Contact-dependent transfer of the galactose-specific lectin of Entamoeba histolytica to the lateral surface of enterocytes in culture

Abstract

In a study to investigate early interactions of Entamoeba histolytica with epithelial cell monolayers, we found that a monoclonal antibody (MAb), CD6, against an ameba surface antigen recognized the lateral surface of epithelial cells after coculture with trophozoites. Display of the CD6 antigen on the epithelial cells necessitated contact with active trophozoites. It was found neither at 4؇C, nor with prefixed trophozoites, nor with trophozoite-conditioned media, nor when a filter prevented direct contact. Monolayers exposed to amebic sonicates or detergent lysates showed random immunostaining. Access to the antigenic site was limited, as immunostaining occurred predominantly with subconfluent monolayers. CD6 epithelial cell binding was first observed after 5 min of coculture; trophozoite-mediated target cell lysis was not detected until 15 min following coculture. Epithelial cell immunostaining occurred with some other ameba-specific antibodies but not with MAbs raised against the 170-kDa subunit of the galactose-N-acetylgalactosamine (Gal/GalNAc)-specific lectin. The CD6 MAb as well as some other ameba-specific antibodies immunoprecipitated from trophozoite lysates the same bands as the MAbs against the cysteine-rich domain of the 170-kDa subunit of the Gal/GalNAcspecific lectin. Why the latter MAbs failed to stain epithelial cells in the vicinity of attached trophozoites is presently unknown. We concluded that E. histolytica trophozoites transferred the intact amebic Gal/GalNAcspecific lectin or a portion of it to the lateral surface of epithelial cells. This juxtacrine transfer preceded killing of target cells. MATERIALS AND METHODSTrophozoites. Trophozoites of E. histolytica HM1:IMSS (provided by H. Vermeersch, Laboratory of Pharmaceutical Technology, University of Ghent, Ghent, Belgium) were grown axenically at 35.5ЊC in 15-ml screw-cap tubes with TYI-S-33 medium (6), supplemented with 500 g of piperacillin per ml and 125 g of amikacin per ml. Trophozoites were harvested at the end of the logarithmic growth phase (72 h) by chilling at 4ЊC. They were concentrated at 200 ϫ g for 5 min and used immediately. For some experiments, freshly harvested trophozoites were fixed for 20 min in 3% paraformaldehyde (PF), washed in phosphatebuffered saline (PBS; pH 7.4) containing 0.9 mM Ca 2ϩ and 0.334 mM Mg 2ϩ (PBS Dϩ ), and quenched for 10 min with NH 4 Cl (50 mM in PBS E [Eisen formulation]). Thereafter, they were handled in the same way as live trophozoites. Conditioned medium was prepared from (i) logarithmic-growth-phase cultures in TYI-S-33; (ii) TYI-S-33, with or without serum, that had been incubated for 1 h with the total number of cells from an equal volume of a logarithmic-phase culture; and (iii) cocultures of trophozoites with mammalian cells as described below. Trophozoite cell extracts were prepared by one of the following methods: (i) intermittent sonication (Vibra Cell VC50; Sonics & Materials, Danbury, Conn.) of 2 ϫ 10 6 live trophozoites in 0.5 ml of PBS Dϩ on ice for a total of 45 s or (ii) vortexing...

Entamoeba histolytica disturbs the tight junction complex in human enteric T84 cell layers

“…It is clear that the tight junction complex is localized apically from the CD6 signal that reveals the transferred lectin. The latter immunosignal rather matches the honeycomb-like staining pattern of E-cadherin, the transmembrane molecule of the adherens junction complex situated basally from the tight junction complex as illustrated previously (4).…”

“…Neither could the effect of trophozoites on TER be neutralized by treatment with any of the following protease inhibitors at concentrations nontoxic for trophozoites (4) and not affecting TER in control cultures: N-ethylmaleimide at 20 M; aprotinin at 1 g/ml; E-64 at 200 M; soybean trypsin inhibitor at 1 mg/ml (our unpublished results). These results and the intact patterns of occludin in immunoblots argue against specific proteolytic attack as described for the Der p1 allergen from fecal pellets of the house dust mite (40).…”

“…In a previous study (4) we found that the amebic 170 kDa subunit of the Gal/GalNAc-specific lectin was transferred to the enteric cell layer and concentrated at enteric cell-cell borders as evidenced by confocal laser scanning after immunostaining with the monoclonal antibody CD6. To find out whether or not tight junction proteins may serve as binding sites for the transferred lectin, we did double immunostaining with antibodies against molecules of the tight junction complex and with the CD6 antibody (data not shown).…”

“…Is there a place for the Gal/GalNAc-specific lectin in such pathway? This multifunctional amebic lectin is also involved in attachment of the trophozoites to the enteric cells (41) and it is transferred to areas of contact between enteric cell (4). The enteric receptor of this lectin, however, is still unknown.…”

“…Its onset is marked by invasion of trophozoites into the enteric mucosa (1). A major role in the pathogenesis of invasive amebiasis has been attributed to the multifunctional Gal/GalNAc-specific amebic lectin, which also mediates trophozoite adhesion and is transferred from the trophozoites to the enterocytes (2)(3)(4). Clinical manifestations of the disease range from mild diarrhea to severe dysentery, due mainly to loss of the barrier function of the intestinal mucosa.…”