Abstract Microbiome research has increasingly highlighted contributions of individual microbiota members to health and disease. Accumulating evidence suggests that microbes influence host physiology and pathology in part through microbial metabolites. To understand the roles of diverse microbial metabolites in host pathophysiology, most studies focus on manipulating individual bacterial strains’ metabolite production with genetic knockout or over- expression to interrogate the causality between microbes, microbial metabolites and host processes. However, this strategy has its own limitations in that certain microbial metabolites are derived from multiple microbial species harboring conserved gene clusters. One example is colibactin, a bacterial secondary metabolite that has garnered increasing attention due to its implications in colorectal cancer and gut microbiota composition and function. Colibactin is a hybrid polyketide-nonribosomal peptide produced by different Enterobacteriaceae carrying a highly-conserved polyketide synthase (pks) gene cluster. However, progress in understanding colibactin+ bacteria has been largely limited to manipulating and characterizing individual knockout bacterial strains in cell culture or germ-free mice, while overlooking the fact that multiple different enteric bacteria in a native environment can produce colibactin to impact the host through the acquisition of the conserved pks island. Furthermore, no strategy has been developed to target colibactin+ bacteria for cancer intervention in light of the accumulating evidence that colibactin promotes host DNA damage, senescence and carcinogenesis. To address the limitations in understanding and targeting colibactin+ bacteria, two complementary and highly integrated approaches will be developed to inhibit colibactin. The first approach is enzymatic inactivation through hijacking an anti-colibactin enzyme employed by diverse colibactin+ bacteria to prevent self-DNA damage by colibactin. Bacterial surface display of the anti-colibactin enzyme will be explored to maximize the catalytic inactivation of colibactin at the bacteria-host interface. In parallel, the second strategy is genetic inhibition, where the conserved pks island coding for colibactin will be inhibited by two different CRISPR systems delivered by a self-transmissible broad-host-range conjugative plasmid. While CRISPR-Cas9 (CRISPR knockout) eliminates colibactin+ bacteria via direct DNA cleavage, CRISPR-dCpf1 (CRISPR interference) suppresses colibactin biosynthesis without inducing bacterial death and selection pressure for evasion. Both enzymatic and genetic inhibition systems will be delivered by genetically tractable native E. coli isolates that have been demonstrated for efficient colonization in the gut. In vitro bacteria-host cell coculture, polymicrobial communities, and mouse models will be employed to compare and validate the efficacy of enzymatic and genetic approaches. While this application focuses on colibactin, if successful, it will pioneer methodologies to directly manipulate microbial metabolites at the cellular, tissue and organismal levels to accelerate fundamental and translational studies.

Project Narrative The majority of studies in establishing causal associations between microbial metabolites and host processes center around manipulating individual bacterial strains' metabolite production and investigating the subsequent impact on the host physiology. This strategy has limitations in that certain metabolites can be derived from multiple microbial species resulting from conserved metabolic pathways. Here, we will develop two complementary and highly integrated methodologies to achieve direct in situ perturbation of microbial metabolites, which will pave the way for both fundamental and applied studies relevant to metabolite-mediated microbe-host interactions.