Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis.
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.
Rollins
Sean M
SM
Massachusetts General Hospital, Boston, Massachusetts, United States of America. srollins1@partners.org
Peppercorn
Amanda
A
Young
John S
JS
Drysdale
Melissa
M
Baresch
Andrea
A
Bikowski
Margaret V
MV
Ashford
David A
DA
Quinn
Conrad P
CP
Handfield
Martin
M
Hillman
Jeffrey D
JD
Lyons
C Rick
CR
Koehler
Theresa M
TM
Calderwood
Stephen B
SB
Ryan
Edward T
ET
D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001408:Bacillus anthracis / Q000235:genetics / Q000276:immunology*; D020869:Gene Expression Profiling; D008253:Macaca mulatta; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000942:Antigens, Bacterial; D012333:RNA, Messenger