Expression of Epithelial Membrane Protein 2 (EMP–2) Controls Chlamydia Infectivity

Abstract

Abstract: : Purpose: Epithelial membrane protein–2 (EMP2), a 4–transmembrane protein, is widely expressed in the eye, reproductive tract, and lung, associates with lipid rafts and is believed to play an important role in trafficking of specific cell surface proteins. Recently, infectivity of Chlamydia, which is responsible for significant ocular morbidity, has been variably linked to lipid raft components. In this study we investigate the possible role for EMP2 in an in vitro Chlamydia infection model. Methods: An epithelial cell line with increased EMP2 expression was created through stable transfection and selection. The control cell line expressed the vector alone. Two different epithelial cell lines were created with decreased EMP2 expression: one with an EMP–2 specific ribozyme and the other which expresses siRNA. These different cell lines were used in 24 hour infection studies with two different Chlamydia preparations (Human serovar strains D–K and mouse strain MoPn). Chlamydia inclusion bodies were identified using a Chlamydia–specific antibody and fluorescein–conjugated secondary antibody. Evans Blue was used as a counterstain to identify the total number of cells. Inclusion bodies were quantified using an Olympus Provis AX70 confocal fluorescent microscope. A total of 500–1000 cells were counted by both fluorescence and light microscopy in random fields and the percent infection was calculated. Additional experiments used an antibody against the second extracellular loop of EMP2 to determine if this would block infection. Results: Chlamydia infectivity correlated with the levels of EMP2 expression. The EMP2 over–expressing cell–line showed a marked increase in the Chlamydia infection rate, as compared to control group. This increase ranged from 20 to 40% and was statistically significant (P<0.05). Cells with decreased EMP2 expression, both through the ribozyme and siRNA, showed decreased Chlamydia infection (P<0.05). Utilization of antibody against EMP2 successfully blocked infection (P<0.05). Conclusions: This data suggests that EMP2 may play an important role in the pathogenesis of chlamydial infections. Elucidation of specific mechanisms of early chlamydial infectivity may reveal potential areas for therapeutic intervention or strategies for vaccine development.

Keywords: bacterial disease • conjunctiva