Reciprocal Expression of Integration Host Factor and HU in the Developmental Cycle and Infectivity ofLegionella pneumophila

Abstract

ABSTRACTLegionella pneumophilais an intracellular parasite of protozoa that differentiates late in infection into metabolically dormant cysts that are highly infectious. Regulation of this process is poorly understood. Here we report that the small DNA binding regulatory proteins integration host factor (IHF) and HU are reciprocally expressed over the developmental cycle, with HU expressed during exponential phase and IHF expressed postexponentially. To assess the role of these regulatory proteins in development, chromosomal deletions were constructed. Single (ihfAorihfB) and double deletion (Δihf) IHF mutants failed to grow inAcanthamoeba castellaniiunless complemented intranswhen expressed temporally from theihfApromoter but not under Ptac(isopropyl-β-d-thiogalactopyranoside). In contrast, IHF mutants were infectious for HeLa cells, though electron microscopic examination revealed defects in late-stage cyst morphogenesis (thickened cell wall, intracytoplasmic membranes, and inclusions of poly-β-hydroxybutyrate), and were depressed for the developmental marker MagA. Green fluorescent protein promoter fusion assays indicated that IHF and the stationary-phase sigma factor RpoS were required for full postexponential expression ofmagA. Finally, defects in cyst morphogenesis noted for Δihfmutants in HeLa cells correlated with a loss of both detergent resistance and hyperinfectivity compared with results for wild-type cysts. These studies establish IHF and HU as markers of developmental stages and show that IHF function is required for both differentiation and full virulence ofL. pneumophilain natural amoebic hosts.