Transcript Example of a scientific poster - SPUR

The Helicobacter pylori Effector Protein CagA Induces Cell Proliferation in the Drosophila Gut Through the Gut
Microbiota
Elisabeth Dewailly1 Allison Banse,Ph.D2 Anica Wandler,PhD2 Karen Guillemin, Ph.D2
University of Oregon Summer Program of Undergraduate Research 2012
1 Harvard University, Cambridge, Massachusetts, 2 University of Oregon Institute of Molecular Biology, Eugene, Oregon
Results
Does the CagA protein alter the intestinal
bacterial community of Drosophila? Is the
increase in cell proliferation in transgenic
CagA Drosophila dependent upon the
presence of its microbiota?
Methods
esg-GFP
esg-GFP, CagA
Mannitol
Agar
The gastric pathogen Helicobactor pylori is associated
with stomach cancer in humans. This pathogen injects
the cytotoxin-associated gene A (CagA) protein into host
cells, and CagA is a major risk factor for disease
development. The Guillemin lab has developed a
transgenic Drosophila model to examine the effects of
CagA on the intestinal epithelium of live Drosophila. We
found that CagA expression enhanced cell proliferation in
the intestinal epithelium. We next tested whether the
intestinal bacteria community differed between wild type
and CagA expressing flies. We dissected and plated out
homogenized CagA and control guts. We observed no
significant increase in the proportion of Lactobacillus
bacteria in CagA flies as compared to control flies,
although the data suggested some difference in
proportion. Based on these results, we hypothesize that
the increase in cell proliferation in CagA Drosophila is
dependent upon the microbiota and is incurred because
of an altered intestinal bacterial community. To test this
hypothesis, we measured rates of cell proliferation in the
intestinal epithelium of conventionally reared (CR) and
germ-free (GF) CagA and control Drosophila.
Surprisingly, we observed a significant difference
between CR CagA and GF CagA Drosophila, with the GF
CagA levels being identical to the level in GF wild type
Drosophila. This result suggests that all of the effects of
CagA are mediated through the intestinal microbiota.
Based on our characterization of the microbiota
composition, we hypothesize that either Lactobacillus is
involved in increasing cell proliferation or Acetobacter is
involved in decreasing cell proliferation in the intestinal
epithelium.
Problem
There was no
statistically
significant
increase in the
proportion of
Lactobacillus
in CagA flies
from control
flies.
Average Proportion of Bacteria
Abstract
For the plating experiment, I dissected 11 CagA fly guts and 13
control fly guts, homogenized them, and plated them on mannitol
agar. There were only two major types of Drosophila-associated
bacteria identified: Acetobacter bacteria were identified as the yellow
colonies, while the Lactobacillus bacteria were identified as the white
colonies.
Conclusion and Future Work
commensal microbes
The CagA Drosophila used were the progeny of a cross between
enhancer-trap esg-Gal4;UAS-GFP flies and UAS-Cag A flies, where
the expression of CagA is limited to the intestinal stem cells of the
CagA
progeny. The control Drosophila
used were the progeny of a cross
between enhancer-trap esg-Gal4;UAS-GFP flies and UAS-Dr/Tm3,
Sb flies. We derived both fly strains germ-free (GF) or reared them
(CR) . To derive germ-free flies, eggs were collected and
sterilized and put in germ-free bottles. We then dissected
intestines from adult flies, which were plated for bacterial
counts or immunostained using an antibody against
PhosphoHistone 3 (PH3) immunostaining to identify proliferating
cells.
esg-GFP
H. pylori
esg-GFP, CagA
PH3 immunostained guts
The CagA Drosophila used were the progeny of a cross between
enhancer-trap esg-Gal4;UAS-GFP flies and UAS-Cag A flies, where
the expression of CagA is limited to the intestinal stem cells of the
progeny. The control Drosophila used were the progeny of a cross
between enhancer-trap esg-Gal4;UAS-GFP flies and UAS-Dr/Tm3,
Sb flies. We derived both fly strains germ-free (GF) or reared them
(CR) . To derive germ-free flies, eggs were collected and
sterilized and put in germ-free bottles. We then dissected
intestines from adult flies, which were plated for bacterial
counts or immunostained using an antibody against
host intestinal epithelium
PhosphoHistone 3 (PH3) immunostaining to identify proliferating
cells.
Surprisingly, we observed a
significant difference in cell
proliferation between CR CagA
and CR control flies, a
significant difference between
CR CagA and GF CagA flies,
and no significant difference
between GF CagA and GF
control flies.
This work implies that all of the increase in cell
proliferation in CagA flies is dependent upon the
presence of the microbiota. More plating data would
need to be collected in order to determine whether
the Lactobacillus Drosophila bacterial group is
involved in increasing cell proliferation or
Acetobacter is involved in decreasing cell
proliferation. Future directions would explore how
specific bacterial subgroups are responsible for the
increased rates of cell proliferation, by measuring
cell proliferation in flies mono-associated with
particular bacterial strains. These results suggest
that Helicobacter pylori infection in humans could
increase gastric cancer risk by altering the stomach
microbiota.
References
Funding: NIH-1R25HD070817, NICHD Summer Research Program
at the University of Oregon
PH3 Mannitol Agar images courtesy of Anica Wandler