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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 Short Hairpin RNA against PATMK Reduced PATMK Protein Levels and the Rate of Amebic Ingestion of Erythrocytes(A) Short hairpin RNAs (shRNA) were created using a two-step PCR strategy that utilized overlapping 3′ primers to create a hairpin loop controlled by an RNA polymerase III (U6) promoter.(B) The shRNAs corresponded to nucleotides 325–354 (325), 2273–2302 (2273), and 3552–3581 (3552), as well as a control which contained the same nucleotide makeup of 3552 in random order (scrambled).(C) Amebic cell lysate (105 cells per lane) was separated on an 8% SDS polyacrylamide gel, transferred to PVDF, and blotted with anti-PATMK or anti-Lgl serum (as a loading control).(D) Phagocytosis of calcium-treated erythrocytes by amebae transfected with the shRNAs were assayed in M199S (black bars) or M199S competed with 55 mM D-galactose (hatched bars). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to scrambled controls (*, p < 0.039, compared to Scrambled 3552; #, p < 0.021 compared with Scrambled 3552 in 50 mM D-galactose, n = 6).
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ppat-0040008-g005: Expression of Short Hairpin RNA against PATMK Reduced PATMK Protein Levels and the Rate of Amebic Ingestion of Erythrocytes(A) Short hairpin RNAs (shRNA) were created using a two-step PCR strategy that utilized overlapping 3′ primers to create a hairpin loop controlled by an RNA polymerase III (U6) promoter.(B) The shRNAs corresponded to nucleotides 325–354 (325), 2273–2302 (2273), and 3552–3581 (3552), as well as a control which contained the same nucleotide makeup of 3552 in random order (scrambled).(C) Amebic cell lysate (105 cells per lane) was separated on an 8% SDS polyacrylamide gel, transferred to PVDF, and blotted with anti-PATMK or anti-Lgl serum (as a loading control).(D) Phagocytosis of calcium-treated erythrocytes by amebae transfected with the shRNAs were assayed in M199S (black bars) or M199S competed with 55 mM D-galactose (hatched bars). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to scrambled controls (*, p < 0.039, compared to Scrambled 3552; #, p < 0.021 compared with Scrambled 3552 in 50 mM D-galactose, n = 6).

Mentions: In order to further address the role of PATMK in phagocytosis, RNA interference using a novel short hairpin RNA (shRNA) system was used to knock down its expression. ShRNAs were stably expressed in amebae using the E. histolytica U6 promoter (RNA polymerase III) (Figure 5A). ShRNAs were expressed to three regions of PATMK (Figure 5B). Knockdown of PATMK protein was seen upon expression of shRNA to two regions of PATMK, nucleotides 2273–2302, and 3552–3581, but not to a scrambled shRNA of identical composition to nucleotides 3552–3581 (Figure 5C). Knockdown of PATMK inhibited erythrophagocytosis: in the absence of galactose, ingestion of calcium-treated erythrocytes was statistically significantly reduced by 48.3% by the 3552–3581 corresponding hairpin (58.0% ± 9.8% vs. 30.0% ± 2.6%, p ≤ 0.0176) and 35.6% by the 2273–2302 hairpin (58.0% ± 9.8% vs. 37.3% ± 6.4%, p ≤ 0.0383) compared with the scrambled control. In the presence of 55 mM galactose, both constructs (2273 and 3552) reduced ingestion of calcium treated erythrocytes by more than 65% (22.0% ± 6.6% (scrambled 3552 control) vs. 5.7% ± 2.5% (3552) or 7.0% ± 2.6% (2273), p ≤ 0.02) (Figure 5D). The amino-terminus most construct (325–354) neither reduced PATMK protein levels nor had a significant affect on ingestion of calcium treated erythrocytes. No off-target effects on mRNA levels were observed with the shRNA technique (Table 2). We concluded both that shRNA is a promising technique for gene knockdown in E. histolytica, and that inhibition of the ingestion of calcium-treated erythrocytes by PATMK knockdown supported a role for PATMK in the ingestion of dead red cells.


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 Short Hairpin RNA against PATMK Reduced PATMK Protein Levels and the Rate of Amebic Ingestion of Erythrocytes(A) Short hairpin RNAs (shRNA) were created using a two-step PCR strategy that utilized overlapping 3′ primers to create a hairpin loop controlled by an RNA polymerase III (U6) promoter.(B) The shRNAs corresponded to nucleotides 325–354 (325), 2273–2302 (2273), and 3552–3581 (3552), as well as a control which contained the same nucleotide makeup of 3552 in random order (scrambled).(C) Amebic cell lysate (105 cells per lane) was separated on an 8% SDS polyacrylamide gel, transferred to PVDF, and blotted with anti-PATMK or anti-Lgl serum (as a loading control).(D) Phagocytosis of calcium-treated erythrocytes by amebae transfected with the shRNAs were assayed in M199S (black bars) or M199S competed with 55 mM D-galactose (hatched bars). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to scrambled controls (*, p < 0.039, compared to Scrambled 3552; #, p < 0.021 compared with Scrambled 3552 in 50 mM D-galactose, n = 6).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211552&req=5

ppat-0040008-g005: Expression of Short Hairpin RNA against PATMK Reduced PATMK Protein Levels and the Rate of Amebic Ingestion of Erythrocytes(A) Short hairpin RNAs (shRNA) were created using a two-step PCR strategy that utilized overlapping 3′ primers to create a hairpin loop controlled by an RNA polymerase III (U6) promoter.(B) The shRNAs corresponded to nucleotides 325–354 (325), 2273–2302 (2273), and 3552–3581 (3552), as well as a control which contained the same nucleotide makeup of 3552 in random order (scrambled).(C) Amebic cell lysate (105 cells per lane) was separated on an 8% SDS polyacrylamide gel, transferred to PVDF, and blotted with anti-PATMK or anti-Lgl serum (as a loading control).(D) Phagocytosis of calcium-treated erythrocytes by amebae transfected with the shRNAs were assayed in M199S (black bars) or M199S competed with 55 mM D-galactose (hatched bars). Data are reported as means ± SD. p Values were determined by a two-tailed t-test compared to scrambled controls (*, p < 0.039, compared to Scrambled 3552; #, p < 0.021 compared with Scrambled 3552 in 50 mM D-galactose, n = 6).
Mentions: In order to further address the role of PATMK in phagocytosis, RNA interference using a novel short hairpin RNA (shRNA) system was used to knock down its expression. ShRNAs were stably expressed in amebae using the E. histolytica U6 promoter (RNA polymerase III) (Figure 5A). ShRNAs were expressed to three regions of PATMK (Figure 5B). Knockdown of PATMK protein was seen upon expression of shRNA to two regions of PATMK, nucleotides 2273–2302, and 3552–3581, but not to a scrambled shRNA of identical composition to nucleotides 3552–3581 (Figure 5C). Knockdown of PATMK inhibited erythrophagocytosis: in the absence of galactose, ingestion of calcium-treated erythrocytes was statistically significantly reduced by 48.3% by the 3552–3581 corresponding hairpin (58.0% ± 9.8% vs. 30.0% ± 2.6%, p ≤ 0.0176) and 35.6% by the 2273–2302 hairpin (58.0% ± 9.8% vs. 37.3% ± 6.4%, p ≤ 0.0383) compared with the scrambled control. In the presence of 55 mM galactose, both constructs (2273 and 3552) reduced ingestion of calcium treated erythrocytes by more than 65% (22.0% ± 6.6% (scrambled 3552 control) vs. 5.7% ± 2.5% (3552) or 7.0% ± 2.6% (2273), p ≤ 0.02) (Figure 5D). The amino-terminus most construct (325–354) neither reduced PATMK protein levels nor had a significant affect on ingestion of calcium treated erythrocytes. No off-target effects on mRNA levels were observed with the shRNA technique (Table 2). We concluded both that shRNA is a promising technique for gene knockdown in E. histolytica, and that inhibition of the ingestion of calcium-treated erythrocytes by PATMK knockdown supported a role for PATMK in the ingestion of dead red cells.

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