Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis

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.

Figure 1. mRNA expression profiles during in vitro and in vivo growth of IVIAT-identified and paralog/similar B. anthracis genes.

Quantitative RT-PCR was performed on RNA recovered from in vitro grown B. anthracis (Ames strain cells grown in BHI and air; late-log phase) and compared to RNA recovered from mice infected with vegetative Ames strain B. anthracis via intravenous injection. RNA from mice was isolated from spleens 18 hr post-infection. Transcript numbers on the Y-axis are normalized against 16S rRNA. Displayed are gene profiles of B. anthracis genes up-regulated in vivo compared to in vitro. In bar graphs, expression levels represent mean values; error bars represent standard deviations (SD). In summary table, expression given as mRNA transcript copies (× 10⁻⁵) per copy of 16S rRNA.

Figure 2. B. anthracis species-specific autolysis effects following addition of exogenous putative autolysins N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446: (A) reduction in A₅₉₅ optical density, (B) reduction in colony forming units, and (C, D) morphological changes of B. cereus (C) or B. anthracis (D).

B. anthracis (Sterne strain), B. cereus (ATCC 14579), B. subtilis (168), and E. coli (DH5α) vegetative cells were resuspended in 20 mM sodium phosphate buffer containing BA0485 and BA2446 (final concentration 2 uM) or buffer alone (BA0485, pH 7.0; BA2446, pH 8.7). (A) A₅₉₅ readings were recorded every 2 min. and reported as a percentage of starting A₅₉₅. Buffer-only controls were not different for cultures containing B. cereus, B. subtilis, or E. coli. (B) Dilutions of cells were plated prior to addition of BA0485 and BA2446 proteins and then again following 20 min of incubation with buffer alone or buffer containing exogenous BA0485 or BA2446, and CFU are reported as percentage of the starting CFU. (C, D) BA0485, BA2446, or buffer alone were added to cell suspensions of B. anthracis, B. cereus, B. subtilis, and E. coli, and incubated for 20 min. Bacterial morphological changes were only evident following protein addition to B. anthracis (D); representative B. cereus samples are shown (C). Gram stains were performed and all images were captured at 1000×.