Orientation within the exosporium and structural stability of the collagen-like glycoprotein BclA of Bacillus anthracis.
Bacillus anthracis spores, which cause anthrax, are enclosed by an exosporium consisting of a basal layer and an external hair-like nap. The filaments of the nap are composed of BclA, a glycoprotein containing distinct N-terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region. In this study, we used immunogold electron microscopy to show that the CTD of BclA forms the distal end of each filament of the hair-like nap, indicating that the NTD is attached to the basal layer. Ten randomly chosen anti-BclA monoclonal antibodies, raised against spores or exosporium, reacted with the CTD, consistent with its exterior location. We showed that recombinant BclA (rBclA), encoded by the B. anthracis Sterne strain and synthesized in Escherichia coli, forms a collagen-like triple helix as judged by collagenase sensitivity and circular dichroism spectroscopy. In contrast, native BclA in spores was resistant to collagenase digestion. Thermal denaturation studies showed that the collagen-like region of rBclA exhibited a melting temperature (T(m)) of 37 degrees C, like mammalian collagen. However, rBclA trimers exhibited T(m) values of 84 degrees C and 95 degrees C in buffer with and without sodium dodecyl sulfate, respectively. CTD trimers exhibited the same T(m) values, indicating that the high temperature and detergent resistances of rBclA were due to strong CTD interactions. We observed that CTD trimers are resistant to many proteases and readily form large crystalline sheets. Structural data indicate that the CTD is composed of multiple beta strands. Taken together, our results suggest that BclA and particularly its CTD form a rugged shield around the spore.
Boydston
Jeremy A
JA
UAB Department of Microbiology, BBRB 409, 1530 3rd Ave. S, Birmingham, AL 35294-2170, USA.
Chen
Ping
P
Steichen
Christopher T
CT
Turnbough
Charles L
CL
D001408:Bacillus anthracis / Q000737:chemistry*; D003094:Collagen / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D003460:Crystallization; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D013696:Temperature
C470593:BclA protein, Bacillus anthracis; D008562:Membrane Glycoproteins; D003094:Collagen; D017364:Collagenases