| dc.description | Legionella pneumophila is a facultative, intracellular parasite of freshwater protozoa that displays a developmental cycle where it alternates between an intracellularly replicating form, and an environmentally resistant, infectious form, termed the mature intracellular form (MIF). Virulent L. pneumophila strains are unable to replicate on BCYE medium that contains physiological concentrations of sodium chloride. Mutations in a number of post-exponential phase regulators, as well as a number of virulence factors, including the Dot/Icm secretion system, alleviate the sodium mediated growth repression. Previous studies revealed expression of the major outer membrane protein, OmpS, is decreased upon exposure to sodium chloride in the growth media during late exponential phase. An unidentified regulatory protein, termed OmpT, whose binding to the ompS promoter is abolished upon sodium chloride challenge, potentially mediates this decrease in expression. The first goal of this study was to identify proteins that bound to this DNA fragment, and to determine the effect of sodium challenge on global protein expression. The results of this study revealed that the sodium sensitive DNA binding protein is present in the L. pneumophila isogenic Lp02 strain, and recognizes multiple sequence elements based on mobility shift experiments using truncated ompS promoter fragments. When late exponential phase bacteria were exposed to equimolar amounts of sodium or potassium chloride, they exhibited the same changes in protein expression profiles. The ompS promoter contains multiple integration host factor (IHF) binding sites, and purified IHF subunits are capable of retarding the ompS amplicon in mobility shift experiments. Exponentially grown IHF deletion mutants display the same mobility shift profile as wild type, indicating IHF is not OmpT, but IHF is required for full retardation of the ompS amplicon in stationary phase. Western blot revealed that IHF is developmentally regulated, with increased expression in stationary phase, and maximal concentration occurring in MIFs. L. pneumophila strains that over expressed IHF, and to a lesser extent the IHF deletion mutant, were resistant to sodium chloride in the growth media. IHF mutants display several protein profile differences as determined by two-dimensional gel electrophoresis, when compared to wild type. IHF is not required for expression of the DotA, DotD or IcmT proteins, nor is it required for attachment, invasion or intracellular multiplication in HeLa cells, or infectivity in L929 cells, although alterations in the Legionella-containing vacuoles were observed. IHF mutants are defective for MIF morphogenesis, as they do not form thickened cell envelopes or cytoplasmic, multilaminated membranes. Poly-beta-hydroxybutyrate synthesis is decreased in the over expressing strain, and the IHF mutant strains, including the complemented strains. In addition to the ultrastructural defects, several proteins were differentially regulated in the mature IHF mutant strains when compared to wild type MIFs. The mature IHF mutants showed slight decreases in the developmental marker MagA production, and some alterations in resistance to detergent and alkalinity, which are previously defined MIF characteristics. This work demonstrates that IHF specifically regulates several genes, and is required for complete MIF morphogenesis, but is dispensible for virulence in the epithelial cell lines used in this study. | en_US |
