Alpha defensins: innate keepers of host-commensal homeostasis

Multiple innate immune mechanisms regulate commensal microbes and promote responses to pathogens. Identification of non-redundant functions of such mechanisms is not a simple task, especially with regulation of complex microbial communities such as intestinal commensals. Commensals are indispensable for the existence of their eukaryotic hosts and provide essential functions) required for the host’s survival. The composition of microbial communities varies greatly from individual to individual and is shaped by multiple factors including the mode of transmission during birth, breastfeeding, alimentary infections and diet. An important question remains unanswered: to what extent and which host’s polymorphic genetic mechanisms are involved in shaping the repertoire of the commensals. Although many polymorphic genes were found to be good potential contributors to shaping commensal communities, a drastic difference in expression of defensins alpha (aDef encoded by Defa genes) was detected between the small intestines of B6 and BALB/c mice. Defensins are anti-microbial peptides that are thought to safeguard the stem cells of the gut epithelium. Defa genes are localized to a chromosomal locus that undergoes a rapid evolution and is characterized by multiple duplications and deletions. In addition, these genes have very high level of homology and there are other defensins with similar functions. There is no consensus whether aDef have some specificity towards different groups of microbes or whether their specificities are very broad. Most of specificity suggestions came from the studies of bactericidal effects in vitro (done with limited variety of Defa-encoded peptides and from the studies of MMP7 KO mice, a metalloprotease that cleaves aDef peptides to activate them), which by today standards cannot be fully accepted as these studies did not exclude cage (legacy) effects. Armed with two state of the art approaches – CRISPR/Cas9 gene editing and germ-free technology – we aim at shedding light on the spectrum of specificities of the aDef peptides, their contribution to host’s genetic polymorphism in shaping the microbiota and resistance to pathogens. The current proposal aims at defining the place of aDef in homeostatic and induced by pathogenic signals innate host defense. Most importantly, it will be done in the most refined and most reliable way. We will pursue the following specific aims: Specific Aim 1. Use the combination of reverse genetic and gnotobiotic approaches to study the homeostatic role of alpha-defensins. Germ-free mice carrying aDef deletions will be colonized with natural or synthetic microbiomes to detect shifts in microbiota composition or gene expression dependent on aDef peptides. Specific Aim 2. Study importance of aDef using infectious and non-infectious stress. Mice lacking Defa will be tested for sensitivity to infections with intestinal pathogens and chemical insult on the intestinal epithelium.