The human gastrointestinal tract houses thousands of resident microbes, termed the gut microbiota, which are critical for development and, in healthy individuals, promote normal physiology. However, an emerging idea is that pathogens can alter microbial communities, contributing to human pathologies. The human pathogen, Helicobacter pylori causes stomach pathologies, such as inflammation and cell proliferation, that can lead to gastric cancer. These pathologies are due at least in part to the action of a translocated virulence protein CagA, and potentially by an altered gut microbiota. Here, we propose to use two transgenic models, Drosophila and zebrafish, expressing CagA in the intestinal epithelium to investigate the cell autonomous effects of CagA expression within the gut epithelium and the non-cell autonomous effects of this expression on the microbiota. Together these models will be used to investigate how pathogen derived factors, such as CagA, influence intestinal microbial ecology and affect intestinal epithelial signaling pathways to promote diseases such as gastrointestinal cancers. Knowledge gained in these experiments will provide valuable insights into the mechanisms by which interactions between bacterial pathogens and resident commensals contribute to human disease, and will provide new ideas for preventing and treating diseases with an infectious etiology. Hypothesis: The H. pylori protein, CagA, promotes intestinal cell proliferation in a cell autonomous fashion and also alters gut microbiota composition, which promotes non-cell autonomous cell proliferation.
Specific Aims :
Aim1 : I will determine if CagA expression induces cell-autonomous proliferation of the gut epithelium.
Aim2 : I will test the hypothesis that CagA expression in the gut epithelium changes the host microbiota and causes cell proliferation in Drosophila.
Aim3 : I will test the hypothesis that CagA transgenic zebrafish harbor an altered microbiota, which is necessary and sufficient to cause CagA-associated gut pathologies, such a hyperplasia. Research design: To examine the cell-autonomous effects of CagA on the intestinal epithelium I will use a mosaic approach in both Drosophila and zebrafish. This approach allows me to directly measure rates of cell proliferation in CagA expressing cells and determine whether these cells exclusively undergo JNK or Wnt pathway activation. Additionally, using reporter lines I will determine whether cell autonomous expression of CagA alter the immune response by inducing cell autonomous expression of antimicrobial peptides. To determine whether expression of CagA alters the gut microbiota and whether this is sufficient to induce cell proliferation in the Drosophila gut, I will overexpress antimicrobial peptides and determine if this is necessary or sufficient to alter the microbiota. Additionally, using transplantation of the entire Drosophila microbiota or through mono association experiments I will determine if the altered microbiota is sufficient to induce cell proliferation and identify the single bacterial species responsible for this effect. Finally, I wil test whether expression of CagA in the zebrafish intestinal epithelium is sufficient to alter the microbiota and immune function. Using transplantation experiments I will also determine whether the microbiota is necessary or sufficient to induce cell proliferation in the zebrafish intestine. The approach outlined here provides a unique opportunity to investigate the conserved role of the H. pylori virulence factory CagA on the intestinal epithelium. The use of two independent model systems will provide valuable insight into the conservation and pathologic nature of this common microbe. This data will be vital for understanding the etiology of pathogen-derived cancers and provide valuable insight into potential avenues for treatment and prevention.
The human body houses many commensal microbial communities that are critical for the normal function of tissues and organ systems, such as the gastrointestinal tract. However, invading pathogens or long-term bacterial residents can contribute to pathologies of these tissues, as is the case with Helicobacter pylori and gastric cancer, and it has become clear that bacterial pathogens exert some of their virulent effects on hosts through alterations in resident microbial communities. My proposal will provide mechanistic insights and determine how the H. pylori virulence factor and carcinogen, CagA, exerts its pro-proliferative effects on the gastrointestinal epithelium through cell autonomous signal versus cell non-autonomous effects on the gut microbial community, and insight into gastrointestinal cancers with a bacterial etiology.
