Adherence of Giardia lamblia trophozoites to Int-407 human intestinal cells

Abstract

Attachment of Giardia lamblia trophozoites to enterocytes is essential for colonization of the small intestine and is considered a prerequisite for parasite-induced enterocyte dysfunction and clinical disease. In this work, coincubation of Giardia with Int-407 cells, was used as an in vitro model to study the role of cytoskeleton and surface lectins involved in the attachment of the parasite. This interaction was also studied by scanning and transmission electron microscopy. Adherence was dependent on temperature and was maximal at 37 degrees C. It was reduced by 2.5 mM colchicine (57%), mebendazole (10 microg/ml) (59%), 100 mM glucose (26%), 100 mM mannose (22%), 40 mM mannose-6-phosphate (18%), and concanavalin A (100 microg/ml) (21%). No significant modification was observed when Giardia was pretreated with cytochalasins B and D and with EDTA. Giardia attachment was also diminished by preincubating Int-407 cells with cytochalasin B and D (5 microg/ml) (16%) and by glutaraldehyde fixation of intestinal cells and of G. lamblia trophozoites (72 and 100%, respectively). Ultrastructural studies showed that Giardia attaches to the Int-407 monolayer predominantly by its ventral surface. Int-407 cells contact trophozoites with elongated microvilli, and both trophozoite imprints and interactions of Giardia flagella with intestinal cells were also observed. Transmission electron microscopy showed that Giardia lateral crest and ventrolateral flange were important structures in the adherence process. Our results suggest a combination of mechanical and hydrodynamic forces in trophozoite attachment; surface lectins also seem to mediate binding and may be involved in specific recognition of host cells.

FIG. 1

Time course of attachment of G. lamblia trophozoites to Int-407 cell monolayers under standard assay conditions (cell ratio 1:1, 37°C, 5% CO₂, and 95% air). Standard errors of the mean (vertical bars) were calculated from data of three experiments (P < 0.01).

FIG. 2

Scanning electron micrographs of G. lamblia attached to Int-407 monolayers. Trophozoites were incubated with Int-407 cells for 2 h (a, c, d, and e) or 24 h (b and f). (a) Most Giardia trophozoites are attached on the Int-407 cell surface, with the ventral disk in contact with the intestinal cell. Note the close interaction of trophozoite flagella with intestinal cells (arrows). (b) Attachment of G. lamblia trophozoites with the dorsal surface. The fimbrial extensions of Int-407 contact the VLF (arrow). (c) The fimbrial extensions (microvilli) of Int-407 cells contact and surround the trophozoite, and the caudal and posterolateral flagella also interact with Int-407 cells (arrows). (d to f) Note the circular imprints of Giardia, after trophozoite detachment, on intestinal cells (arrows).

FIG. 3

Transmission electron micrographs showing the interactions of G. lamblia trophozoites with Int-407 cells after staining with uranyl and citrate leads. (A) The lateral crest was applied to the substrate and the VLF contacts laterally with the Int-407 cell surface plasma membrane. (B and C) In some cases the VLF indented into the fimbrial extension of intestinal cells (arrow). (D) Some extracelular fimbrial extensions of Int-407 cells surrounding the parasite were grossly elongated (arrow). (A to C) Note the arched ventral disk in attached trophozoites. Magnification: ×11,475 (A), ×13,140 (B), ×12,150 (C), ×33,300 (D). Abbreviations. N, nucleus; AD, adhesive disk; LC, lateral crest.

FIG. 3

Transmission electron micrographs showing the interactions of G. lamblia trophozoites with Int-407 cells after staining with uranyl and citrate leads. (A) The lateral crest was applied to the substrate and the VLF contacts laterally with the Int-407 cell surface plasma membrane. (B and C) In some cases the VLF indented into the fimbrial extension of intestinal cells (arrow). (D) Some extracelular fimbrial extensions of Int-407 cells surrounding the parasite were grossly elongated (arrow). (A to C) Note the arched ventral disk in attached trophozoites. Magnification: ×11,475 (A), ×13,140 (B), ×12,150 (C), ×33,300 (D). Abbreviations. N, nucleus; AD, adhesive disk; LC, lateral crest.