BA1952 and BA5427 (lytE; putative endopeptidase). BA1952 and BA5427 have been demonstrated to be antigenic secreted proteins, and both contain P60 and S-layer homology (SLH) domains [31–33]. SLH domains are commonly associated with cell surface proteins and non-covalently anchor proteins to the cell wall polysaccharide, suggesting cell surface localization [34]. BA5427 is homologous to B. subtilis lytE, and LytE (also referred to as CwlF) has been shown to function as a cell wall lysin that affects bacterial cell separation in B. subtilis [35]. Our application of IVIAT to B. anthracis also identified BA5474, encoding a pseudogene that has sequence similarity to several genes encoding N-acetylmuramoyl-L-alanine amidases and an enterotoxin in B. cereus [36]. RT-PCR quantification of in vivo expression of IVIAT- identified and paralog B. anthracis genes during wild type B. anthracis infection To further assess in vivo expression of IVIAT-identified ORF products, we measured mRNA levels of antigens identified by IVIAT. Comparing total bacterial RNA recovered from blood, spleen, and lung of mice 6, 12, or 18 hours after inoculation of vegetative B. anthracis Ames, we detected the highest quantity of bacterial RNA in spleen specimens recovered 18 hours after infection. Based on rRNA 16S concentrations, spleen specimens contained bacterial to mouse RNA in ratios ranging from 1:100 to 1:2,000. Comparing RNA recovered from spleen samples to RNA recovered from in vitro cultured organisms grown under the same conditions used to adsorb sera, we detected up-regulation of five B. anthracis IVIAT-identified genes or paralogs/similar proteins in in vivo samples using RT-PCR (Figure 1): pagA; the alanine-amidase genes, BA3767, BA4073, and amiA (pXO2-42); and the bacterio- phage holin gene BA4074. Within our assay, pagA mRNA was present at approximately 54-fold higher levels in in vivo samples than in vitro samples. mRNA of L-alanine amidases BA4073, BA3767, amiA (pXO2-42), as well as prophage holin BA4074 were also elevated in in vivo samples compared to in vitro samples (33- fold, 24-fold, 16-fold, and 11-fold higher, respectively). Our identification of PA by IVIAT and our confirmation of up- regulation of pagA mRNA in vivo confirms the validity of our approach since PA is preferentially expressed in vivo and is immunogenic [12,14]. Of note, our inducible B. anthracis library was constructed using B. anthracis strains that did not encode LF or EF, and we would, therefore, not expect our screens to have identified these virulence factors. Our documentation of induced expression of mRNA in vivo of only a subset of genes identified by IVIAT (and not the complete set) is also not unexpected, since mRNA quantity is only one of a number of variables that may alter expression or display of a given protein. Indeed, separate proteomic based analysis has recently demonstrated increased protein expression of two additional B. anthracis gene products that we have identified by IVIAT, lipoproteins BA1952 and BA5427, when B. anthracis are grown in high versus low CO2 concentrations (the former conditions thought to mimic the in vivo environment) [31]. In addition, a number of IVIAT identified antigens (BA0314, BA0485, BA1952, BA2805, BA2748, BA3737, BA5427, pXO2-08 and pXO2-42) were also previously identified by Gat et al. using a bioinformatics screening strategy to select B. anthracis ORFs for use in an in vitro transcription/translation assay followed by immunoprecipitation using sera from B. anthracis- infected animals [37]. Identification of these ORF products by both our application of IVIAT to B. anthracis, and by Gat et al.’s screening system, support identified ORF-product immuno- reactivity in vivo. B. anthracis IVIAT Species-specific effects of N-acetyl-L-alanine amidases BA0485 and BA2446 on B. anthracis We were intrigued that a number of the B. anthracis proteins identified in our analysis were predicted to be involved in cell wall- peptidoglycan synthesis or remodeling, and that a number were homologous to known lysins. In order to investigate this further, we focused our efforts on the best characterized family of identified proteins, the L-alanine amidases. We were able to purify recombinant versions of two identified amidases, BA0485 and BA2446. Upon addition of exogenous L-alanine amidases BA0485 (buffered at pH 7.0) or BA2446 (buffered at pH 8.7), the OD595 of a cell suspension made from exponential phase organisms fell specifically for B. anthracis, but not for B. cereus, B. subtilis, or E. coli DH5a (Figure 2A). This drop in OD595 corresponded to a 50% reduction in CFU for B. anthracis grown in the presence of BA0485 or BA2446, and this reduction in CFU was not noted in cultures containing BA0485 or BA2446 and B. cereus, B. subtilis, E. coli, or buffer alone (Figure 2B). B. anthracis vegetative bacteria cultured in the presence of exogenous BA0485 or BA2446 also had decreased chain length compared to B. anthracis cultured in buffer alone (BA0485: 1.06 [mean] +/20.01 [standard error of mean] versus 2.17 +/20.08, p,0.001; BA2446: 1.14 +/20.02 versus 2.43 +/ 20.07, p,0.001; Figure 2C–D), as well as a more swollen appearance. The exogenous addition of BA0485 or BA2446 to cultures of B. cereus, B. subtilis, or E. coli had no measurable effect on bacterial morphology, chain length, or viability. Although our exogenous addition of these proteins to in vitro grown cultures may not represent their in vivo location or activity, our data strongly suggest an effect of these proteins on B. anthracis cell wall integrity, a focus of on-going work in our laboratory. We also noted an optimal pH requirement (pH = 8.7) for the BA2446 product in vitro, and although similar pH dependence has previously been noted for autolysins specific for B. subtilis [38], the physiological significance of this observation in vivo is currently unclear. Although our application of IVIAT to B. anthracis has identified a number of genes warranting additional evaluation, our study has a number of limitations. (1) IVIAT only identifies immuno- reactive proteins (not carbohydrates or lipids). (2) Our inducible protein library was undoubtedly only partial due to the Sau3A partial digestion used to generate library clones, the effect of codon bias on protein expression, and difficulties expressing all antigens (especially membrane associated proteins) equivalently in E. coli based expression libraries. (3) Immuno-reactivity of IVIAT- identified antigens is a marker of expression of proteins in vivo, and does not predict protective humoral or cellular immune responses. (4) Immuno-reactivity of proteins may also reflect immune responses to related antigens, although we have attempted to identify cross-reactivity in these experiments by also analyzing related proteins. (5) Due to genetic cross-talk between the virulence plasmids and the chromosome of B. anthracis [39], our use in the adsorption protocol of attenuated B. anthracis strains that did not contain the complete genome in a single strain may have misrepresented potential gene expression in vitro. (6) Our use of convalescent phase sera from macaques partially immunized against anthrax and then surviving inhalational challenge with virulent B. anthracis, may not exactly mirror events that occur during natural infection in un-immunized humans or animals, and anti-PA responses in macaques may not correlate with overall anti- B. anthracis immunoreactivity. (7) Our use of the intravenously- challenged mouse model to measure bacterial gene expression in vivo may not exactly reflect events that occur during natural infection (since the mouse model involves neither spore germina- tion nor formation); however, using this model, we were still able PLoS ONE | www.plosone.org 4 March 2008 | Volume 3 | Issue 3 | e1824