Application of In Vivo Induced Antigen Technology (IVIAT) to Bacillus anthracis Sean M. Rollins1*, Amanda Peppercorn1, John S. Young 1 , Melissa Drysdale2,3, Andrea Baresch1, Margaret V. Bikowski1, David A. Ashford4, Conrad P. Quinn 5 , Martin Handfield6, Jeffrey D. Hillman 7 , C. Rick Lyons2, Theresa M. Koehler3, Stephen B. Calderwood1,8,9, Edward T. Ryan1,9,10 1 Massachusetts General Hospital, Boston, Massachusetts, United States of America, 2 University of New Mexico Health Science Center, Albuquerque, New Mexico, United States of America, 3 Department of Microbiology and Molecular Genetics, The University of Texas Medical School at Houston, Houston, Texas, United States of America, 4 United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Sao Paulo, Brazil, 5 Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 6 Center for Molecular Microbiology and Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, United States of America, 7 Oragenics Inc, Alachua, Florida, United States of America, 8 Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, United States of America, 9 Department of Medicine, Harvard Medical School, Boston, Massachusetts, United States of America, 10 Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America Abstract In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets. Citation: Rollins SM, Peppercorn A, Young JS, Drysdale M, Baresch A, et al. (2008) Application of In Vivo Induced Antigen Technology (IVIAT) to Bacillus anthracis. PLoS ONE 3(3): e1824. doi:10.1371/journal.pone.0001824 Editor: Abraham L. Sonenshein, Tufts University, United States of America Received November 29, 2007; Accepted February 5, 2008; Published March 19, 2008 Copyright: 2008 Rollins et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by AI053442 and N01-A1-40053 (ETR); U54 AI057159 (SBC); T32 AI07061 (AP), a New England Regional Center of Excellence/Biodefense and Emerging Infectious Disease Career Development Award U54 AI057159 (SMR), DE13523 (MH), U54 AI057156 (TMK and CRL) and AI33537 (TMK). Competing Interests: The authors have declared that no competing interests exist. * E-mail: srollins1@partners.org Introduction In vivo induced antigen technology (IVIAT) is an immuno- screening technique designed to identify immunogenic bacterial genes expressed specifically during infection [1–4]. We hypothe- sized that applying IVIAT to Bacillus anthracis, the cause of anthrax, could lead to increased understanding of bacterial events during infection, improved diagnostic assays, and novel therapeu- tic targets. IVIAT utilizes convalescent sera from patients or animals infected with a pathogen of interest [1–4]. Convalescent sera are pooled from several individuals and adsorbed against the cognate pathogen grown under standard laboratory culture conditions. Extensive adsorptions are performed to remove antibodies that bind bacterial antigens expressed in vitro, while retaining antibodies that recognize bacterial antigens specifically expressed during in vivo growth. Adsorbed serum is then used to probe an Escherichia coli-based inducible protein expression library of the pathogen. IVIAT thus provides a screen to identify proteins that generate a humoral immune response, and are displayed specifically during infection. Thus far, IVIAT has been applied to several pathogenic bacteria including Vibrio cholerae, Vibrio vulnificus, E. coli O157:H7, Salmonella enterica serovar Typhi, Streptococcus pyogenes (Group A), and Mycobacterium tuberculosis, among others [2,3,5–9]. In these applications, IVIAT has identified pathogen genes expressed uniquely in vivo, including antigens previously established to be expressed in vivo, and newly recognized virulence factors [2,3,5– 9]. Identification of up-regulated virulence genes by IVIAT has been confirmed in complementary studies, including gene microarray analysis, quantitative real time PCR (RT-PCR), signature tagged mutagenesis, and recombinase in vivo expressed technologies (RIVET), among others [9–11]. Certain in vitro conditions have been identified that induce expression of B. anthracis genes involved in pathogenesis during anthrax, including expression of toxins and capsule. For instance, growth of B. anthracis in Nutrient Broth Yeast extract medium PLoS ONE | www.plosone.org 1 March 2008 | Volume 3 | Issue 3 | e1824