This proposal seeks continued support for the molecular analysis of the role of the intestine's nonpathogenic bacteria in gut development and function. The applicant has colonized adult germ-free mice with Bacteroides thetaiotaomicron, a genetically manipulatable, prominent member of the normal mouse and human ileal microflora. The investigator has already used high density oligonucleotide-based microarrays, laser capture microdissection (LCM) and real-time quantitative RT-PCR to show that 10 days after colonization, B. thetaiotaomicron affects expression of genes involved in a number of critical intestinal functions (e.g. nutrient processing and absorption, fortification of the mucosal barrier, xenobiotic metabolism, angiogenesis, plus genes associated with the enteric nervous system). Moreover, changes normally associated with the suckling-weaning transition are elicited in adult germ-free mice by B. thetaiotaomicron, suggesting that indigenous bacteria may play an instructive role in postnatal intestinal development.The PI wishes to pursue two specific aims.
Aim 1 seeks to obtain a comprehensive molecular view of the impact of B. thetaiotaomicron on intestinal function and development. They will use a combination of germ-free mice, microarrays, LCM, real-time RT-PCR, in situ hybridization and immunohistochemical methods to examine the effect of B. thetaiotaomicron on intestinal gene expression as a function of time after initial colonization, position along the length of the intestine, and the presence or absence of mucosal T- and B-cells. The specificity of host responses will be explored by colonizing germ-free mice with another prominent member of the ileal microflora, Bifidobacterium infantis, and with the 'complete' ileal microflora of conventionally raised mice. The results will be used to develop, and genetically test specific hypotheses about contribution of selected host responses to normal intestinal development and function.
Aim 2 proposes to identify microbial factors that regulate key host functions. They are in the process of sequencing the entire 4.8 Mb B. thetaiotaomicron genome. The investigators will identify ORFs using a variety of computational methods. Bacterial genes that are preferentially expressed in vivo will then be identified using promoter traps, as well as direct measurements of the levels of microbial mRNAs with custom microarrays. The results will then be used to design and conduct genetic tests of hypotheses about the role of selected B. thetaiotaomicron genes on intestinal responses to colonization. These studies should help determine whether microbes and their products can be used as therapeutic agents to improve nutrition, to promote proper gut development and physiologic functioning, and to prevent or treat various intestinal diseases.
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