P. aeruginosa is an important opportunistic pathogen, causing infections in individuals with cystic fibrosis (CF), burns, eye injuries as well as patients with compromised immune systems.  In some of these diseases, this microbe is believed to form biofilms, and in other settings, interactions with its host are mediated via products secreted by this organism.  Thus, we have become interested in the interactions between P. aeruginosa and the host in two different contexts.  We have been studying these bacterial-host interactions through a collaborative effort with Dr. Bruce Stanton and his group here at Dartmouth.  Dr. Stanton’s expertise is in host cell protein trafficking.  We are interested in three different research topics: 

What are the mechanisms of biofilm formation and biofilm antibiotic resistance on airway epithelial cells?  We have been approaching this problem through the perspective of P. aeruginosa in the context of the CF lung.  There are currently no robust animal models for this disease, thus a few years ago we developed an in vitro tissue culture model to assess P. aeruginosa biofilm formation on CF derived airway cells.

Shown are top-down images of biofilms of GFP-labeled P. aeruginosa (yellow arrow) grown on CF-derived airway cells (grey background). 

                                        From Moreus-Marquis, AJP-LCMP, 2008.

Using this system, we have shown that: (i) Biofilms of P. aeruginosa form very quickly on airway cells, (ii) Biofilm formation on this biotic substratum requires many of the same genes required for biofilm formation on plastic or glass, (iii) These biofilms are >25-fold more resistant to antibiotics than biofilms grown on plastic or glass, (iii) Iron release from the CF cells stimulates biofilm formation and (iv) Chelating iron renders these biofilms much more sensitive to antibiotic resistance.  We are actively studying how these biofilms form and new therapeutic approaches to treat such biofilm infections.

How do P. aeruginosa secreted products impact epithelial cell biology?  To approach this question, we have focused our efforts on a toxin we have discovered called Cif, for CFTR inhibitory factor.  This secreted protein reduces the apical membrane expression of a number of ABC transporter-family proteins, including CFTR, the ion channel mutated in patients with CF.

The left panel shows a Western blot assessing apical membrane CFTR, with Ezrin as a control.  Cif mediates the time-dependent loss of CFTR from the apical membrane of airway epithelial cells.  The graph beneath shows quantitation of the data. From Urban, Am J Physiol Cell Physiol. 2006. On the right is a crystal of the Cif protein. 

We are collaborating with the Stanton and Madden labs here at Dartmouth to study the mechanism by which alters protein trafficking, as well as exploring the biochemical and structural properties of CIF.

How are bacterial secreted proteins delivered to the host?  Many Gram-negative bacteria produce outer membrane vesicles (OMV), which are ~100 nm structures spontaneously released from the OM of bacteria. 

This EM of OMV was taken by Terry Beveridge's group.  Dr. Beveridge was a pioneer in the study of these OMV, and his early work has generated a great deal of interest.  Arrows indicate OMV.  The larger structures are bacteria.

From: Kadurugamuwa & Beveridge, JB, 1995.

OMV often contain bacterial periplasmic and secreted proteins.  We have recently shown that these OMV can deliver multiple bacterial virulence factors, across long distances, and deliver these factors directly to the host cytoplasm.  OMV-mediated delivery of the bacterial secreted products occurs via lipid rafts in the host membrane.  Current work is focusing on host and bacterial factors required for this fusion event, and how bacterial proteins are packaged into the OMV. 

Our current model for delivery of bacterial toxins to OMV is shown here.  OMV are released by P. aeruginosa, a variety of proteins are packaged in the OMV (inset).  Our data suggest the OMV fuse with lipid raft domains and release their contents directly into the host cell cytoplasm.  From, Bomberger, PLOS Pathogens, 2009.

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Here are some links to our recent work regarding P. aeruginosa-host interactions:

Bomberger JM, Maceachran DP, Coutermarsh BA, Ye S, O'Toole GA, Stanton BA. 2009.  Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles. PLoS Pathog. 5:e1000382.

Moreau-Marquis S, O'Toole GA, Stanton BA. Tobramycin and FDA-approved Iron Chelators Eliminate P. aeruginosa Biofilms on Cystic Fibrosis Cells. 2009. Am J Respir Cell Mol Biol. ePub.

Moreau-Marquis S, Bomberger JM, Anderson GG, Swiatecka-Urban A, Ye S, O'Toole GA, Stanton BA. 2008. The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability. Am J Physiol Lung Cell Mol Physiol. 295:L25-37.

Anderson GG, Moreau-Marquis S, Stanton BA, O'Toole GA. 2008.  In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells. 76:1423-33.

MacEachran DP, Ye S, Bomberger JM, Hogan DA, Swiatecka-Urban A, Stanton BA, O'Toole GA. 2007. The Pseudomonas aeruginosa secreted protein PA2934 decreases apical membrane expression of the cystic fibrosis transmembrane conductance regulator. Infect Immun. 75:3902-12.