Table 1. B. anthracis gene products identified by IVIAT. Transport BA0669 (rbsB) Metabolism BA2125 (narG) Functional category Virulence Transport Peptidoglycan architecture Peptidoglycan architecture Peptidoglycan architecture Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical ORF BA0314 pXO1-110 (pagA) Protective antigen Gene product-description, function (reference) Predicted cellular location Gene encoded by Extracellular pXO1 Substrate-binding protein of an ABC transporter Membrane associated Ribose binding protein of a ribose ABC transporter Respiratory nitrate reductase, alpha subunit BA2748 (sleB) Spore cortex lytic protein BA4073 (plyL) N-acetylmuramoyl-L-alanine amidase Extracellular, cell- associated BA4606 (pbpI) Penicillin binding protein pXO2-08 BXB0048 BA0698 BA4074 BA5474 NLP/P60 lipoprotein family Hypothetical protein Hypothetical protein Prophage holin Degenerative enterotoxin Membrane associated Unknown Unknown Extracellular, cell- associated Extracellular, cell- associated Unknown Unknown Unknown Membrane associated Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome pXO2 pXO2 Chromosome Chromosome # of clones identified by primary screening/# of independent clones identified by primary screening 3/3 1/1 1/1 3/1 1/1 8/4 1/1 1/1 1/1 8/4 4/1 28/11 B. anthracis IVIAT Comments Known virulence factor [45,46] Predicted ABC metal ion transporter; contains lipoprotein domain Contains membrane- associated signal peptide sequence May assist organism to persist in anaerobic conditions [47]. See text See text [17] See text See text See text See text See text See text doi:10.1371/journal.pone.0001824.t001 (NBY) in the presence of 5–20% CO2 and sodium bicarbonate at 37uC (selected to mimic the physiology of blood) is associated with increased expression of protective antigen (PA) and capsule compared to that observed during growth in standard laboratory media such as Brain Heart Infusion (BHI) in the presence of air (0.03% CO2) at 37uC [12]. Although PA itself is non-toxic, PA binds specific host cell receptors and forms a pre-pore, which binds either edema factor (EF) or lethal factor (LF) to create edema toxin (ET) and lethal toxin (LT), respectively [13]. Anti-PA immune responses correlate with protection from anthrax, and PA is a primary immunogen in existing anthrax vaccines [14]. Full virulence of B. anthracis also correlates with presence of B. anthracis capsule, which is comprised of poly-D-glutamic acid. The presence of capsule impedes opsonophagocytosis and clearance of B. anthracis organisms from infected hosts [15]. Un-encapsulated strains of B. anthracis are markedly attenuated in both humans and animals, despite their ability to express fully functional LT and ET complexes. Beneath the capsule is a proteinaceous S-layer, composed of two paracrystalline proteins (extractable antigen 1 [EA1] and surface array protein [SAP]) that cover the bacterial cell surface [16]. Beneath the S-layer is a peptidoglycan layer comprising the cell wall. B. anthracis contains two large virulence plasmids: genes encoding toxin proteins PA (pagA), LF (lef), and EF (cya) are on the large virulence plasmid pXO1 (182 kb; 143 ORFs), while genes encoding proteins involved in encapsulation are on the large virulence plasmid, pXO2 (96 kb; 85 ORFs). Many B. anthracis genes on pXO1, pXO2, and the single large chromosome have not been characterized, and it is possible that additional factors induced during infection and not under typical in vitro culture conditions contribute to B. anthracis pathogenesis. Identification of such gene products could improve our understanding of host- pathogen interactions during anthrax, and could assist further development of appropriate therapeutic, diagnostic, or preventa- tive measures for anthrax. To identify antigenic B. anthracis proteins displayed specifically during B. anthracis infection, we applied IVIAT using convalescent sera from partially immunized macaques surviving aerosol challenge with wild type B. anthracis Ames spores, quantified expression of identified genes in a mouse model of virulent B. anthracis Ames infection, and performed preliminary functional analysis on a subset of identified B. anthracis gene products. Results and Discussion Identification of B. anthracis proteins expressed in vivo by IVIAT Using IVIAT, we immuno-screened an approximately 125,000 clone inducible B. anthracis expression library in E. coli BL21(DE3) (17,000 pXO1 clones, 7,500 pXO2 clones, and 100,000 chromosomal clones). We identified 60 immuno-reactive clones, representing 25 distinct B. anthracis genes or gene fragments. Of these, we constructed full length constructs and confirmed immuno-reactivity of ten (Table 1). Significant immuno-reactivity PLoS ONE | www.plosone.org 2 March 2008 | Volume 3 | Issue 3 | e1824