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Motile cilia project from the airway apical surface and directly interface with inhaled external environment. Due to cilia’s nanoscale dimension and high beating frequency, quantitative assessment of their motility remains a sophisticated task. Here we described a robust approach for reproducible engineering of apical-out airway organoid (AOAO) of defined size. Propelled by exterior-facing cilia beating, the mature AOAO exhibited stable rotational motion when surrounded by Matrigel. We developed a computational framework leveraging computer vision algorithms to quantify AOAO rotation and validated its correlation with direct measurement of cilia motility. We further established the feasibility of using AOAO rotation to recapitulate and measure defective cilia motility caused by chemotherapy-induced toxicity and by CCDC39 mutations in cells from primary ciliary dyskinesia patient. We expect our rotating AOAO model and the associated computational pipeline to offer a generalizable framework to expediate modeling of and therapeutic development for genetic and environmental ciliopathies.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection., Author summary Bacteria must overcome host defenses to cause infection. This is especially true for corneal infections where bacteria must penetrate the epithelium in order to gain access to the stroma where bacteria can rapidly multiply, induce inflammation, and cause vision loss. Members of the Enterobacteriaceae commonly cause contact lens associated infections, but the mechanisms by which they damage corneal cells are largely unknown. Here we present evidence that Serratia marcescens and Proteus mirabilis are able to induce dramatic morphological changes in mammalian corneal cells that correlates with rapid cellular death. Secretion of ShlA-like cytolysins via type V secretion was responsible for this phenotype, and this effect was regulated by the conserved Rcs phosphorelay stress response system, including IgaA-family protein GumB. This study provides a model for stress-mediated regulation of cytolysins that induce epithelial damage and promote ocular infection.