Induced conformational changes in the activation of the Pseudomonas aeruginosa type III toxin, ExoU.

ExoU is a 74-kDa, water-soluble toxin injected directly into mammalian cells through the type III secretion system of the opportunistic pathogen, Pseudomonas aeruginosa. Previous studies have shown that ExoU is a Ca(2+)-independent phospholipase that requires a eukaryotic protein cofactor. One protein capable of activating ExoU and serving as a required cofactor was identified by biochemical and proteomic methods as superoxide dismutase (SOD1). In these studies, we carried out site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the effects of SOD1 and substrate liposomes on the structure and dynamics of ExoU. Local conformational changes within the catalytic site were observed in the presence of substrate liposomes, and were enhanced by the addition of SOD1 in a concentration-dependent manner. Conformational changes in the C-terminal domain of ExoU were observed upon addition of cofactor, even in the absence of liposomes. Double electron-electron resonance experiments indicated that ExoU samples multiple conformations in the resting state. In contrast, addition of SOD1 induced ExoU to adopt a single, well-defined conformation. These studies provide, to our knowledge, the first direct evidence for cofactor- and membrane-induced conformational changes in the mechanism of activation of ExoU.

Identification of Francisella tularensis Himar1-based transposon mutants defective for replication in macrophages.

Francisella tularensis, the etiologic agent of tularemia in humans, is a potential biological threat due to its low infectious dose and multiple routes of entry. F. tularensis replicates within several cell types, eventually causing cell death by inducing apoptosis. In this study, a modified Himar1 transposon (HimarFT) was used to mutagenize F. tularensis LVS. Approximately 7,000 Km(r) clones were screened using J774A.1 macrophages for reduction in cytopathogenicity based on retention of the cell monolayer. A total of 441 candidates with significant host cell retention compared to the parent were identified following screening in a high-throughput format. Retesting at a defined multiplicity of infection followed by in vitro growth analyses resulted in identification of approximately 70 candidates representing 26 unique loci involved in macrophage replication and/or cytotoxicity. Mutants carrying insertions in seven hypothetical genes were screened in a mouse model of infection, and all strains tested appeared to be attenuated, which validated the initial in vitro results obtained with cultured macrophages. Complementation and reverse transcription-PCR experiments suggested that the expression of genes adjacent to the HimarFT insertion may be affected depending on the orientation of the constitutive groEL promoter region used to ensure transcription of the selective marker in the transposon. A hypothetical gene, FTL_0706, postulated to be important for lipopolysaccharide biosynthesis, was confirmed to be a gene involved in O-antigen expression in F. tularensis LVS and Schu S4. These and other studies demonstrate that therapeutic targets, vaccine candidates, or virulence-related genes may be discovered utilizing classical genetic approaches in Francisella.