Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family

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.

Figure 1. 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”.

Figure 2. 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×.

Figure 3. PATMK Co-Localizes with Carboxylate-Modified 2.0-mm Beads during Phagocytosis

(A) Anti-PATMK did not directly bind to beads in the absence of amebae. 2 μm carboxylate-modified fluorescent beads were stained with anti-PATMK and RPE-conjugated goat-anti-rabbit antibodies as described and representative images are shown. (B) Ingested beads did not co-localize with anti-rabbit IgG:PE in a permeabilized cell. Ameba were allowed to ingest beads, fixed and stained with RPE-conjugated goat-anti rabbit antibodies. (C) PATMK co-localized with an ingested bead at the surface of an unpermeabilized cell. (D) PATMK co-localized with ingested beads in a permeabilized cell. Amebae were allowed to ingest beads, left unpermeabilized (C) or permeabilized (D) and stained as described with anti-PATMK and secondary antibody.

Figure 4. 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×.

Figure 5. 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).

Figure 6. 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.