An intact surface and mucosal epithelium is one of the most crucial preventives of infection, therefore it is one of the most important aspects of our innate immune system. Mucosal epithelia provide both a physical and chemical barrier to invading bacteria, while allowing the colonization of the mucosal surface by species of bacteria which aid in the prevention of infection by pathogenic organisms. We are interested in one aspect of the chemical barrier, the enteric defensins, and their role in the establishment of intestinal commensal bacteria. The human enteric defensins are produced by Paneth cells at the bases of the small intestinal crypts. They are cysteine rich peptides with broad spectrum antibiotic activity, and a subset of the larger group of defensins originally described in neutrophils. To study the expression and function of human enteric defensins, we have developed a transgenic mouse model, by generating three human defensin 5 (HD5) transgenic mouse lines, using a fragment of genomic DNA that spans the HD5 gene and includes 1.5Kb of 5'UTR and flanking region. Characterization of the HD5 transgenic lines has demonstrated expression of the transgene to be (a) specific to mouse Paneth cells, (b) developmentally appropriate; and (c) the HD5 peptide is produced and packaged in the same manner observed in human Paneth cells. Preliminary evidence suggests that there is a significant reduction in the quantity of colonizing bacteria in the intestines of the transgenic mice, compared to wild type age matched litter mates.
In Specific Aim 1 we propose to expand and clarify this observation. First we will use the technique of flow cytometric in situ hybridization with a eubacterial probe for bacterial ribosomal RNA to accurately quantitate the differences in small and large intestinal commensal bacteria between wild type and transgenic mice. Second, we will develop a series of bacterial ribosomal RNA probes specific to mouse commensal bacteria that will be used to determine any variation in the types of colonizing bacteria.
In Specific Aim 2 we seek to understand the role of HD5 in the establishment of commensal flora in the absence of the acquired immune system. To achieve this we will breed the HD5 transgenic onto a SCID background, and evaluate the expression of the transgene in this system. Finally we will investigate the establishment of commensal flora in these mice at various stages of post natal development. These studies will be incorporated into an overall training plan, including seminars, journal clubs, and interactions with more junior and senior scientists, to prepare the principal investigator to become a productive, independent investigator in immunology.
| Salzman, Nita H; Chou, Margaret M; de Jong, Hendrik et al. (2003) Enteric salmonella infection inhibits Paneth cell antimicrobial peptide expression. Infect Immun 71:1109-15 |
| Salzman, Nita H; de Jong, Hendrik; Paterson, Yvonne et al. (2002) Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria. Microbiology 148:3651-60 |
