The immunogenicity and protective efficacy of overlapping regions of the protective antigen (PA) polypeptide, cloned and expressed as glutathione S-transferase fusion proteins, have been assessed. Results show that protection can be attributed to individual domains and imply that it is domain 4 which contains the dominant protective epitopes of PA

The highly virulent intracellular pathogenFrancisella tularensisis a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis ofFrancisellavirulence in the Fischer 344 rat, we have constructed anF. tularensisSchu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. A transposon-directed insertion site sequencing (TraDIS) approach was used to identify 453 genes essential for growthin vitro. Many of these essential genes were mapped to key metabolic pathways, including glycolysis/gluconeogenesis, peptidoglycan synthesis, fatty acid biosynthesis, and the tricarboxylic acid (TCA) cycle. Additionally, 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen. Thisin vivoselection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-typeF. tularensisSchu S4 strain.IMPORTANCEThe intracellular bacterial pathogenFrancisella tularensiscauses a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches.F. tularensisis one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important forF. tularensisunderin vitroandin vivoconditions, providing candidates that can be evaluated for vaccine or antibacterial development.

The a-toxin produced by the type strain of Clostridium perfringens (NCTC 8237) was shown to differ from the a-toxins produced by most strains of C. perfringens isolated from man and from calves with respect to reactivity with a neutralizing monoclonal antibody (DY2F5D11). The difference in antibody binding correlated with three differences in the deduced amino acid sequence (Ala174 to Asp, , ; Thr177 to Ala177; Ser335 to Pr o, , , ) of the a-toxins. Using octapeptides synthesized on the basis of the amino acid sequences from these regions of variability, it was shown that the Ala174 to Asp, , change had the greatest effect on reducing the binding of monoclonal antibody DY2F5Dll to the a-toxin. These differences did not affect the enzymic or toxic properties of the protein. However, the phospholipase C activity of the a-toxin produced by strain NCTC 8237 was more susceptible to inactivation by chymotrypsin. The changes in amino acid sequence did not affect the ability of a C-terminal domain vaccine, derived from the a-toxin of strain NCTC 8237, to induce protection against the a-toxin from a bovine enteric strain of C. perfringens.

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