Structure and Function of the PiuA and PirA Siderophore-Drug Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii

Abstract

The outer membrane of Gram-negative bacteria presents an efficient barrier to the permeation of antimicrobial molecules. One strategy pursued to circumvent this obstacle is to hijack transport systems for essential nutrients, such as iron. BAL30072 and MC-1 are two monobactams conjugated to a dihydroxypyridone siderophore that are active against Pseudomonas aeruginosa and Acinetobacter baumannii Here, we investigated the mechanism of action of these molecules in A. baumannii We identified two novel TonB-dependent receptors, termed Ab-PiuA and Ab-PirA, that are required for the antimicrobial activity of both agents. Deletion of either piuA or pirA in A. baumannii resulted in 4- to 8-fold-decreased susceptibility, while their overexpression in the heterologous host P. aeruginosa increased susceptibility to the two siderophore-drug conjugates by 4- to 32-fold. The crystal structures of PiuA and PirA from A. baumannii and their orthologues from P. aeruginosa were determined. The structures revealed similar architectures; however, structural differences between PirA and PiuA point to potential differences between their cognate siderophore ligands. Spontaneous mutants, selected upon exposure to BAL30072, harbored frameshift mutations in either the ExbD3 or the TonB3 protein of A. baumannii, forming the cytoplasmic-membrane complex providing the energy for the siderophore translocation process. The results of this study provide insight for the rational design of novel siderophore-drug conjugates against problematic Gram-negative pathogens.

Keywords: Acinetobacter baumannii; Pseudomonas aeruginosa; siderophore-drug receptor.

FIG 1

Chromosomal loci of piuA and pirA genes in A. baumannii, P. aeruginosa, and E. coli. The TBDR gene piuA is colocalized with the Fe(II)-dependent oxidoreductase gene piuC, while the pirA loci differ among the three organisms. Only genes relevant to the study context are shown.

FIG 2

Structure-based sequence alignment of PiuA and PirA proteins from P. aeruginosa and A. baumannii. Secondary-structure elements of Ab-PiuA are illustrated above the alignment. Regions predicted to be involved in the binding of the siderophores are boxed in red. Residues spatially equivalent to those involved in substrate binding in FhuA, FptA, and FpvA are highlighted in green. Key residues of the secondary binding site of FepA and conserved in the PirA structure are shown in yellow. Positions of residues of the nonconserved initial binding site are indicated by orange triangles. The plug domain is boxed in cyan. The alignment was drawn using Aline (71).

FIG 3

Crystal structures of PiuA and PirA from P. aeruginosa and A. baumannii. β-Barrels are colored green, and N-terminal plug domains are blue. Loops NL1, NL2, and NL3 of the plug domains are shown in red. Residues likely to be part of the siderophore substrate binding site are shown as yellow sticks. The last solved residues of the flexible loops are represented as spheres.

FIG 4

Putative binding sites in PiuA of P. aeruginosa and A. baumannii and PirA of A. baumannii. The color scheme is the same as that in Fig. 3. The phosphate ion present in the Ab-PiuA structure is colored orange and red, and residues Asp303 and Glu73, which coordinate it, are represented as green and white sticks.

FIG 5

Growth of spontaneous mutants with decreased susceptibility to BAL30072. The strains were grown in MH broth (MHB) with or without Fe(II)Cl₂ supplementation (20 μM final concentration). Growth was followed in a plate reader with regular OD₆₀₀ readings. The data represent the average OD₆₀₀ value for duplicate cultures. Experiments were repeated on three different occasions, yielding similar results. Data from one representative experiment are shown. The OD₆₀₀ values, measured after 18 h for each of the three mutants, were statistically different from that of the wild type (Student t test; P < 0.05) in the absence, but not in the presence, of iron, as indicated by asterisks (top).