Summary The mucosal epithelium consists of polarized cells with distinct apical and basolateral membranes that serve as functional and physical barriers to the organisms’ exterior. 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 leads 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 form de novo underneath bacterial aggregates and involve the apical recruitment of a Par3/Par6α/aPKC/Rac1 signaling module for a robust, spatially localized host NFκB response. Our data reveal an unanticipated role for spatio-temporal epithelial polarity changes in the activation of innate immune responses.

Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studied in vitro on glass or plastic surfaces, much less is known about biofilm formation at the epithelial barrier. We have previously shown that when added to the apical surface of polarized epithelial cells, P. aeruginosa rapidly forms cell-associated aggregates within 60 minutes of infection. By confocal microscopy we now show that cell-associated aggregates exhibit key characteristics of biofilms, including the presence of extracellular matrix and increased resistance to antibiotics compared to planktonic bacteria. Using isogenic mutants in the type III secretion system, we found that the translocon, but not the effectors themselves, were required for cell-associated aggregation on the surface of polarized epithelial cells and at early time points in a murine model of acute pneumonia. In contrast, the translocon was not required for aggregation on abiotic surfaces, suggesting a novel function for the type III secretion system during cell-associated aggregation. Supernatants from epithelial cells infected with wild-type bacteria or from cells treated with the pore-forming toxin streptolysin O could rescue aggregate formation in a type III secretion mutant, indicating that cell-associated aggregation requires one or more host cell factors. Our results suggest a previously unappreciated function for the type III translocon in the formation of P. aeruginosa biofilms at the epithelial barrier and demonstrate that biofilms may form at early time points of infection.

Children with MDR-TB and LTBI were best cared for in public health settings. A multicenter registry for pediatric MDR-TB and MDR-LTBI would be desirable to obtain accurate rates of toxicity and cure.

While human or animal models are often considered the gold standard experimental system for defining virulence factors, cell culture-based infection models have proven useful for identifying important virulence factors and for examining the interactions between pathogens and the epithelial barrier. The first step in infections for most mucosal pathogens involves binding (adhesion) to the epithelial cells that line the mucosa. Successful pathogens can then penetrate the barrier by (1) inducing their uptake (i.e., “entry” or “invasion”) into epithelial cells, (2) crossing the barrier by inducing epithelial cell death, and/or (3) penetrating between cells. This chapter describes growth conditions to form polarized cultures, either two-dimensional monolayers or three-dimensional cysts, of various immortalized epithelial cell lines. It describes assays to measure key early interactions between P. aeruginosa and host cells, including binding, invasion, and cytotoxicity. Many virulence factors defined by these criteria have been shown to be important for pathogenesis of P. aeruginosa infections in animals or humans. These methods are also applicable to other pathogens.