Novel transmembrane receptor involved in phagosome transport of lysozymes and β-hexosaminidase in the enteric protozoan Entamoeba histolytica

Abstract

Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes.

Figure 1. Localization of CPBF8 in E. histolytica.

(A) Phagosome localization of CPBF8. Amoebae were incubated with Cell Tracker Blue-stained CHO cells (blue) for 10 (top row) or 60 minutes (bottom row), fixed, and reacted with anti-HA antibody (green). Bar, 10 µm. (B) Lysosomes localization of CPBF8. Amoebae were labeled with LysoTracker Red (red) and subjected to immunofluorescence assay with anti-HA antibody (green). Bar, 10 µm. (C) Colocalization of EhPNT and CPBF8. The cells were fixed, and reacted with anti-EhPNT (red) and anti-HA antibody (green). Bar, 10 µm.

Figure 2. Isolation and identification of CPBF8-binding proteins.

Lysates of CPBF8-HA and control (“HA”) transformants were mixed with anti-HA-antibody-conjugated agarose, washed, and eluted with HA peptide. Immunoprecipitated samples were separated on SDS-PAGE and silver stained. Apparent molecular weight of standards (kDa) are indicated on the left. Six bands excised for protein identification are marked (A–F).

Figure 3. Specific repression of CPBF8 gene in CPBF8gs strain.

(A) RT-PCR analysis. A 200 bp long partial CPBF8 gene was amplified using cDNA from control and CPBF8gs strains. (B) DNA microarray analysis of CPBF genes (CPBF1-11). The raw fluorescence data of triplicates is shown.

Figure 4. Enzymatic activities in total lysates, culture supernatant, and phagosomes and immuno blot analysis of total lysates and phagosomes derived from control and CPBF8gs strains.

After the amoebas were incubated in fresh medium for 2 h, trophozoites and culture supernatant were separated by brief centrifugation. After the supernatant was removed (“sup”), the cell pellet was resuspended and solublized in lysis buffer (“ppt”). To isolate phagosomes (E), the amoebas were incubated with latex beads, and, after brief centrifugation, the cell pellet was resuspended, and mechanically homogenized. The phagosomes were isolated by ultracentrifugation on a sucrose gradient. Enzymatic activities of culture supernatant, whole lysate (A–D), and phagosomes (E) are shown. (A–D) Enzymatic activities of β-hexosaminidase activity toward MUGS (A) and MUG (B), lysozyme activity (C), and amylase activity (D) in the cell pellet and culture supernatant. (E) Enzymatic activities of β-hexosaminidase toward MUGS, lysozyme, and amylase in phagosomes. Data shown are the means ± standard deviations of three independent experiments. “*” or “**” represents statistical significance at p<0.01 or p<0.05, respectively. (F) Immuno blot analysis of the cell pellet and the phagosome fraction. Approximately 20 µg of the cell pellet and 2 µg of the phagosome fraction were electrophoresed. CPBF1 and CP5 are phagosomal proteins, while cysteine synthase 3 (CS3) is a cytosolic marker. Abbreviations are: ppt, pellet fraction; sup, culture supernatant; phagosome, phagosome fraction.

Figure 5. Effects of CPBF8 repression on the digestion of C. perfringens and destruction of CHO monolayers.

(A) Micrographic images of control and CPBF8gs strains coincubated with C. perfringens. Approximately 1.5×10⁴ cells of the control and CPBF8gs strains were incubated with 1.5×10⁶ C. perfringens, pretreated with 10 µM of SYTO-59. After 4 h co-incubation, amoebas were washed and microscopically examined. Bar, 10 µm. Arrows indicate representative round shape “deformed” or “damaged” C. perfringens. Arrowheads indicate representative rod-shaped “intact” C. perfringens. (B) Quantitative analysis of rod-shaped and round C. perfringens in the control and CPBF8gs strains. Data shown are the means ± standard deviations of 20 independent cells. (C, D) Kinetics of CHO cell destruction by the control and CPBF8gs strains. Approximately 5×10⁴ cells of the control and CPBF8gs strains, untreated (C) or pretreated with 200 µM of E-64 for 2 h and washed with PBS (D), were added to a monolayer of confluent CHO cells in 24 wells and incubated at 35°C for the indicated times. Data shown are the means ± standard deviations of four independent experiments. Monolayer destruction is expressed as the percentage of destroyed CHO cells. Data shown are the means ± standard deviations of four independent experiments.

Figure 6. The serine-rich region of CPBF8 is involved in the binding with β-hexosaminidase α-subunit and lysozymes.

(A) Schematic diagram of the serine-rich region and transmembrane domain in CPBF8. Numbers indicate amino acid positions from the amino terminus. The filled or hatched box depicts the serine rich region or the transmembrane domain, respectively. (B) Comparison of the carboxyl-terminal region of CPBF proteins. Boxes indicate the putative transmembrane domain. The serine-rich region is underlined. (C) The amino acid sequences of the wild-type and mutated serine-rich regions (SRR). Note that the entire SRR was deleted in CPBF8ΔSRR-HA. The first or second stretch of three serine residues within SRR were substituted with alanines in CPBF8AAA1-HA and CPBF8AAA2-HA, respectively. (D) Localization of CPBF8ΔSRR-HA to phagosomes. Amoebae were incubated with Cell Tracker Blue-stained CHO cells (blue) for 60 min, fixed, and reacted with anti-HA antibody (green). Bar, 10 µm. (E–F) Isolation and identification of binding proteins of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA. Lysates of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA transformants were mixed with anti-HA-antibody-conjugated agarose, washed, and eluted with HA peptide. Immunoprecipitated samples were separated on SDS-PAGE and silver stained (The upper and lower arrow indicated that β-hexosaminidase α-subunit and lysozymes, respectively. (E), or blotted and reacted with anti-HA, β-hexosaminidase α-subunit and lysozyme2 antibody (F).

Figure 7. Post-translational modification of CPBF8.

CPBF8-HA and CPBF8ΔSRR-HA was immunoprecipitated with anti-HA antibody from the lysates of CPBF8-HA and CPBF8ΔSRR-HA transformants, treated (+) or untreated (−) with TFA for 10 min. The samples were separated on SDS-PAGE and silver-stained (bottom panel), or blotted and reacted with anti-HA (top panel) or anti-lysozyme 2 antibody (middle panel). The apparent molecular weight (kDa) of standards are indicated on the left.