Identification of Mutasynthetic Inhibitors of Yersiniabactin Production in Uropathogenic E. coli

Abstract

E. coli urinary tract infections (UTIs) are among the most common infectious diseases and account for a substantial proportion of antibiotic prescriptions. The continuous increase of multi‐drug resistance (MDR) among E. coli motivates efforts to identify new therapeutic approaches to UTIs. One strategy is to diminish the pathogenic potential of E. coli by pharmacologically inactivating key virulence factors. The Yersinia High Pathogenicity Island (HPI), which encodes a distinctive metallophore system implicated in passivation and trafficking of Fe (III), Cu (II) and Ni (II) ions, is one such candidate drug target in uropathogenic E. coli (UPEC). Key to this system is yersiniabactin (Ybt), a small molecule chelator which is biosynthesized from salicylic acid and other precursor molecules by a hybrid nonribosomal peptide synthetase and polyketide synthase (NRPS/PKS). With the knowledge that exogenous salicylic acid can be taken up and utilized by the model uropathogen UTI89, we hypothesized that some chemically modified forms of salicylic acid may be incorporated into the Ybt biosynthetic pathway and inhibit native Ybt biosynthesis. We created a mass spectrometric screen to monitor Ybt production by UPEC in the presence of salicylate analogs. Counter‐screens monitored bacterial growth and biosynthesis of enterobactin, a chemically distinct siderophore produced by E. coli. For inhibitory compounds, production of chemically altered (“mutasynthesized”) forms of Ybt pathway were identified using LC‐MS constant neutral loss (CNL) scans based upon detection of the putatively conserved, carboxy terminal fragment of Ybt. Among over 40 substrate analogs tested, we identified four that inhibited Ybt production without affecting bacterial growth or enterobactin biosynthesis. Each of these substrates was associated with production of varying levels of mutasynthetic Ybt. These results demonstrate that a mutasynthetic strategy may identify useful modulators of Ybt in growing bacteria. These compounds will serve as distinctive biochemical probes of the Ybt metallophore system in intact bacteria. Moreover, these suggest a mechanistically distinctive therapeutic strategy to diminish the pathogenic potential of uropathogenic E. coli and related Enterobacteriaceae.