The expansive microbial community in the colon interacts with the host in important ways, such as maintenance of barrier function, break-down and energy utilization of otherwise non-digestible foods, and education of the host immune system. However, host/microbe interactions are complex, and microbes may also play a major role in causing conditions such as inflammatory bowel diseases (IBD). Through recent advances in cultivation-independent technologies and bioinformatics, we are gaining a much better understanding of the complexities of microbial communities. We are beginning to understand the ways that the host senses and interacts with microbes through pattern recognition receptors ( such as toll like receptors) and the ways in which microbes interact with the host via products such as the short chain fatty acid butyrate, which regulates host cell proliferation and differentiation. I propose to study the ways in which microbes affect the host's gene expression through microRNAs. microRNAs (miRNAs) are ~22 nucleotide, non-coding RNAs that decrease gene expression. miRNAs play a role in microbial actions on immune and inflammatory cells. However, little is known of the effects of microbes on gene expression in the normal colon via microRNAs. I have chosen to investigate whether miRNAs are involved in the microbial regulation of intestinal heat shock proteins (Hsps). Hsps protect the intestinal epithelium from stressors such as heat (fever), inflammation, starvation, and infection. Heat shock protein expression is known to be regulated by the 3'untranslated region (3'UTR) of its messenger RNA, the same region that microRNAs are known to target. I hypothesize that the regional distribution of the bacteria in the colon influences intestinal epithelial miRNA expression, and that miRNAs mediate the regulation of Hsp70 by microbes. I will first determine if the enteric bacteria alter miRNA profiles in the mouse colonic epithelium though comparison of the microRNA expression in germ free mice to the expression in germ free mice that have been fed the stool of conventional mice using microarrays. I will also treat young adult mouse colon (YAMC) cells with mouse stool to measure microRNA expression. After finding which microRNAs are affected by exposure to bacteria, I will determine whether these microRNAs are amongst the group predicted to target Hsp70. I will also use quantitative PCR to determine whether miRNAs with predicted binding sites on Hsp70 have altered expression in stool- transplanted germ-free mice or mouse stool exposed YAMC cells. I will confirm targeting of these miRNAs to Hsp70 with the use of microRNA mimics and luciferase reporter constructs that emit a light signal when the microRNA of interest binds a region of Hsp70 that has been engineered into the construct. Establishing the link that microbes affect host gene expression through modulation of miRNAs that target key regulatory proteins such as Hsp70 will provide the essential foundation needed for further studies linking the environment and gene expression to the pathogenesis of IBD and other diseases.
The microbes that inhabit the surface of the GI tract contain at least 100 times as many genes as the host, and provide important functions such as utilization of otherwise non-digestible food as an energy source, and immune system development. I will study the ways in which the colonic bacteria affect the host's gene expression through regulatory microRNA molecules. I have chosen to look at the possibility that miRNAs are involved in the microbial regulation of intestinal heat shock protein 70, a protein that protects the intestinal cells from stressors such as heat (fever), inflammation, starvation, and infection. Establishing this link will provide the essential foundation needed for further studies linking the environment and gene expression to diseases such as Crohn's and ulcerative colitis, which result from an abnormal immune response to the environment in the setting of genetic susceptibility.
