Apical spore phagocytosis is not a significant route of infection of differentiated enterocytes by Encephalitozoon intestinalis

Abstract

Encephalitozoon intestinalis is a microsporidian species that infects the intestinal mucosal epithelium, primarily in immunodeficient individuals. The present study employed undifferentiated and differentiated human colonic carcinoma cell lines to determine if this parasite species infected polarized epithelial cells by spore phagocytosis or by impalement with the deployed spore polar tube. Apical surface spore attachment differed between cell lines such that SW480>HT-29>Caco-2>HCT-8, with attachment being greater to undifferentiated Caco-2 cells than differentiated cells and greater to partially differentiated HCT-8 cells than differentiated HCT-8 cells. Attachment was inhibited by chondroitin sulfate A, suggesting that it was mediated by host cell sulfated glycoaminoglycans. Infection rates 3 days postinfection paralleled spore attachment in the various cell lines. The undifferentiated cell line SW480 and undifferentiated Caco-2 and HCT-8 cells exhibited modest spore phagocytosis while the more differentiated cell line HT29 and differentiated Caco-2 and HCT-8 cells did not. All cell lines were impaled by the polar tubes of germinating spores. When normalized to the number of spores attached to the apical membrane, such impalement was greatest in the more differentiated Caco-2 and HCT-8 cells. The host cell apical surface influenced parasite spore germination, as in populations of large undifferentiated Caco-2 cells to which >3 spores had attached, the frequency distribution of the percentages of spores germinated per cell was bimodal, indicating that the surface of some cells favored germination, while others did not. This study suggests that phagocytosis is not a biologically significant mode of infection in differentiated intestinal epithelial cells.

FIG. 1.

Immunofluorescent images of undifferentiated Caco-2 cells showing host cell tight junction ZO-1 (green), extracellular E. intestinalis stages (yellow), and intracellular E. intestinalis stages (red). (A) Cells infected for 5 days showing intracellular parasite stages within parasitophorous vacuoles (arrows). (B) Cells exposed to spores for 4 h showing multiple spores attached to the cell surface and one phagocytized spore (arrow). (C) Cells exposed to spores for 4 h showing four spores, three of which have clearly germinated and impaled a large undifferentiated cell (arrowheads). Extracellular spore and polar tube (yellow) can be distinguished from intracellular segments of the polar tube and sporoplasm (red). Bar, 2 μm.

FIG. 2.

Scanning electron microscope images of the apical surface of HCT-8 and Caco-2 cells categorized on the basis of x-y surface area and location on the monolayer as undifferentiated, intermediate, and differentiated cells. The density of microvilli increased in both cell lines as the cells became more columnar and differentiated. Bar, 1 μm.

FIG. 3.

Spore attachment, spore germination, host cell impalement by germinating spores (hatched bars), and host cell spore phagocytosis (solid bars) in cultures of SW480 and HT-29 cells exposed to E. intestinalis spores for 4 h. Different from SW480: *, P < 0.05.

FIG. 4.

Spore attachment, spore germination, host cell impalement by germinating spores (hatched bars), and host cell spore phagocytosis (solid bars) in fields of undifferentiated, intermediate, and differentiated Caco-2 cells exposed to E. intestinalis spores for 4 h. Different from undifferentiated group: *, P < 0.05; **, P < 0.01.

FIG. 5.

Spore attachment, spore germination, host cell impalement by germinating spores (hatched bars), and host cell spore phagocytosis (solid bars) in fields of undifferentiated, intermediate, and differentiated HCT-8 cells exposed to E. intestinalis spores for 4 h. Different from other group(s): *, P < 0.01; **, P < 0.01.

FIG. 6.

Percentage of undifferentiated Caco-2 cells on which germinated spores were either none (solid bars) or all (hatched bars) of the attached spores. Numbers represent the number of cells to which three, four, five, six, seven, or eight spores had attached.

FIG. 7.

Scanning electron microscopic image of a germinated E. intestinalis spore impaling an undifferentiated Caco-2 cell (arrow). Bar, 1 μm.