Download An investigation of the mechanism of destruction by mammalian

An investigation of the mechanism of destruction by mammalian immune cells of azidelabeled Helicobacter pylori
Jennifer Helble, class of 2014
Helicobacter pylori is a Gram-negative bacterial species that colonizes the gastric tract of
over 50% of the world’s population and is one of the leading causes of gastric cancer and peptic
ulcers.1 As with many other species of bacteria, certain H. pylori strains have started to build up
antibiotic resistance to current treatment methods.1 Thus, there is an essential need for alternative
therapeutics to slow the infection rate around the world.
H. pylori pathogenesis is directly linked to its ability to glycosylate proteins.2 These
glycoproteins are not present in mammalian cells, and are therefore viable targets for selectively
labeling H. pylori in a mammalian host. A technique called metabolic oligosaccharide
engineering (MOE) can be used to label only H. pylori glycoproteins by using an unnatural sugar
that mammalian cells are unable to process.2 Once H. pylori’s glycoproteins have been labeled
using this unnatural sugar, they can be linked to an immune stimulant such that the introduction
of antibodies and mammalian immune effector cells allow for the destruction of the bacteria.3
This summer, I set out to optimize this approach by investigating varying concentrations of
azido-sugar and phosphine warhead. Effectiveness of eradication was determined using a livedead assay, which essentially stains live cells green and dead cells red. The live versus dead cell
populations were then counted using an instrument called a flow cytometer, which detects
cellular fluorescence. Data generated from the instrument were further analyzed for the
percentage of dead cells. My results indicate that there were no significant differences between
the original conditions (as used by the Dube lab previously) and the various experimental
conditions I explored. Thus, I concluded the original conditions used for prior experiments were
appropriate for future experiments.
In addition to attempting to optimize the killing conditions of H. pylori, I also set out to
investigate the exact immune system mechanism of destruction of the bacteria. There are two
possible immune-mediated ways by which H. pylori cell death can occur: antibody-dependent
cell mediated cytotoxicity (ADCC) and antibody-dependent cell mediated phagocytosis (ADCP).
ADCC has been previously shown to be a valid mechanism of destruction but leads to short term
immunity.3 The presence of ADCP has yet to be determined, but if found to occur, could lead to
long-term immunity and prevention against future infections. Unfortunately, due to
complications with the ADCP assay, I have not yet determined if this mechanism of destruction
occurs.
Faculty Mentor: Danielle Dube
Funded by the Kibbe Science Fellowship
References
1. McColl, K. E. L. (2010) Helicobacter pylori Infection. New England Journal of Medicine 362,
1597-1604.
2. Dube, D. H., Champasa, K., and Wang, B. (2011) Chemical tools to discover and target
bacterial glycoproteins. Chemical Communications 47, 87-101.
3. Kaewsapsak, P., Esonu, O., and Dube, D. H. (2013) Recruiting the Host's Immune System to
Target Helicobacter pylori's Surface Glycans. ChemBioChem 14, 721-726.