Escherichia coli enterobactin synthesis and uptake mutants are hypersensitive to an antimicrobial peptide that limits the availability of iron in addition to blocking Holliday junction resolution

Abstract

The peptide wrwycr inhibits Holliday junction resolution and is a potent antimicrobial. To study the physiological effects of wrwycr treatment on Escherichia coli cells, we partially screened the Keio collection of knockout mutants for those with increased sensitivity to wrwycr. Strains lacking part of the ferric-enterobactin (iron-bound siderophore) uptake and utilization system, parts of the enterobactin synthesis pathway, TolC (an outer-membrane channel protein) or Fur (an ironresponsive regulator) were hypersensitive to wrwycr. We provide evidence that the DtolC mutant was hypersensitive to wrwycr due to its reduced ability to efflux wrwycr from the cell rather than due to its export of newly synthesized enterobactin. Deleting ryhB, which encodes a small RNA involved in iron regulation, mostly relieved the wrwycr hypersensitivity of the fur and ferricenterobactin uptake mutants, indicating that the altered regulation of a RyhB-controlled gene was at least partly responsible for the hypersensitivity of these strains. Chelatable iron in the cell, measured by electron paramagnetic resonance spectroscopy, increased dramatically following wrwycr treatment, as did expression of Fur-repressed genes and, to some extent, mutation frequency. These incongruous results suggest that while wrwycr treatment caused accumulation of chelatable iron in the cell, iron was not available to bind to Fur. This is corroborated by the observed induction of the suf system, which assembles iron-sulfur clusters in low-iron conditions. Disruption of iron metabolism by wrwycr, in addition to its effects on DNA repair, may make it a particularly effective antimicrobial in the context of the low-iron environment of a mammalian host.

A Bacterial Mutant Library as a Tool to Study the Attack of a Defensin Peptide

“…The plates were incubated at 37 °C for 1 h in a shaking incubator (150 rpm). After that time, a 50-µL aliquot of 2x AMA media (adapted from Orchard et al , [33] Supporting Information) was added and the plates were sealed with parafilm. The plates were incubated first at 37 °C for 2 to 3 h, then at 30 °C overnight in a shaking incubator (150 rpm).…”

Similarities between Exogenously- and Endogenously-Induced Envelope Stress: The Effects of a New Antibacterial Molecule, TPI1609-10

“…We have shown that wrwycr causes accumulation of intracellular iron, transient loss of membrane potential, and leakage of potassium from cells ([56]; Naili, Rostron and Segall, ms. submitted). Like wrwycr, SM10 also causes loss of membrane potential (Figure 4C).…”

Novel antimicrobial peptide prevents C. rodentium infection in C57BL/6 mice by enhancing acid-induced pathogen killing

“…More recent studies have expanded the list of potential intrabacterial targets of the peptide, based on how bacterial cells respond to peptide treatment. It is clear that the peptide in some way causes cells to phenotypically starve for iron and to damage the cell membrane in ways that lead to the induction of the envelope stress response (Orchard et al, 2012). Many of the effects of the peptide are similar to those of a small molecule, TPI1609-10 (Yitzhaki et al, 2012.…”

“…The peptide wrwycr was synthesized with a C-terminal amide group, purified to >95 % purity at SigmaGenosys or Biosynthesis and dissolved in 50 % dimethyl sulphoxide (DMSO), as described previously (Gunderson & Segall, 2006). A wrwycr stock solution (10 mM) was maintained at À20 C in 50 or 100 % DMSO and the degree of dimerization was checked by HPLC, as described previously (Orchard et al, 2012). The stock used comprised at least 85 % dimerized wrwycr; for ease, however, concentrations are expressed as if the solution was composed of monomer.…”