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

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.Ninety-five percent of all infectious agents enter through mucosal surfaces of the gastrointestinal, genitourinary, and respiratory tracts (reviewed in reference 35). These mucosal surfaces are usually lined by a single layer of epithelial cells, which serves as the primary barrier against the entry of most infectious agents and can be considered a primary component of the innate immune system. Epithelial cells form highly polarized cell layers with apical (AP) and basolateral (BL) surfaces that exhibit distinct protein, lipid, and glycoconjugate compositions. Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen of humans that exploits injured mucosa to cause acute and chronic infections with high morbidity and mortality (reviewed in references 26 and 31). In the setting of epithelial injury and immunocompromise, this Gram-negative pathogen causes serious infections in patients with extensive burns, corneal trauma, or catheter-related bladder injury or in those on ventilators. In addition, P. aeruginosa chronically colonizes the lungs of patients with cystic fibrosis (CF) (4), leading to severe pulmonary damage and death. Despite aggressive antibiotic therapy, the fatality rate for many P. aeruginosa infections is 40%, and new approaches to treatment are even more critical now that antibiotic resistance...

Mechanisms of phagocytosis and host clearance ofPseudomonas aeruginosa

“…These data were later supported by evidence that demonstrated that P. aeruginosa binding affects cell polarity and subsequent membrane composition, allowing the bacteria to preferentially invade the basolateral side of Madin-Darby canine kidney cells (67). Bacterial binding may be facilitated through cellular heparan sulfate proteoglycans and N-glycans, though the role of these receptors in subsequent invasion is still fairly preliminary (17,18,117). These collective observations are particularly intriguing since P. aeruginosa is not known to exhibit specific adaptations for intracellular survival and, as noted below, does employ potent toxins that efficiently kill host cells.…”

<em>In vitro</em> Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

“…The in vitro coculture assay offers a larger degree of complexity providing versatility in experimental design 16,37,38 . Polarized epithelial cells grown on the underside of permeable transwell filters creates an apical compartment that faces the bottom chamber when placed in a 24-well plate and a basolateral compartment that faces the inside top well of the transwell 14,17 .…”

Human Metapneumovirus (HMPV) Binding and Infection Are Mediated by Interactions between the HMPV Fusion Protein and Heparan Sulfate

“…Some studies have shown that cell surface HS is primarily located on the basolateral side of differentiated ciliated airway epithelial cells (10,39,71). Thus, binding to HSPG may be used to infect nonciliated cells in the respiratory tract or to promote cell-to-cell spreading from the basolateral side.…”

“…Moreover, the small amount of HSPG present on the apical side (10, 13) could be enough for HMPV to establish an initial infection in the upper respiratory tract. Since apical levels of HSPG are upregulated upon injury and basolateral HSPGs can be exposed when the monolayer is disrupted (including during tissue regeneration) (10), this initial infection would in turn increase the accessibility of HSPGs to the virus.…”