Legionella pneumophila, the gram-negative species that causes legionnaires'disease and other acute respiratory diseases, replicates within a wide range of phagocytic host cells. During legionnaires'disease, L. pneumophila replicates in alveolar macrophages. In the environment, it can replicate in an extraordinary range of protozoan hosts. Replication of L. pneumophila in protozoa is thought to occur in both planktonic and biofilm conditions. Biofilms are thought to be a primary source of the organism during outbreaks in both community-acquired and nosocomial infections. Little is known about the molecular basis for the ability of L. pneumophila to survive and multiply within such a wide range of environmental settings. Several investigators have proposed that L. pneumophila undergoes a developmental switch between distinct forms to account for the observed versatility in changing lifestyles. The availability of the complete L. pneumophila genome sequence and a microarray containing all known L. pneumophila genes, open reading frames and intergenic regions will permit the evaluation of several hypotheses that may account for these different physiologic states. In particular we propose to: (1) Test the hypothesis that distinct patterns of gene expression accompany discrete events during intracellular survival and growth of L. pneumophila;RNA from human macrophages will be isolated at various times after infection and the global pattern of Legionella gene expression will be determined using a whole genome Legionella microarray;(2)Test the hypothesis that distinct patterns of gene expression account for the observed phenotypic differences between axenically-grown and protozoan-grown L. pneumophila;RNA will be isolated from Legionella grown in protoza and the pattern of global gene expression will be compared to that of Legionella grown in axenic media as well as to Legionella that have been exposed to environmental stresses;(3) Test the hypothesis that specific patterns of gene expression in the presence of protozoan hosts promote the formation of Legionella-containing biofilms; RNA will be isolated from Legionella containing biofilms and the patterns of gene expression will be compared to the bacteria in the planktonic phase;(4) Develop model networks that describe relevant patterns of gene expression from the different conditions that have been examined to define specific reporters for each Legionella lifestyle or environmental condition.
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