DESCRIPTION (provided by applicant): Legionella neumophila (Lp) is the agent of Legionnaires' disease. It is ubiquitous in natural and man-made water systems, infecting humans after aerosol inoculation. In aquatic habitats, Lp survives in biofilms and as an intracellular parasite of protozoa, and in the lung, it flourishes as an intracellular parasite of alveolar macrophages and epithelia. Iron is vital to Lp growth in extra- and intracellular niches and its ability to cause disease. For twenty years, my laboratory has been involved in deciphering mechanisms by which Lp causes disease and, for fifteen of those years, has served a leadership role in the study of Lp iron acquisition. Previously, we documented that Lp expresses both siderophore (legiobactin)-mediated ferric iron uptake and FeoB-mediated ferrous iron assimilation and that each of these is required for optimal infection of the lung. In the course of screening for genes involved in legiobactin production, we determined that the secreted brown pigment of Lp, a pyomelanin derived from homogentisic acid (i.e., HGA-melanin), confers ferric reductase activity. In light of these data, we hypothesize i) that secreted HGA-melanin promotes Lp iron acquisition by providing ferrous iron for import into the bacterial cell and ii) that this pyomelanin is required for Lp growth within the infected lung. The pursuit of this hypothesis would be the first test of whether HGA-melanin mediated iron reduction is a (general) mechanism for bacterial iron uptake as well as the first direct assessment of the importance of a bacterial melanin, Lp or otherwise, in an animal model of disease. In this R21 , we will utilize purified HGA-melanin and Lp mutants specifically lacking HGA-melanin to i) determine whether or not secreted pyomelanin can mediate iron acquisition and promote bacterial growth under low-iron conditions, ii) determine the importance of pyomelanin for bacterial growth, inflammation, and tissue damage within Lp-infected mouse lungs. The impact of the proposed studies is significant for multiple reasons. First, it will increase our understanding of Lp, which is an important public health concern throughout the world. Second, since many other important pathogens as well as other important environmental microbes secrete HGA-melanin, it will elucidate new paradigms for both iron assimilation and bacterial pathogenesis. Third, it will potentially provide a new type of target for disease diagnosis, treatment, and/or prevention.

PUBLIC HEALTH RELEVANCE: We have determined that the secreted pigment (known as pyomelanin or HGA- melanin) of Legionella pneumophila confers a ferric reductase activity. We now hypothesize that the secreted pigment is an alternate mechanism for bacterial iron acquisition and an important but heretofore unappreciated virulence factor of Legionella; pyomelanin thus represents a potential target for disease diagnosis, prevention, or therapy. To examine this hypothesis, we will i) use biochemical and genetic means to determine the contribution of HGA-melanin to Legionella iron acquisition, and ii) define the importance of the secreted pyomelanin in the mouse model of lung infection by L. pneumophila.