Gut permeability, its interaction with gut microflora and ...
The Helicobacter pylori Effector Protein CagA Induces Cell Proliferation in the Drosophila Gut...
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The Helicobacter pylori Effector Protein CagA Induces Cell Proliferation in the Drosophila Gut Through the Gut Microbiota Elisabeth Dewailly 1 Allison Banse,Ph.D 2 Anica Wandler,PhD 2 Karen Guillemin, Ph.D 2 University of Oregon Summer Program of Undergraduate Research 2012 1 Harvard University, Cambridge, Massachusetts , 2 University of Oregon Institute of Molecular Biology, Eugene, Oregon Abstract 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 Methods Results Conclusion and Future Work 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? esg-GFP, CagA esg-GFP Mannitol Agar 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 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. esg-GFP esg-GFP, CagA PH3 immunostained guts 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 in humans could increase gastric cancer microbiota. 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. host intestinal epithelium CagA H. pylori commensal microbes References Funding: NIH-1R25HD070817, NICHD Summer Research Program at the University of Oregon PH3 Mannitol Agar images courtesy of Anica Wandler There was no statistica lly significan t increase in the proportion of Lactobacillus in CagA flies from control flies. 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 PhosphoHistone 3 (PH3) immunostaining to identify proliferating cells. Average Proportion of Bacteria
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Transcript of The Helicobacter pylori Effector Protein CagA Induces Cell Proliferation in the Drosophila Gut...
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
Abstract
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
Methods
Results
Conclusion and Future Work
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?
esg-
GF
P, C
agA
esg-
GF
P
Man
nito
l A
gar
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 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.
esg-GFP esg-GFP, CagA
PH3 immunostained guts 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.
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.
host intestinal epithelium
CagA
H. pylori
commensal microbes
References
Funding: NIH-1R25HD070817, NICHD Summer Research Program at the University of Oregon
PH3 Mannitol Agar images courtesy of Anica Wandler
There was no statistically significant increase in the proportion of Lactobacillus in CagA flies from control flies.
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 PhosphoHistone 3 (PH3) immunostaining to identify proliferating cells.
Ave
rage
Pro
port
ion
of B
acte
ria
