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kinase, chemoattractant-induced migration, and TLR-induced
proliferation. Moreover, we found that TLR-stimulated antibody
responses by B-1 and MZ B cells, as well as the localization of
MZ B cells in the spleen, depend on p110delta activity. Finally,
we show that the in vivo production of natural antibodies
requires p110delta and that p110delta inhibitors can reduce in
vivo autoantibody responses. Thus targeting p110delta may be a
novel approach for regulating innate-like B cells and for treating
antibody-mediated autoimmune diseases.
The Biosynthesis of CMP-N,N’-
Diacetyllegionaminic Acid from UDP-
N,N’-Diacetylbacillosamine in Legionella
pneumophila
Pavel A. Glaze 1 , David C. Watson 2 , N. Martin Young 2
and Martin E. Tanner 1
1
Department of Chemistry, University of British Columbia,
2
Institute for Biological Sciences, National Research Council of
Canada, Ottawa, Ontario
Legionaminic acid is a nine carbon alpha-keto acid that is similar
in structure to other members of the sialic acid family that
include neuraminic acid and pseudaminic acid. It is found as a
component of the lipopolysaccharide in several bacterial species,
and is perhaps best known for its presence in the O-antigen
of the causative agent of Legionnaires’ disease, Legionella
pneumophila. In this work, the enzymes responsible for the
biosynthesis and activation of N,N’-diacetyllegionaminic acid are
identified for the first time. A cluster of three L. pneumophila
genes bearing homology to known sialic acid biosynthetic genes
(neuA,B,C) were cloned and overexpressed in Escherichia
coli. The NeuC homolog was found to be a hydrolyzing
UDP-N,N’-diacetylbacillosamine 2-epimerase that converts
UDP-N,N’-diacetylbacillosamine into 2,4-diacetamido-2,4,6-
trideoxymannose and UDP. Stereochemical and isotopic labeling
studies showed that the enzyme utilizes a mechanism involving
an initial anti-elimination of UDP to form a glycal intermediate,
and a subsequent syn-addition of water to generate product. The
NeuB homolog was found to be a N,N’-diacetyllegionaminic acid
synthase that condenses 2,4-diacetamido-2,4,6-trideoxymannose
with phosphoenolpyruvate (PEP), although the in vitro activity
of the recombinant enzyme (isolated as a MalE-fusion protein)
was very low. The synthase activity was dependent on the
presence of a divalent metal ion and the reaction proceeded via
a C-O bond cleavage process, similar to the reactions catalyzed
by the sialic acid and pseudaminic acid synthases. Finally,
the NeuA homolog was shown to possess the CMP-N,N’-
diacetyllegionaminic acid synthetase activity that generates the
activated form of legionaminic acid used in lipopolysaccharide
biosynthesis. Together, the three enzymes constitute a pathway
that converts a UDP-linked bacillosamine derivative into a CMPlinked
legionaminic acid derivative.
Identification of Cpn60.2 as a surface
ligand of Mycobacterium tuberculosis
that mediates bacterial binding to
macrophages via CD43
T. B. M. Hickey 1 , H. J. Ziltener 1 , D. P. Speert 1,2,3 , R. W.
Stokes 1,2,3
1
Department of Pathology & Laboratory Medicine, University of
British Columbia
2
Department of Pediatrics, BC Children’s Hospital
3
Department of Microbiology & Immunology, University of British
Columbia
Mycobacterium tuberculosis bacilli initially contact host cells with
elements of their outer cell wall, or capsule. We have shown that
capsular proteins from the surface of M. tuberculosis competitively
inhibit the non-opsonic binding of whole M. tuberculosis bacilli to
macrophages in a dose-dependent manner that is not acting through
a global inhibition of macrophage binding. CD43 is a large
sialylated glycoprotein that is found on the surface of macrophages
and has been shown in previous studies to be necessary for efficient
macrophage binding and immunological responsiveness to M.
tuberculosis. Using capsular proteins and recombinant CD43,
we have employed affinity chromatography to show that Cpn60.2
(Hsp65, GroEL), and to a lesser extent DnaK, bind to CD43. We
subsequently demonstrated that both Cpn60.2 and DnaK can be
identified on the surface of M. tuberculosis bacilli. Furthermore,
we performed recombinant protein competitive inhibition and
polyclonal F(ab’)2 antibody-mediated epitope masking studies to
show that Cpn60.2, but not DnaK, acts as a mycobacterial adhesin
for macrophage binding. We then compared M. tuberculosis
binding of CD43+/+ versus CD43-/- macrophages and found that
the adhesin function of Cpn60.2 is CD43-dependent. Additionally,
the binding between Cpn60.2 and CD43 can be saturated; however
the binding affinity is comparatively weak in the micromolar range.
We also showed that the ability of Cpn60.2 to competitively inhibit
M. tuberculosis binding to macrophages is shared by the Escherichia
coli homologue, GroEL, but not by the mouse and human Hsp60
homologues. These findings add to a growing field of research that
implicates molecular chaperones as having extracellular functions,
including bacterial adherence to host cells, distinct from their welldescribed
protein folding activities within the cytosol.
Impact of an antimicrobial stewardship
program on treatment of critically
ill patients with Gram-positive
bloodstream infections
Amanda Israel 1 , Andrew M. Morris MD SM (Epi)
FRCPC 2
1
Medical Surgical ICU, Mount Sinai Hospital, Interdepartmental
Division of Critical Care Medicine, University of Toronto
2
Antimicrobial Stewardship Program, Mount Sinai Hospital,
Division of Infectious Diseases, Department of Medicine,
University of Toronto
UBCMJ | APRIL 2010 1(2) | www.ubcmj.com 3