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ABSTRACT The inflammasome is essential for host defense against intracellular bacterial pathogens, including Legionella pneumophila, the causative agent of the severe pneumonia Legionnaires’ disease. Inflammasomes recruit and activate caspases, which promote IL-1 family cytokine release and pyroptosis to restrict infection. In mice, interferon (IFN) signaling promotes inflammasome responses against L. pneumophila and other bacteria, in part, through inducing a family of IFN-inducible GTPases known as guanylate-binding proteins (GBPs). Within murine macrophages, IFN promotes the rupture of the L. pneumophila-containing vacuole (LCV), while GBPs are dispensable for vacuole rupture. Instead, GBPs facilitate the lysis of cytosol-exposed L. pneumophila. In contrast, the functions of IFN-γ and GBPs in human inflammasome responses to L. pneumophila are poorly understood. Here, we show that IFN-γ enhances caspase-1- and caspase-4-dependent inflammasome responses to L. pneumophila in human macrophages. We find that human GBP1 is required for these IFN-γ-driven inflammasome responses. Furthermore, we find that GBP1 co-localizes with L. pneumophila and/or LCVs in a type IV secretion system (T4SS)-dependent manner and facilitates damage to the LCV, resulting in increased bacterial access to the host cell cytosol. Our findings reveal species- and pathogen-specific differences in how GBPs function during infection. Importance Inflammasomes are essential for host defense against intracellular bacterial pathogens like Legionella, as they activate caspases, which promote cytokine release and cell death to control infection. In mice, interferon (IFN) signaling promotes inflammasome responses against bacteria by inducing a family of IFN-inducible GTPases known as guanylate-binding proteins (GBPs). Within murine macrophages, IFN promotes the rupture of the Legionella-containing vacuole (LCV), while GBPs are dispensable for this process. Instead, GBPs facilitate the lysis of cytosol-exposed Legionella. In contrast, the functions of IFN and GBPs in human inflammasome responses to Legionella are poorly understood. We show that IFN-γ enhances inflammasome responses to Legionella in human macrophages. Human GBP1 is required for these IFN-γ-driven inflammasome responses. Furthermore, GBP1 co-localizes with Legionella and/or LCVs in a type IV secretion system (T4SS)-dependent manner and promotes damage to the LCV, which leads to increased exposure of the bacteria to the host cell cytosol. Thus, our findings reveal species- and pathogen-specific differences in how GBPs function to promote inflammasome responses.

ABSTRACT Microbiology courses are often designed as either a lecture class with a laboratory component or a seminar-style class. Each type of course provides students with unique learning opportunities. Lab courses allow students to perform simple experiments that relate to fundamental concepts taught in the corresponding lectures, while seminar courses challenge students to read and discuss primary literature. Microbiology courses offering a combination of seminar-style discussions and laboratory procedures are rare. Our goal in the “Microbial Diversity and Pathogenesis” undergraduate course is to integrate experiences of a seminar class with those of a discovery-driven lab course, thereby strengthening students’ learning experiences through diversified didactic approaches. In the first half of the course, students read and discuss published peer-reviewed articles that cover major topics in both basic and applied microbiology, including antibiotic resistance, pathogenesis, and biotechnology applications. Complementing this primary literature, students perform microbiology experiments related to the topics covered in the readings. The assigned readings, discussions, and experiments provide a foundation in the second half of the course for inquiry-based exploratory research using student-designed transposon screens and selections. The course culminates in each student drafting a hypothesis-driven research proposal based on their literature review, their learned experimental techniques, and the preliminary data generated as a class. Through such first-hand experimental experience, students gain fundamental lab skills that are applicable beyond the realm of microbiology, such as sterile technique and learning how to support conclusions with scientific evidence. We observed a tremendous synergy between the seminar and lab aspects of our course. This unique didactic experience allows students to understand and connect primary literature to their experiments, while the discovery-driven aspect of this approach fosters active engagement of students with scientific research.