Project Summary Bacteria within the human microbiome typically benefit the host. However, an imbalance of some bacteria groups such as Bacteroidales can lead to diseases like obesity and inflammatory bowel disease. A current grand challenge in the microbiome field is to identify products produced by microbiome bacteria that tip this health-to-disease balance. The lack of knowledge about these products is a significant barrier to improving therapies that modulate our microbiome (e.g. fecal transplants)—a current area of intense investigation. The objective of my laboratory is to identify products from normal microbiome bacteria that promote health or disease and determine their mechanisms of action. To this end, we discovered a previously undescribed family of Contractile Injection System that bacteria use to naturally promote the development of an invertebrate host (a tubeworm called Hydroides elegans). Contractile Injection Systems work by injecting stimulatory effector proteins into host cells that either promote development or elicit disease. This new family of Contractile Injection System is unique because they exclusively target eukaryotic organisms (e.g. amoeba, insects, tubeworms). Until now, this family of Injection System was not known to occur in human-associated bacteria. However, we recently discovered that the gut microbiomes of nearly all human adults (>99%) from the United States and Europe carry a closely related, yet previously undescribed Contractile Injection System. Moreover, our in vitro experiments show that Contractile Injection Systems in Bacteroides bacteria promote human cell line proliferation and inflammatory responses. In this proposal, we will lay the foundation for determining the impact of these newly discovered Contractile Injection Systems on human health. Our central hypothesis that this new Injection System family comprise a key means of interaction between microbiome bacteria and host, promoting both development and disease depending on the effectors deployed and context of interaction. Efforts for the next five years focus on (Project 1) investigating the functions of Subtype-4 CIS effector proteins and link effector activity with the host’s cellular responses; (Project 2) determining how this class of CIS binds to eukaryotic cells using fibers that structurally resemble eukaryotic cadherin proteins; and (Project 3) elucidating the structure-function relationship of Subtype-4 CIS from human gut Bacteroidales using cryo- electron tomography and fluorescence microscopy. Our results will lay a foundation for technology employing Bacteroidales bacteria and their Contractile Injection Systems as probiotics to modulate the human microbiome and host health.