POSTERS - BLAST X - University of Utah

BLAST X Poster #11
THE COMPLEX HOOK BASAL BODY STRUCTURE OF THE LYME DISEASE SPIROCHETE
BORRELIA BURGDORFERI
K. Miller 1 , R. Duda 2 , R. Hendrix 2 , M. Motaleb 1,3 , and N.W. Charon 1
1
West Virginia University Health Sciences Center, Microbiology and Immunology Department,
Box 9177, Room 2077, Morgantown, WV 26506
2
University of Pittsburgh, Department of Biological Sciences, 4249 Fifth Avenue, 357A,
Crawford Hall, Pittsburgh, PA 15260
3
East Carolina University, Department of Microbiology and Immunology, Greenville, NC 27834
FlgE is the structural protein of the flagellar hook of B. burgdorferi. Previous western blot
analysis using polyclonal FlgE antibody indicated that the majority of the hook migrated as a
high molecular weight complex in SDS-PAGE gels instead of at the position of monomeric FlgE
(46 kDa). Both the high molecular complex and a small amount of the monomer were present in
wild-type and complemented cells but absent in flgE mutants. These data suggest that FlgE
may be cross-linked into oligomers in a manner similar to that found in bacteriophage capsid
proteins and in some bacterial pili. High molecular weight FlgE complexes have been observed
in other spirochete species (Treponema phagedenis and Treponema denticola), suggesting that
this property is conserved. We are interested in determining whether or not FlgE is cross-linked,
and what amino acids are involved in the cross-linking.
The hook-basal body complexes were purified using a refined version of methods
previously reported (Sal, et al, 2008). Following purification of periplasmic flagella that contained
attached hook-basal body complexes, the filament portions were depolymerized by acidic
conditions or the use of urea. Hook-basal body complexes were then isolated by pelleting using
an ultracentrifuge recovered in a neutralizing buffer. Electron microscopy of these preparations
showed that acidic conditions resulted in more intact hook-basal body complexes than urea
treatment, although the high molecular weight forms of FlgE were found using both methods. In
order to determine results from each experiment, the experimental samples were run on 10%
SDS-PAGE gels. We are using proteolytic finger printing and peptide mapping methods to
compare the high molecular weight complexes of FlgE with the monomeric form (histidinetagged
recombinant). Hook-basal body complexes and recombinant FlgE were treated with
proteases or hydroxylamine at various temperatures (37, 45, and 65°C) and run in SDS-PAGE
gels. Western blotting was used to assess the stability of the FlgE complex as well as to
determine the size of (antigenic) peptide fragments resulting from proteolytic digestion. Digest
patterns were compared between recombinant FlgE and the isolated hook-basal body
complexes.
Refining the hook-basal body isolation procedure resulted in improved hook-basal body
preparations. The FlgE complex is stable under harsh conditions (both urea and acid treatment).
Differences and similarities in hydroxylamine digest patterns between recombinant FlgE and
purified hook-basal body complexes supports the hypothesis that FlgE is cross-linked.
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