Investigations of the Biosynthesis and Structure of Colibactin, a Cytotoxin Made by Human-Associated Escherichia Coli

Abstract

Humans exist in symbiosis with trillions of bacteria that are collectively referred to as the human microbiota. While commensal microbes are essential for health, some resident microbes can promote disease. Certain strains of human-associated Escherichia coli cause double-strand breaks in host DNA through the production of colibactin, a genotoxin of unknown structure. To broaden our understanding of the chemistry of the human microbiota, we sought to elucidate the structure of colibactin and characterize its biosynthetic pathway. We first characterized the self-resistance mechanism in colibactin biosynthesis (Chapter 2). We found that the enzyme that initiates the assembly line pathway is ClbN, a non-ribosomal peptide synthetase (NRPS). Biochemical assays showed that ClbN biosynthesizes an N-terminal prodrug motif consisting of an N-myristoyl-D-asparagine residue that is proposed to mask the reactivity of colibactin. We performed bioinformatic analyses to identify the enzyme that acts after ClbN in the assembly line. In vitro reconstitution assays revealed that ClbB, a NRPS/ polyketide synthase (PKS) hybrid, elongates the prodrug motif produced by ClbN. In addition, we demonstrated that the periplasmic peptidase ClbP cleaves the prodrug motif from synthesized model substrates and that the membrane domain of ClbP is required for full activity. Next, we sought to isolate precolibactin, the prodrug-containing precursor to the active genotoxin (Chapter 3). Metabolite profiling of the extracted metabolome of ΔclbP and wild-type colibactin-producing strains led to the identification of several candidate precolibactins. We isolated one of these metabolites, which we named Metabolite B. This metabolite contains an unusual azaspiro[2.4] bicyclic ring system that has not been observed previously in a natural product scaffold. The structure of Metabolite B suggested that the colibactin assembly line pathway may utilize novel biosynthetic logic and pointed to a possible mechanism through which colibactin damages DNA. Finally, we describe the isolation of other pks-associated metabolites and provide biosynthetic hypotheses for these pathway intermediates (Chapter 4). We also present our attempts to characterize the next enzymatic steps in the colibactin biosynthetic pathway. As part of these efforts, the PKS module of ClbB was characterized in vitro and several other colibactin biosynthetic enzymes were examined using genetic studies. The challenges associated with studying a biosynthetic pathway for which the final product is unknown are highlighted. Overall, the work presented here contributes significantly to our knowledge of the biosynthesis and structure of a microbial genotoxin.