozoites. Immunoprecipitations were performed using anti-FLAG binding resin, and Western blots revealed that both PATMK1279, and PATMKΔ932 were expressed (Figure 6B). Interestingly, the truncated form of PATMKΔ932 was co-immunoprecipitated with native PATMK, as indicated by western blots of the immunoprecipitate indicating interaction of the truncated and wild type proteins (Figure 6B, lane 3). The expression of the carboxy-terminal truncated form of PATMK reduced erythrophagocytosis by 67% (11.33 ± 2.52 vs. 34.33 ± 4.93, p ≤ 0.002), and by 81% in the presence of 55 mM D-galactose (3.33 ± 2.08 vs. 17.67 ± 3.06, p ≤ 0.003) compared with the empty vector control (Figure 6C). Expression of PATMK1279 had no statistical impact on erythrophagocytosis. To ensure that the expression of PATMKΔ932 did not dramatically alter the amebic surface, transfectants were stained with anti-Gal/GalNAc lectin heavy subunit polyclonal serum, and analyzed using flow cytometry (Figure 6D). There were no differences observed in lectin staining between any of the transfectants. We concluded that the ability of the truncated form of PATMK to inhibit erythrophagocytosis was consistent with all of the previous experiments implicating PATMK in ingestion of the dead red cell. The co-immunoprecipitation with anti-FLAG antibody of the carboxy-truncated PATMK with the native full-length PATMK suggested that the truncated protein was interfering with PATMK via a direct interaction.


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)

Expression of Carboxy-Truncated PATMK Reduced Ingestion of Eythrocytes by E. histolytica(A) Two constructs were assembled by PCR and cloned behind the cysteine synthase promoter in the vector pEhEx and transfected into HM1:IMSS trophozoites: a full-length, carboxy Flag epitope tagged PATMK (PATMK1279), and a truncation at residue 932, with a carboxy Flag epitope tag (PATMK_932).(B) Amebic lysates (107 cells of PATMK1279, PATMK_932, or empty vector) were subjected to immunoprecipitation using anti-Flag resin. Proteins from the IP were separated on an 8% polyacrylamide gel, transferred to PVDF and blotted with anti-PATMK or pre-immune serum. (In every lane, the heavy chain from the immunoprecipitating antibody appears at ∼50 kDa).(C) Phagocytosis of calcium-treated erythrocytes by amebae expressing PATMK_932, PATMK1279, and empty vector controls were assayed in M199S (hatched bars) or M199S competed with 55 mM D-galactose (black bars). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to empty vector controls (*, p < 0.003, n = 6).(D) Amebic surface staining was performed on non-permeablized fixed E. histolytica trophozoites using pre-immune (bold line) or anti-Gal/GalNAc Hgl specific serum (thin line) and analyzed by flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040008-g006: Expression of Carboxy-Truncated PATMK Reduced Ingestion of Eythrocytes by E. histolytica(A) Two constructs were assembled by PCR and cloned behind the cysteine synthase promoter in the vector pEhEx and transfected into HM1:IMSS trophozoites: a full-length, carboxy Flag epitope tagged PATMK (PATMK1279), and a truncation at residue 932, with a carboxy Flag epitope tag (PATMK_932).(B) Amebic lysates (107 cells of PATMK1279, PATMK_932, or empty vector) were subjected to immunoprecipitation using anti-Flag resin. Proteins from the IP were separated on an 8% polyacrylamide gel, transferred to PVDF and blotted with anti-PATMK or pre-immune serum. (In every lane, the heavy chain from the immunoprecipitating antibody appears at ∼50 kDa).(C) Phagocytosis of calcium-treated erythrocytes by amebae expressing PATMK_932, PATMK1279, and empty vector controls were assayed in M199S (hatched bars) or M199S competed with 55 mM D-galactose (black bars). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to empty vector controls (*, p < 0.003, n = 6).(D) Amebic surface staining was performed on non-permeablized fixed E. histolytica trophozoites using pre-immune (bold line) or anti-Gal/GalNAc Hgl specific serum (thin line) and analyzed by flow cytometry.
Mentions: Because gene replacement is not currently possible in E. histolytica, we sought a fourth (in addition to shRNA knockdown, anti-PATMK antibodies and proteomics identification of PATMK in the early phagosome) independent approach to test the role of PATMK in erythrophagocytosis. PATMK1279 (full length carboxy-FLAG epitope-tagged PATMK), PATMKΔ932 (a truncated, carboxy-FLAG epitope-tagged PATMK), and empty vector control (Figure 6A) were stably transfected into trophozoites. Immunoprecipitations were performed using anti-FLAG binding resin, and Western blots revealed that both PATMK1279, and PATMKΔ932 were expressed (Figure 6B). Interestingly, the truncated form of PATMKΔ932 was co-immunoprecipitated with native PATMK, as indicated by western blots of the immunoprecipitate indicating interaction of the truncated and wild type proteins (Figure 6B, lane 3). The expression of the carboxy-terminal truncated form of PATMK reduced erythrophagocytosis by 67% (11.33 ± 2.52 vs. 34.33 ± 4.93, p ≤ 0.002), and by 81% in the presence of 55 mM D-galactose (3.33 ± 2.08 vs. 17.67 ± 3.06, p ≤ 0.003) compared with the empty vector control (Figure 6C). Expression of PATMK1279 had no statistical impact on erythrophagocytosis. To ensure that the expression of PATMKΔ932 did not dramatically alter the amebic surface, transfectants were stained with anti-Gal/GalNAc lectin heavy subunit polyclonal serum, and analyzed using flow cytometry (Figure 6D). There were no differences observed in lectin staining between any of the transfectants. We concluded that the ability of the truncated form of PATMK to inhibit erythrophagocytosis was consistent with all of the previous experiments implicating PATMK in ingestion of the dead red cell. The co-immunoprecipitation with anti-FLAG antibody of the carboxy-truncated PATMK with the native full-length PATMK suggested that the truncated protein was interfering with PATMK via a direct interaction.

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