PROJECT SUMMARY / ABSTRACT Legionella pneumophila (Lp) is the agent of Legionnaires disease, an oft-fatal form of pneumonia that is increasing in incidence. In natural and man-made aquatic habitats, Lp flourishes as an intracellular parasite of amoebae, often in biofilms. Following the inhalation of Lp-contaminated water droplets, the pathogen grows primarily in alveolar macrophages and secondarily in lung epithelia. Relatively little is known about Lp pili or the bases of Lp attachment to human cells and other surfaces. Early electron microscopy revealed different filaments emanating from the Lp surface, suggesting that Lp expresses “long” and “short” pili. Genomic analysis done by others and us gradually found that Lp has all the genes that typically encode a type IV pilus (T4P) apparatus affirming that at least some Lp pili are T4P. Recently, we uncovered pilA2, a previously unrecognized gene predicted to encode a protein with similarity to major building blocks (type IVa major pilins) of other T4P-expressing bacteria. Immunofluorescence microscopy has revealed that PilA2 exists prominently across the entire length of visualized Lp pili, affirming its role as a major pilin. Intriguingly, we discovered adjacent to pilA2 a second type IVa major pilin gene, pilA1. From our RNA-Seq work, both pilA1 and pilA2 are expressed by Lp growing in liquid and solid media. Moreover, based on the behavior of a pilA1 pilA2 double mutant, pilA1 and pilA2 each promote twitching, a form of surface associated motility. However, whereas a pilA2 mutant was impaired for DNA uptake and infection of Acanthamoeba castellanii (Ac) but hyper- aggregative, a pilA1 mutant was impaired for auto-aggregation but normal for DNA uptake and Ac infection. Thus, we hypothesize that i) PilA2 majorly comprises T4aP that promote competence, twitching, and infection of Ac, but are inhibitory to Lp aggregation and ii) that PilA1 forms distinct T4aP that promote aggregation as well as twitching motility but are not needed for DNA uptake or Ac infection. Thus, overturning long-standing perspectives, our work provides the first genetic evidence that Lp elaborates more than one type of functional T4P, compatible with the “old” EM data that had shown two sizes of pili on the Lp surface. This proposal aims to i) confirm if PilA1 and PilA2 assemble, as major pilins, into distinct Lp T4aP and ii) discern if PilA1-T4aP and PilA2-T4aP are functionally different in the context of adherence to and infection of human macrophages and epithelia, infection of various amoebae that help transmit Lp to humans, and biofilm formation, which is another key attribute of Lp in vivo and in the environment. This work will i) increase our knowledge of Lp, a growing clinical problem, ii) define new forms of T4P and infection pathways, iii) have implications for other pathogens that use T4P or are intracellular parasites, and iv) suggest new targets for disease treatment or prevention.