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. 2004 Feb;15(2):411-9.
doi: 10.1091/mbc.e03-08-0559. Epub 2003 Oct 31.

Epithelial cell polarity alters Rho-GTPase responses to Pseudomonas aeruginosa

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Epithelial cell polarity alters Rho-GTPase responses to Pseudomonas aeruginosa

Barbara I Kazmierczak et al. Mol Biol Cell. 2004 Feb.

Abstract

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.

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Figures

Figure 1.
Figure 1.
LatA and Toxin B have different effects on P. aeruginosa entry into nonpolarized versus polarized epithelial cells. (A and B) HeLa cells were infected with PA103::pscJTn5 (MOI 10-20) for 2 h. The number of internalized bacteria per well was determined as described in MATERIALS AND METHODS. Bars represent the mean ± SD. of triplicate samples from a representative experiment. (C and D) MDCK cells were plated on 12-mm Transwell filters at 1.5 × 106 cells/well 3 d before infection with PA103::pscJTn5, Invasin-expressing E. coli (MC4100 pRI203), or S. typhimurium (SL1344) for 2 h. Bars show the mean ± SD of triplicate samples from a representative experiment. In some instances, error bars are too small to see on the log scale used in these graphs. (A and C) Latrunculin A or DMSO was added to cells 30 min before addition of bacteria and was present for the 2 h of cocultivation with bacteria. (B and D) Cells were pretreated with Toxin B for 4 h before the addition of bacteria.
Figure 2.
Figure 2.
Sensitivity to inhibitors of actin polymerization is affected by cell polarity. MDCK cells were plated on 12-mm Transwell filters at 1.5 × 106 cells/well and allowed to polarize for 1 or 3 d. Cells were pretreated with LatA for 30 min (A) or Toxin B for 4 h (B) before infection with PA103::pscJTn5 at an MOI of 10-20 for 2 h. Bars show mean ± SD of triplicate samples from a representative experiment. In some instances, error bars are too small to see on the log scale used in these graphs.
Figure 5.
Figure 5.
Increasing the number of bacteria bound to polarized MDCK monolayers does not lead to increased bacterial internalization nor to RhoA activation. (A and B) MDCK cells were plated to 12-mm Transwell filters at 1.5 × 106 cells/well and allowed to polarize for 1 or 3 d before infection with PA103ΔUΔT at MOIs of 10-20 (1×), 40-60 (3×), or 100-150 (10×). All determinations were carried out in quadruplicate; bars indicate the average number of adherent or internalized organisms (±SD). In some instances, error bars are too small to see on the log scale used in these graphs. (C) MDCK cells were plated to 7-cm Transwell filters at confluent density (2.0 × 107 cells/dish) and allowed to polarize for 3 d before infection with PA103ΔUΔT at an MOI of ∼200 (10×). RhoA-GTP and total RhoA were determined as described previously at various times postinfection. The gel shown is representative of four independent determinations.
Figure 3.
Figure 3.
Basolateral internalization decreases with increasing MDCK polarity and becomes insensitive to Toxin B inhibition. MDCK cells were plated on 12-mm Transwell filters (3-μm pore size) at confluent density (1.5 × 106 cells/well). PA103::pscJTn5 was grown overnight in LB with shaking, diluted in MEM to A600 0.6. The filter was placed on a 40-μl drop (MOI 50) for 2 h in a humid chamber to allow infection to occur. Invasion assays were performed as described in Figure 1. The treated MDCK cells were exposed to Toxin B for 4 h before infection. Bars show mean ± SD of triplicate samples from a representative experiment. In some instances, error bars are too small to see on the log scale used in these graphs.
Figure 4.
Figure 4.
MDCK cell polarization is accompanied by a switch from RhoA activation to Cdc42 activation in response to infection. MDCK cells were plated to 7-cm Transwell filters at confluent density (2.0 × 107 cells/dish) and allowed to polarize for 1 or 3 d before infection with PA103ΔUΔT at an MOI of 20. Cells were lysed at indicated times, and aliquots of cell lysates were incubated with GST-TRBD bound to glutathione-Sepharose 4B beads (A and B) or GST-hPAK3 (C and D) as described in MATERIALS AND METHODS, allowing selective precipitation of GTP-bound RhoA or Cdc42, respectively. Both affinity-precipitated samples (GTP-bound) and aliquots of un-precipitated lysates were Western blotted with anti-RhoA (A and B) or anti-Cdc42 (C and D) antibodies. The gels shown are representative of two to four independent experiments carried out in duplicate.
Figure 6.
Figure 6.
Exposure of polarized MDCK cells to EDTA increases internalization without activating RhoA. MDCK cells were plated to 7-cm Transwell filters at confluent density (2.0 × 107 cells/dish) and allowed to polarize for 3 d. Immediately before infection, cells were washed with Hanks' Ca2+Mg2+-free BSS; indicated samples were treated with EDTA (2.5 mM) for 15 min. All samples were then returned to calcium and magnesium-replete tissue culture media before infection with PA103ΔUΔT for 1.5 h. Samples were lysed and total RhoA versus GTP-bound RhoA levels determined as described previously. The gel illustrates a representative experiment. Bars indicate the mean percentage of GTP-bound versus total RhoA, normalized to the uninfected/untreated control (100%); error bars show the SD for five separate determinations.

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