Angewandte
Chemie
DOI: 10.1002/anie.201302303
Brucellosis Detection
Design and Synthesis of a Universal Antigen to Detect Brucellosis**
Julie Guiard, Eugenia Paszkiewicz, Joanna Sadowska, and David R. Bundle*
In memory of Malcolm Perry
Brucellosis is a highly contagious zoonosis primarily caused
by ingestion of unsterilized milk or meat from animals
infected by members of the genus Brucella.[1] Bovine brucel-
losis is caused by B. abortus, less frequently by B. melitensis,
and occasionally by B. suis. B. abortus in cattle causes
premature abortion in pregnant cows. In humans, Brucella
species cause a nonfatal but debilitating disease. Infection is
widespread globally and in those parts of the world where
malaria is endemic it would be useful to have a simple
diagnostic test to differentiate between malaria and Brucella
or other microbes as the cause of febrile fever.[2,3] Presump-
tive diagnosis depends on detection of antibodies to Brucella
A and M antigens and is confirmed by microbiological
culture.[4] The A- and M-antigenic determinants are expressed
Figure 1. Structures of the A and M antigenic determinants of B. abor-
simultaneously on the O-antigen polysaccharide domain of
tus and B. melitensis, respectively.
Brucella smooth lipopolysaccharides (sLPS), and this sLPS is
used to detect antibodies present in sera of animals or humans
suspected of being infected. Brucella is a virulent pathogen
requiring level 3 containment, rendering the production of
diagnostic O-antigens a demanding and specialized task. An
alternative diagnostic, the O-antigen of Yersinia enterocolitica
O:9 (level 2 containment) is an exclusively A antigen devoid
of M epitopes.
Brucella sLPS is resistant to partial degradation methods
that would permit isolation of pure A- or M-antigenic
determinants in quantities for practical application. A well-
defined synthetic antigen that incorporates both A and M
epitopes combined with a versatile tether that allows attach-
ment to surfaces, particles, and polymers could replace the
native O-antigen in diagnostic tests, including those required
for simple, on-site tests in remote locations.[5]
The O-antigen of Brucella sLPS is a homopolymer of the
rare sugar 4,6-dideoxy-4-formamido-a-d-mannose (a-d-
Rha4NFo).[6] Three Brucella antigenic phenotypes A+MÀ,
AÀM+, and A+ M+ were characterized using NMR spectros-
copy[6] and monoclonal antibodies[7] and confirmed that
a single O-polysaccharide molecule incorporates both A-
and M-antigenic determinants (Figure 1).[6e,7b] The A epitope
is dominant in most strains of B. abortus while the M epitope
is characteristic of B. melitensis. Prototypical A structures
consist predominantly of a1,2-linked d-Rha4NFo residue-
s.[6a,7b] In its highly expressed form, the M-antigenic determi-
nant was first proposed to contain one a1,3 linkage for every
four a1,2-linked residues.[6c,7b] A revised structure was
reported, where the ratio of 1,2 to 1,3 linkages is 3:1.[7c]
Even in Brucella strains defined as A+MÀ, an a1,3-linked
d-Rha4NFo is present and generally occurs at least once in
every 50 residues.[7b] This irregular structural feature is
consistent with the biosynthesis of these polysaccharides by
the ABC-type translocation pathway.[8] Two genes, wzm and
wzt, which typically form the ABC transporter/exporter of the
ABC-2 subfamily, are present in the B. melitensis genome.[9]
The precise structure of the M-antigenic determinant which is
devoid of any A activity remains uncertain.
Our work with monoclonal antibodies and synthetic
oligomers established that the antigenic determinant of the
B. abortus A antigen is most likely a tetrasaccharide of
contiguous a1,2-linked d-Rha4NFo residues.[6e,7b] While the
M antigen could in principle be characterized as an a1,3-
linked d-Rha4NFo disaccharide, this antigenic determinant is
more likely to be defined as a larger oligosaccharide with
adjacent a1,2-linked d-Rha4NFo residues that are sufficiently
short to preclude recognition by A-specific antibodies.[7b]
Pentasaccharide 1 (Figure 2) was selected as the largest
sized antigen that might selectively exhibit M-type character-
istics with limited cross reaction with A-specific antibodies.
Nonasaccharide 2 contains two A- and one M-type epitopes
(Figure 2) and should serve as a universal antigen to detect
antibodies in animals or humans infected by B. abortus,
B. melitensis, and B. suis.
[*] Dr. J. Guiard, Dr. E. Paszkiewicz, J. Sadowska, Prof. D. R. Bundle
Department of Chemistry, University of Alberta
Edmonton, Alberta T6G 2G2 (Canada)
E-mail: dave.bundle@ualberta.ca
[**] This work was made possible by a Natural Science and Engineering
Research Council of Canada Discovery grant to D.R.B.
The large size of the target oligosaccharides 1 and 2, and
the incorporation of an internal 1,3 linkage and a tether for
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2013, 52, 7181 –7185
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7181