Figure 3. Identification of escherichelin as HPTT-COOH, a metallophore inhibitor of Pseudomonas iron uptake.

(A) Multiple chemical characterizations identify escherichelin as HPTT-COOH, a compound shown to inhibit pyochelin-mediated iron uptake in Pseudomonas. The positions of ¹³C salicylate incorporation are indicated in green, while MS/MS neutral losses are indicated as dashed lines. (B) Mass spectra at the retention time for escherichelin from WT UTI89, UTI89ΔybtS, and ¹³C₆-SA–supplemented UTI89ΔybtS culture supernatants revealed that 6 carbons in escherichelin are derived from salicylate. (C) MS/MS product ion spectrum of escherichelin. The 46-Da neutral loss indicates the presence of a carboxylic acid group, and the 87- and 104-Da losses correspond to fragmentation within the thiazoline ring. (D) UV-visible absorption spectra of purified escherichelin incubated with equimolar FeCl₃ or CuSO₄. (E and F) DFT simulations of escherichelin bound to Fe(III) in a 1:1 (E) and 2:1 (F) complex. The simulations predict stable Fe(III)-escherichelin complexes with hexacoordinate octahedral geometry. (G) UTI89 growth curve in the presence or absence of a ferric ion chelator (0.5 mM EDDHA) and/or ferric ion supplementation (1 μM FeCl₃). Growth was monitored by OD at 600 nm (OD₆₀₀). Data represent the mean of 3, with SD plotted. (H) PW5011 (pvdA-E02::ISlacZ/hah), a P. aeruginosa strain MPAO1 transposon mutant (55, 56) expressing pyochelin as its sole siderophore, was grown in succinate medium for 20 hours in the presence of increasing escherichelin concentrations relative to vehicle control. Bacterial growth was quantified by enumerating CFU/ml and is expressed as a percentage of the vehicle control. Data represent the mean of 3, with the SEM plotted. *P < 0.01 and **P < 0.001, by 1-way ANOVA with Dunnett’s multiple comparisons test.