Zebrafish (Danio rerio) have a number of strengths as a host model for infection, including genetic tractability, a vertebrate immune system similar to that of mammals, ease and scale of laboratory handling, which allows analysis with reasonable throughput, and transparency, which facilitates visualization of the infection. With these advantages in mind, we examined whether zebrafish could be used to study Pseudomonas aeruginosa pathogenesis and found that infection of zebrafish embryos with live P. aeruginosa (PA14 or PAO1) by microinjection results in embryonic death, unlike infection with Escherichia coli or heat-killed P. aeruginosa, which has no effect. Similar to studies with mice, P. aeruginosa mutants deficient in type three secretion (pscD) or quorum sensing (lasR and mvfR) are attenuated in zebrafish embryos infected at 50 h postfertilization (hpf), a developmental stage when both macrophages and neutrophils are present. In contrast, embryos infected at 28 hpf, when only macrophages are initially present, succumb to lethal challenge with far fewer P. aeruginosa cells than those required for embryos infected at 50 hpf, are susceptible to infection with lasR and pscD deletion mutants, and are moderately resistant to infection with an mvfR mutant. Finally, we show that we can control the outcome of infection through the use of morpholinos, which allow us to shift immune cell numbers, or small molecules (antibiotics), which rescue embryos from lethal challenge. Thus, zebrafish are a novel host model that is well suited for studying the interactions among individual pathogenic functions of P. aeruginosa, the role of individual components of host immune defense, and small-molecule modulators of infection.Pseudomonas aeruginosa, one of the most common causes of nosocomial infections in the United States, typically infects injured, burned, and immunocompromised patients and is the primary cause of mortality among cystic fibrosis patients. It is a ubiquitous, gram-negative bacterium adapted to a variety of niches, including water and soil in associations with other eukaryotic organisms. A number of evolutionarily divergent model hosts have been used to examine P. aeruginosa pathogenesis, including amoebae, plants, nematodes, insects, and rodents (25, 36, 37). While much has been learned about P. aeruginosa pathogenesis from these models, each model has different strengths and weaknesses. Invertebrate model hosts such as Caenorhabditis elegans offer greater genetic tractability than rodent models. Moreover, the size and life cycle of organisms like C. elegans enable experiments such as comprehensive genetic screens that require large numbers of animals, in contrast to rodent models, where such studies are often simply unfeasible due to cost and space requirements. The drawback to modeling human infections in invertebrate hosts is the dissimilarity between vertebrate and invertebrate immune responses. Invertebrate model hosts such as Drosophila melanogaster and C. elegans do not possess adaptive immunity, a true ...

Two-component sensors are widely used by bacteria to sense and respond to the environment. Pseudomonas aeruginosa has one of the largest sets of two-component sensors known in bacteria, which likely contributes to its unique ability to adapt to multiple environments, including the human host. Several of these two-component sensors, such as GacS and RetS, have been shown to play roles in virulence in rodent infection models. However, the role and function of the majority of these two-component sensors remain unknown. Danio rerio is a recently characterized model host for pathogenesis-related studies that is amenable to higher-throughput analysis than mammalian models. Using zebrafish embryos as a model host, we have systematically tested the role of 60 two-component sensors and identified 6 sensors that are required for P. aeruginosa virulence. We found that KinB is required for acute infection in zebrafish embryos and regulates a number of virulenceassociated phenotypes, including quorum sensing, biofilm formation, and motility. Its regulation of these phenotypes is independent of its kinase activity and its known response regulator AlgB, suggesting that it does not fit the canonical two-component sensor-response regulator model.