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Vol. 15, Issue 2, 411-419, February 2004

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Epithelial Cell Polarity Alters Rho-GTPase Responses to Pseudomonas aeruginosa

Barbara I. Kazmierczak ^* , Keith Mostov   ^||, and Joanne N. Engel ^* ¶ || #

^* Department of Medicine, University of California, San Francisco, San Francisco, California 94143; ^¶ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California 94143; Department of Anatomy, University of California, San Francisco, San Francisco, California 94143; Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ^|| Department of Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 94143

Submitted August 5, 2003; Revised October 3, 2003; Accepted October 7, 2003
Monitoring Editor: Suzanne Pfeffer

Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues. Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface. We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen. Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA. In contrast, P. aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation. There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly. Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases. Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P. aeruginosa infection. Nevertheless, polarized epithelial cells still sense the presence of apically infecting P. aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.

Abbreviations used: LatA, latrunculin A; LDH, lactate dehydrogenase; MOI, multiplicity of infection.

Present address: School of Medicine, Yale University, 333 Cedar St., New Haven CT 06520-8022.

^# Corresponding author. E-mail address: jengel{at}medicine.ucsf.edu.

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