<i>Legionella pneumophila</i> IrsA, a novel, iron-regulated exoprotein that facilitates growth in low-iron conditions and modulates biofilm formation

To discover new factors that are involved in iron acquisition by <i>Legionella pneumophila</i>, we used RNA-Seq to identify the genes that are most highly induced when virulent strain 130b is cultured in a low-iron chemically defined medium. Among other things, this revealed <i>14915</i>, a heretofore uncharacterized gene that is predicted to be transcriptionally regulated by Fur and to encode a novel, ~15 kDa protein. <i>14915</i> was present in all <i>L. pneumophila</i> strains examined and had homologs in a subset of the other <i>Legionella</i> species. Compatible with it containing a classic signal sequence, the 14915 protein was detected in bacterial culture supernatants in a manner dependent upon the <i>L. pneumophila</i> type II secretion system. Thus, we designated 14915 as IrsA for <u>ir</u>on-<u>r</u>egulated, <u>s</u>ecreted protein <u>A</u>. Based on mutant analysis, the <i>irsA</i> gene was not required for optimal growth of strain 130b in low-iron media. However, after discovering that the commonly used laboratory-derived strain Lp02 has a much greater requirement for iron, we uncovered a growth-enhancing role for IrsA after examining an Lp02 mutant that lacked both IrsA and the Fe²⁺-transporter FeoB. The <i>irsA</i> mutant of 130b, but not its complemented derivative, did, however, display increased biofilm formation on both plastic and agar surfaces, and compatible with this, the mutant hyper-aggregated. Thus, IrsA is a novel, iron-regulated exoprotein that modulates biofilm formation and, under some circumstances, promotes growth in low-iron conditions. For this study, we determined and deposited in the database a complete and fully assembled genome sequence for strain 130b.IMPORTANCEThe bacterium <i>Legionella pneumophila</i> is the principal cause of Legionnaires' disease, a potentially fatal form of pneumonia that is increasing in incidence. <i>L. pneumophila</i> exists in many natural and human-made water systems and can be transmitted to humans through inhalation of contaminated water droplets. <i>L. pneumophila</i> flourishes within its habitats by spreading planktonically, assembling into biofilms, and growing in larger host cells. Iron acquisition is a key determinant for <i>L. pneumophila</i> persistence in water and during infection. We previously demonstrated that <i>L. pneumophila</i> assimilates iron both by secreting a non-protein iron chelator (siderophore) and by importing iron through membrane transporters. In this study, we uncovered a novel, secreted protein that is highly iron-regulated, promotes <i>L. pneumophila</i>'s growth in low-iron media, and impacts biofilm formation. We also identified uncharacterized, IrsA-related proteins in other important human and animal pathogens. Thus, our results have important implications for understanding iron assimilation, biofilm formation, and pathogenesis.