Identification and characterization of novel and potent transcription promoters of Francisella tularensis.

Two alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potent Francisella tularensis promoters. Of the 26,000 F. tularensis strain LVS gfp library clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr).

Novel and unique diagnostic biomarkers for Bacillus anthracis infection.

A search for bacterium-specific biomarkers in peripheral blood following infection with Bacillus anthracis was carried out with rabbits, using a battery of specific antibodies generated by DNA vaccination against 10 preselected highly immunogenic bacterial antigens which were identified previously by a genomic/proteomic/serologic screen of the B. anthracis secretome. Detection of infection biomarkers in the circulation of infected rabbits could be achieved only after removal of highly abundant serum proteins by chromatography using a random-ligand affinity column.

Characterization of Bacillus anthracis iron-regulated surface determinant (Isd) proteins containing NEAT domains.

Three iron-regulated surface determinant (Isd) proteins, containing NEAr Transporter (NEAT) domains (GBAA4789-7), constitute part of an eight-member Bacillus anthracis operon. GBAA4789 (IsdC), previously characterized by others as a haem-binding protein, and two novel Isd proteins characterized in this study, GBAA4788 (IsdJ) and GBAA4787 (IsdK) proteins, can be translated from two alternative overlapping transcriptional units. The three NEAT-containing Isd proteins are shown to be expressed in vivo during B.

In vitro screen of bioinformatically selected Bacillus anthracis vaccine candidates by coupled transcription, translation, and immunoprecipitation analysis.

The availability of the Bacillus anthracis genome sequence allowed for in silico selection of a few hundred open reading frames (ORFs) as putative vaccine candidates. To screen such a vast number of candidate ORFs, without resorting to laborious cloning and protein purification procedures, methods were developed for generation of PCR elements, compatible with in vitro transcription-translation and immunoprecipitation, as well as with their evaluation as DNA vaccines. Protocols will be provided for application of these methods to analyze the anti-B.

Identification of in vivo-expressed immunogenic proteins by serological proteome analysis of the Bacillus anthracis secretome.

In a previous comparative proteomic study of Bacillus anthracis examining the influence of the virulence plasmids and of various growth conditions on the composition of the bacterial secretome, we identified 64 abundantly expressed proteins (T. Chitlaru, O. Gat, Y.