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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: 4.16). 12/2009; 78(3):939-53. DOI: 10.1128/IAI.01215-09
Source: PubMed

ABSTRACT 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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    • "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.
    Frontiers in Microbiology 05/2011; 2:114. DOI:10.3389/fmicb.2011.00114 · 3.94 Impact Factor
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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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    Cellular Microbiology 05/2011; 13(8):1212-22. DOI:10.1111/j.1462-5822.2011.01611.x · 4.82 Impact Factor
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    • "Tissue injury or epithelial damage increases apical surface expression of N-glycans leading to increased P. aeruginosa attachment. Using incompletely polarized cells as a model of injured tissue, it was shown that HSPGs are also upregulated at the apical surface further increasing P. aeruginosa binding and subsequent tissue damage (Bucior et al. 2010 "
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    ABSTRACT: To cause infections, microbial pathogens elaborate a multitude of factors that interact with host components. Using these host–pathogen interactions to their advantage, pathogens attach, invade, disseminate, and evade host defense mechanisms to promote their survival in the hostile host environment. Many viruses, bacteria, and parasites express adhesins that bind to cell surface heparan sulfate proteoglycans (HSPGs) to facilitate their initial attachment and subsequent cellular entry. Some pathogens also secrete virulence factors that modify HSPG expression. HSPGs are ubiquitously expressed on the cell surface of adherent cells and in the extracellular matrix. HSPGs are composed of one or several heparan sulfate (HS) glycosaminoglycan chains attached covalently to specific core proteins. For most intracellular pathogens, cell surface HSPGs serve as a scaffold that facilitates the interaction of microbes with secondary receptors that mediate host cell entry. Consistent with this mechanism, addition of HS or its pharmaceutical functional mimic, heparin, inhibits microbial attachment and entry into cultured host cells, and HS-binding pathogens can no longer attach or enter cultured host cells whose HS expression has been reduced by enzymatic treatment or chemical mutagenesis. In pathogens where the specific HS adhesin has been identified, mutant strains lacking HS adhesins are viable and show normal growth rates, suggesting that the capacity to interact with HSPGs is strictly a virulence activity. The goal of this chapter is to provide a mechanistic overview of our current understanding of how certain microbial pathogens subvert HSPGs to promote their infection, using specific HSPG–pathogen interactions as representative examples.
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