Escherichia coli cytotoxic necrotizing factor 1 effaces microvilli and decreases transmigration of polymorphonuclear leukocytes in intestinal T84 epithelial cell monolayers

Abstract

Cytotoxic necrotizing factor type 1 (CNF1), a 110-kDa toxin-like protein from pathogenic Escherichia coli strains, induces an actin cytoskeleton reorganization consisting of the formation of prominent stress fibers by permanent activation of the small GTP-binding protein Rho. Since p21Rho regulates tight-junction permeability and perijunctional actin reorganization in epithelial intestinal cells (A. Nusrat, M. Giry, J. R. Turner, S. P. Colgan, C. A. Parkos, E. Lemichez, P. Boquet, and J. L. Madara, Proc. Natl. Acad. Sci. USA 92:10629-10633, 1995), we used polarized T84 epithelial intestinal cell monolayers to examine whether CNF1 could affect microvillus structure, transepithelial resistance, and polymorphonuclear leukocyte (PMN) transmigration. Incubation of T84 cells with CNF1 did not influence transepithelial resistance, suggesting that barrier function and surface polarity were not affected by the toxin. However, CNF1 effaced intestinal cell microvilli and induced a strong decrease of PMN transepithelial migration in either the luminal-to-basolateral or the basolateral-to-luminal direction. CNF1 could thus be a virulence factor exhibiting a new type of combined activity consisting of effacing of microvilli and occlusion of the epithelial barrier to PMNs. Attenuated transepithelial migration of PMNs could result in the enhanced growth and protection of luminal bacteria.

FIG. 1

F-actin and ZO-1 fluorescent staining of T84 cell monolayers incubated with or without CNF1. (A and C) F-actin contents of apical and basolateral faces, respectively, of control cells. (B and D) F-actin contents of apical and basolateral faces, respectively, of cells treated with CNF1 (10⁻¹⁰ M) for 16 h. The arrow in panel B shows the absence of F-actin microvilli, whereas the arrowhead in panel A shows F-actin present in microvilli. (E and F) Distribution of the junctional protein ZO-1 in T84 control cells and in T84 cells exposed to CNF1 (10⁻¹⁰ M) for 16 h, respectively. Bars, 10 μm.

FIG. 2

Electron microscopic study of T84 cell monolayers incubated in the presence or absence of CNF1. (A) Section of a T84 monolayer not treated with CNF1; (B) section of a T84 monolayer incubated with CNF1 (10⁻¹⁰ M) for 16 h. The arrows indicate positions of tight junctions. Bar, 5 μm.

FIG. 3

Measurements of transepithelial resistances in T84 cell monolayers after 10 and 16 h of incubation with either 1 × 10⁻¹⁰ M CNF1 or 2 × 10⁻³ mM EDTA. Incubation of confluent monolayers with CNF1 (open squares) showed no effect on transepithelial resistance during the course of incubation compared to results for controls (solid diamonds). Monolayers responded normally to EDTA (solid squares) by decreasing junctional resistances. Results are presented as means ± SEM for five experiments.

FIG. 4

Apical-to-basolateral transmigration of 2 × 10⁶ PMNs across confluent T84 monolayers in the presence (solid diamonds) or absence (open squares) of 10⁻¹⁰ M CNF1. Monolayers incubated with CNF1 demonstrated less of a decrease in the transepithelial resistance during the course of transmigration than did controls. Results are presented as means ± SEM for 6 to 12 monolayers.

FIG. 5

Myeloperoxidase assay of PMNs in the monolayers (black bars) and reservoirs (hatched bars) after 2 h of transmigration. Grey bars, total. T84 monolayers were incubated with no CNF1 (bars 0) or with 10⁻¹⁰ M (bars 1), 10⁻¹¹ M (bars 2), or 10⁻¹² M (bars 3) CNF1. (A) Transmigration of PMNs in the physiological (basolateral-to-apical) direction was diminished in a concentration-dependent manner in the presence of CNF1. (B) Transmigration of PMNs in the nonphysiological (apical-to-basolateral) direction was also attenuated in a concentration-dependent manner in monolayers incubated with CNF1. Data are pooled from 6 to 12 individual monolayers for each condition, and results are expressed as means ± SEM.

FIG. 6

Myeloperoxidase assay of PMNs in the monolayers (black bars) and reservoirs (hatched bars) after 2 h of transmigration (apical-to-basolateral direction). Grey bars, total. PMNs were incubated for 4 h prior to transmigration either without CNF1 (bars 0) or with 10⁻¹⁰ M (bars 1), 10⁻¹¹ M (bars 2), or 10⁻¹² M (bars 3) CNF1. Results are presented as means ± SEM for 6 to 12 monolayers.