TonB-Dependent Receptor Repertoire of Pseudomonas aeruginosa for Uptake of Siderophore-Drug Conjugates

Abstract

The conjugation of siderophores to antimicrobial molecules is an attractive strategy to overcome the low outer membrane permeability of Gram-negative bacteria. In this Trojan horse approach, the transport of drug conjugates is redirected via TonB-dependent receptors (TBDR), which are involved in the uptake of essential nutrients, including iron. Previous reports have demonstrated the involvement of the TBDRs PiuA and PirA from Pseudomonas aeruginosa and their orthologues in Acinetobacter baumannii in the uptake of siderophore-beta-lactam drug conjugates. By in silico screening, we further identified a PiuA orthologue, termed PiuD, present in clinical isolates, including strain LESB58. The piuD gene in LESB58 is located at the same genetic locus as piuA in strain PAO1. PiuD has a similar crystal structure as PiuA and is involved in the transport of the siderophore-drug conjugates BAL30072, MC-1, and cefiderocol in strain LESB58. To screen for additional siderophore-drug uptake systems, we overexpressed 28 of the 34 TBDRs of strain PAO1 and identified PfuA, OptE, OptJ, and the pyochelin receptor FptA as novel TBDRs conferring increased susceptibility to siderophore-drug conjugates. The existence of a TBDR repertoire in P. aeruginosa able to transport siderophore-drug molecules potentially decreases the likelihood of resistance emergence during therapy.

Keywords: Pseudomonas aeruginosa; TonB-dependent receptor; siderophore-drug conjugate.

FIG 1

Siderophore-drug conjugates used in this study. BAL30072 and MC-1 contain a dihydroxypyridone as a siderophore, while cefiderocol contains a chlorinated catechol group.

FIG 2

Expression analysis of pirA and piuA in PAO1 and pirA and piuD in LESB58. RNA was extracted from cells grown to late exponential phase in MHB. qPCR was performed using target-gene-specific primers. Expression of pirA is 3-fold lower than piuA in a PAO1 background, while piuD and pirA expression levels are comparable in strain LESB58. Values are the expression ratios of the target gene divided by the rpsL housekeeping gene. The expression of piuA and piuD was set to 1 (100%) in the respective strain. Values show the means from three independent experiments performed in duplicates. Error bars indicate standard deviations. *, P < 0.05 by Student t test; ***, P < 0.001 by analysis of variance (ANOVA); ns, not significant.

FIG 3

Crystal structure of PiuD from P. aeruginosa. Side (A) and extracellular (B) views of PiuD. The 22-stranded transmembrane β-barrel is colored in green. β-Sheets of the plug domain are colored in yellow, loops in green, and helices in red. (C) Structural comparison between PiuA (light blue) and PiuD (green).

FIG 4

Proteomic analysis of TBDRs from P. aeruginosa PAO1. Protein expression levels were compared between cells grown for 20 h in MHB or Chelex-treated MHB. The dashed line indicates the 2-fold induction threshold level.