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.

Cysteine Protease-Binding Protein Family 6 Mediates the Trafficking of Amylases to Phagosomes in the Enteric Protozoan Entamoeba histolytica

“…We further demonstrated that CPBF1 is essential for the processing and lysosomal transport of EhCP-A5. On the other hand, CPBF8 binds to ␤-hexosaminidase ␣-subunit and lysozymes and transports them to phagosomes (19). We further showed that repression of CPBF8 by gene silencing decreased degradation of the Gram-positive bacillus Clostridium perfringens and in vitro cytopathic activity against mammalian cells.…”

“…Images were further analyzed using LSM510 software. We defined CPBF6-positive vacuoles based on criteria described elsewhere (19).…”

“…Furthermore, immuno-affinity pulldown of a putative M6PR (EHI_096320) failed to coprecipitate any cargo protein, suggesting that putative M6PRs are not likely receptors/carriers of lysosomal hydrolases (K. Nakada-Tsukui, unpublished data). We recently discovered a novel class of a single-transmembrane carrier/receptor family, designated the cysteine protease-binding protein family (CPBF), that specifically binds to various lysosomal hydrolytic enzymes and regulates their trafficking in E. histolytica (19,20). CPBF consists of 11 proteins (CPBF1 to -11) with significant mutual identity and structural conservation: the signal peptide at the amino terminus, a single transmembrane domain close to the carboxyl terminus, and the Yxx⌽ motif at the carboxyl terminus.…”

Entamoeba mitosomes play an important role in encystation by association with cholesteryl sulfate synthesis

“…The cell-growth assay was performed as described previously (36) except that the starting cell number was set at 3,000 trophozoites/mL, and growing cells were counted at 48, 72, and 96 h. Cytopathic Activity Assay. The level of destruction of CHO cell monolayers was assessed as described previously (37), with minor modifications. Briefly, confluent CHO cells prepared by overnight culture in 24-well plates at 37°C and in 5% CO 2 were washed with Opti-MEM (Life Technologies) containing 1 mg/mL ascorbic acid and 5 mg/mL cysteine.…”

“…The plates were incubated under anaerobic conditions at 37°C for 60 min. After the plates were placed on ice for 10 min to detach trophozoites from the CHO cell monolayer, the number of CHO cells remaining in each well was measured using WST-1 reagent (Roche Diagnostics) as described previously (37).…”