No abstract

Francisella tularensis, the highly virulent etiologic agent of tularemia, is a low-dose intracellular pathogen that is able to escape from the phagosome and replicate in the cytosol. Although there has been progress in identifying loci involved in the pathogenicity of this organism, analysis of the genome sequence has revealed few obvious virulence factors. We previously reported isolation of an F. tularensis subsp. tularensis strain Schu S4 transposon insertion mutant with a mutation in a predicted hypothetical lipoprotein, FTT1103, that was deficient in intracellular replication in HepG2 cells. In this study, a mutant with a defined nonpolar deletion in FTT1103 was created, and its phenotype, virulence, and vaccine potential were characterized. A phagosomal integrity assay and lysosome-associated membrane protein 1 colocalization revealed that ⌬FTT1103 mutant bacteria were defective in phagosomal escape. FTT1103 mutant bacteria were maximally attenuated in the mouse model; mice survived, without visible signs of illness, challenge by more than 10 10 CFU when the intranasal route was used and challenge by 10 6 CFU when the intraperitoneal, subcutaneous, or intravenous route was used. The FTT1103 mutant bacteria exhibited dissemination defects. Mice that were infected by the intranasal route had low levels of bacteria in their livers and spleens, and these bacteria were cleared by 3 days postinfection. Mutant bacteria inoculated by the subcutaneous route failed to disseminate to the lungs. BALB/c or C57BL/6 mice that were intranasally vaccinated with 10 8 CFU of FTT1103 mutant bacteria were protected against subsequent challenge with wild-type strain Schu S4. These experiments identified the FTT1103 protein as an essential virulence factor and also demonstrated the feasibility of creating defined attenuated vaccines based on a type A strain.

This pilot study suggests that a prolonged multidisciplinary rehabilitation programme in early-to-middle-stage HD is feasible, well-tolerated and associated with therapeutic benefit. Further explorative, larger studies are warranted.

Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds β first and then opens the clamp. Mutation of conserved “arginine fingers” in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.

Background: RFC and Rad24-RFC are clamp loaders with different roles in DNA replication. Results: RFC-PCNA binding is faster than PCNA opening, and PCNA opening by Rad24-RFC is about 10-fold weaker than by RFC. Conclusion: RFC binds PCNA prior to opening PCNA, and the Rfc1 subunit missing in Rad24-RFC is important for PCNA opening. Significance: These results provide insight into the mechanisms of these AAAϩ protein complexes.