Yersiniabactin is a Recyclable Copper Importer in Pathogenic E. coli

During an infection, the host manipulates metal ion concentrations in an effort to thwart microbial pathogens. In response, microbial pathogens use multiple strategies to control extracellular metal ions. Siderophores, small molecule chelators that competitively bind iron(III) for nutritional use, exemplify this concept. Yersiniabactin (Ybt), a virulence‐associated siderophore of uropathogenic E. coli (UPEC), was recently appreciated to bind copper ions during human urinary tract infections. Copper ions can act both as toxins and nutrients to bacteria, depending on concentration, redox state, and cellular demands. Within the copper‐toxic environment of the phagolysosome, Ybt‐mediated copper sequestration outside bacteria is a valuable adaptation. In physiologic environments characterized by low copper, however, extracellular Ybt‐mediated copper sequestration would be expected to block nutritional access to this metal. Although E. coli are widely considered to lack a copper import system, we hypothesized that uropathogenic E. coli are able to import copper‐yersiniabactin complexes as a nutritional copper source. We monitored trafficking and metabolism of exogenous Cu(II)‐Ybt complexes with a quantitative LC‐MS/MS method using stable isotope‐labeled standards. The role of different membrane proteins in metal‐Ybt complex transport was assessed through ectopic expression or genetic knockouts in UTI89, a model uropathogenic E. coli strain. We observed that UTI89 consumed copper‐Ybt and secreted metal‐free Ybt. This process was dependent upon the inner membrane ATPase YbtPQ. Use of the radioactive copper isotope, 64 Cu, showed that copper is retained by the cells following 64 Cu‐Ybt import. To determine whether copper from Ybt can be used as a nutrient source, we monitored activity of the cuproprotein TynA in UTI89. TynA is a well‐characterized E. coli amine oxidase that specifically catalyzes phenylethylamine oxidation in a copper‐dependent manner. By measuring the product of this reaction using LC‐MS/MS we can monitor cellular copper delivery to TynA. Using this experimental system we found that Cu‐Ybt greatly enhances TynA activity in a YbtPQ‐dependent manner. These findings are consistent with Ybt as a true metallophore, capable of binding and importing both iron and copper ions for nutritional purposes. Copper abundance and scarcity appear to play key roles in pathogen fitness, suggesting future routes for antivirulence agent development. Support or Funding Information National Institute of Diabetes and Digestive and Kidney Diseases grant R01DK099534 (JPH), NSF Graduate Research Fellowship DGE‐1143954 (AER), and the Mr. and Mrs. Spencer T. Olin Fellowship for Women in Graduate Study (AER)General scheme of metallophore import and recycling.