Prophage-mediated regulation of antimicrobial production

The mammalian intestinal tract is inhabited by trillions of bacteria. Approximately half of the bacteria contain viral DNA—prophages—in their genome. Nearly exclusively studied in pathogens, the long-term co-evolutionary relationship between these so-called lysogenic bacteria and their prophages has led to beneficial and detrimental consequences to the bacterial host. However, we know virtually nothing to what extent, and by which mechanisms, prophages modulate the metabolism of a probiotic gut symbiont. Until we have filled these voids in our knowledge base, we will not be able to develop rational selection approaches for (engineered) probiotics and, consequently, the impact of probiotic-encoded prophages on human health will be vastly overlooked. Our long-term goal is to gain mechanistic insight into the interplay between probiotic bacteria and their prophages. The objectives of this research program are (1) to elucidate the mechanism by which prophages modulate the production of a probiotic effector molecule, reuterin, in the probiotic gut symbiont Limosilactobacilus reuteri; and (2) to determine the ecological ramifications of phage-mediated reuterin production in vivo. The overarching hypothesis is that prophage-mediated regulation of cellular metabolism promotes probiosis. We base our hypothesis on a synthesis of published findings and exciting preliminary data, which are that (a) the pdu operon produces the broad-spectrum antimicrobial reuterin; (b) reuterin is not detected upon deletion of both prophages; (c) reuterin production is restored in the prophage-deletion strain by expression of a single prophage-derived protein, hereafter referred to as reuterin-modulation protein A (RmpA); (d) we established that in vivo reuterin production reduces pathogenic E. coli. The rationale of the work proposed is that its successful completion will result in a paradigm shift in our understanding of how prophage-microbe interplay can be intertwined with human health. To accomplish the objectives for this project, we will pursue the following specific aims: (1) To characterize the interplay between RmpA and TpiA in relation to reuterin production; (2) To characterize RmpA and identify its interacting partners; (3) To determine to what extent prophages impact probiosis via reuterin production. This research is innovative because we apply sophisticated genome editing tools in an important gut symbiont species. Also, our research question is innovative as we expect to uncover fundamental knowledge on the mechanisms by which prophages modulate microbial metabolism in the gut ecosystem. The research is significant because its successful completion is expected to result in a paradigm shift in how we view the role of prophages in (probiotic) gut symbionts.