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.
Highlights
In vivo induced antigen technology (IVIAT) is an immunoscreening technique designed to identify immunogenic bacterial genes expressed during infection [1,2,3,4]
Significant immuno-reactivity of clones containing in-frame protein-coding fragments of BXB0048 and BA2125 and lacking gene fragments of other B. anthracis genes was detected; immunoreactivity could not be confirmed in clones containing the complete ORFs of these gene fragments, possibly suggesting inefficient expression or display of full-length products in E. coli
Since a number of identified seroreactive ORF products were predicted to be cell wall active amidases or hydrolases, to evaluate whether immuno-reactivity was unique to a given IVIATidentified ORF, we evaluated immuno-reactivity of paralogs of IVIAT-identified genes BA4073 and pXO2-08, and identified eight additional gene products with immuno-reactivity (Table 2)
Summary
In vivo induced antigen technology (IVIAT) is an immunoscreening technique designed to identify immunogenic bacterial genes expressed during infection [1,2,3,4]. To identify antigenic B. anthracis proteins displayed 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.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
