Pseudomonas aeruginosa-Mediated Damage Requires Distinct Receptors at the Apical and Basolateral Surfaces of the Polarized Epithelium

Department of Medicine, Microbial Pathogenesis and Host Defense Program, University of California, San Francisco, CA 94143, USA.
Infection and immunity (Impact Factor: 3.73). 12/2009; 78(3):939-53. DOI: 10.1128/IAI.01215-09
Source: PubMed


Pseudomonas aeruginosa, an important opportunistic pathogen of humans, exploits epithelial damage to establish infection. We have rigorously explored
the role of N-glycoproteins and heparan sulfate proteoglycans (HSPGs) in P. aeruginosa-mediated attachment and subsequent downstream events at the apical (AP) and basolateral (BL) surfaces of polarized epithelium.
We demonstrate that the N-glycan chains at the AP surface are necessary and sufficient for binding, invasion, and cytotoxicity
to kidney (MDCK) and airway (Calu-3) cells grown at various states of polarization on Transwell filters. Upregulation of N-glycosylation
enhanced binding, whereas pharmacologic inhibition of N-glycosylation or infection of MDCK cells defective in N-glycosylation
resulted in decreased binding. In contrast, at the BL surface, the HS moiety of HSPGs mediated P. aeruginosa binding, cytotoxicity, and invasion. In incompletely polarized epithelium, HSPG abundance was increased at the AP surface,
explaining its increased susceptibility to P. aeruginosa colonization and damage. Using MDCK cells grown as three-dimensional cysts as a model for epithelial organs, we show that
P. aeruginosa specifically colocalized with HS-rich areas at the BL membrane but with complex N-glycans at the AP surface. Finally, P. aeruginosa bound to HS chains and N-glycans coated on plastic surfaces, showing the highest binding affinity toward isolated HS chains.
Together, these findings demonstrate that P. aeruginosa recognizes distinct receptors on the AP and BL surfaces of polarized epithelium. Changes in the composition of N-glycan chains
and/or in the distribution of HSPGs may explain the enhanced susceptibility of damaged epithelium to P. aeruginosa.

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    • "We have previously used P. aeruginosa infection of filtergrown epithelial cells to model host-pathogen interactions at the mucosal barrier (Bucior et al., 2010, 2012; Kazmierczak et al., 2001). When grown for several days on semiporous filters (Transwells), Madin-Darby canine kidney (MDCK) epithelial cells form well-polarized, confluent monolayers with distinct apical and basolateral surfaces (Mostov, 1995). "
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    ABSTRACT: The mucosal epithelium consists of polarized cells with distinct apical and basolateral membranes that serve as functional and physical barriers to external pathogens. The apical surface of the epithelium constitutes the first point of contact between mucosal pathogens, such as Pseudomonas aeruginosa, and their host. We observed that binding of P. aeruginosa aggregates to the apical surface of polarized cells led to the striking formation of an actin-rich membrane protrusion with inverted polarity, containing basolateral lipids and membrane components. Such protrusions were associated with a spatially localized host immune response to P. aeruginosa aggregates that required bacterial flagella and a type III secretion system apparatus. Host protrusions formed de novo underneath bacterial aggregates and involved the apical recruitment of a Par3/Par6α/aPKC/Rac1 signaling module for a robust, spatially localized host NF-κB response. Our data reveal a role for spatiotemporal epithelial polarity changes in the activation of innate immune responses.
    Full-text · Article · May 2014 · Cell host & microbe
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    • "Finally, some of the epithelial cells, including MDCK cells and primary mouse alveolar type II cells, can be grown as three-dimensional (3D) cysts when cultured on extracellular matrix in which the basolateral surface faces outward (Bryant and Mostov, 2008). These models may more closely mimic organs; in addition, they facilitate the examination of interactions of pathogens with the basolateral surface in the absence of the porous filter support (Barrila et al., 2010; Bucior et al., 2010). These reductionist systems provide a platform to analyze host–pathogen interactions, which can then be further validated in animal studies. "
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    ABSTRACT: The lumenal surfaces of human body are lined by a monolayer of epithelia that together with mucus secreting cells and specialized immune cells form the mucosal barrier. This barrier is one of the most fundamental components of the innate immune system, protecting organisms from the vast environmental microbiota. The mucosal epithelium is comprised of polarized epithelial cells with distinct apical and basolateral surfaces that are defined by unique set of protein and lipid composition and are separated by tight junctions. The apical surface serves as a barrier to the outside world and is specialized for the exchange of materials with the lumen. The basolateral surface is adapted for interaction with other cells and for exchange with the bloodstream. A wide network of proteins and lipids regulates the formation and maintenance of the epithelium polarity. Many human pathogens have evolved virulence mechanisms that target this network and interfere with epithelial polarity to enhance binding to the apical surface, enter into cells, and/or cross the mucosal barrier. This review highlights recent advances in our understanding of how Pseudomonas aeruginosa, an important opportunistic human pathogen that preferentially infects damaged epithelial tissues, exploits the epithelial cell polarization machinery to enhance infection.
    Preview · Article · May 2011 · Frontiers in Microbiology
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    • "Pseudomonas aeruginosa strain K was routinely grown shaking overnight in Luria-Bertani broth at 37°C. For fluorescence microscopy studies P. aeruginosa carrying plasmids containing either m-Cherry (Mougous et al., 2007) or GFP (Bucior et al., 2010) genes were used. Stationary phase bacteria were co-cultivated with MDCK monolayers at the indicated moi. "
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    ABSTRACT: Growing evidence is pointing to the importance of multicellular bacterial structures in the interaction of pathogenic bacteria with their host. Transition from planktonic to host cell-associated multicellular structures is an essential infection step that has not been described for the opportunistic human pathogen Pseudomonas aeruginosa. In this study we show that P. aeruginosa interacts with the surface of epithelial cells mainly forming aggregates. Dynamics of aggregate formation typically follow a sigmoidal curve. First, a single bacterium attaches at cell-cell junctions. This is followed by rapid recruitment of free-swimming bacteria and association of bacterial cells resulting in the formation of an aggregate on the order of minutes. Aggregates are associated with phosphatidylinositol 3,4,5-trisphosphate (PIP3)-enriched host cell membrane protrusions. We further show that aggregates can be rapidly internalized into epithelial cells. Lyn, a member of the Src family tyrosine kinases previously implicated in P. aeruginosa infection, mediates both PIP3-enriched protrusion formation and aggregate internalization. Our results establish the first framework of principles that define P. aeruginosa transition to multicellular structures during interaction with host cells.
    Full-text · Article · May 2011 · Cellular Microbiology
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