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 Gramnegative pathogens.

TonB‐dependent uptake of β‐lactam antibiotics in the opportunistic human pathogen Stenotrophomonas maltophilia

“…WGS confirmed shortening of the proline‐rich region in Smlt0009 in KLTV (Figure a). TonB mutations are known to reduce susceptibility to siderophore‐conjugated antimicrobials in other species but have never previously been reported to affect β‐lactam susceptibility (Hassett et al, ; Moynie et al, ; Tomaras et al, ). Interestingly, S. maltophilia smlt0009 mutants do not have reduced susceptibility to the non‐siderophore‐conjugated parent lactivicin, LTV‐13 (Table ), even though this γ‐lactam is structurally related to the β‐lactams (Starr et al, ).…”

“…in KLTV (Figure 2a). TonB mutations are known to reduce susceptibility to siderophore-conjugated antimicrobials in other species but have never previously been reported to affect β-lactam susceptibility (Hassett et al, 1996;Moynie et al, 2017;Tomaras et al, 2013).…”

The structure of the iron-catecholate transporter Fiu suggests substrate import occurs via a 2-step mechanism

“…In this study, we show that while Fiu and its homologues PiuA and PiuD function in the import of catechol-siderophores [25,30], they are evolutionarily distinct from the well-studied catecholate siderophore transporters FepA/PfeA and Cir. In order to investigate the substrate import mechanism of the Fiu/Piu TBDT subgroup, we solved the crystal structure of Fiu.…”

“…To resolve this question, we performed phylogenetic analysis on these transporters in the context of a panel of diverse TBDTs of known structure and/or function. This analysis revealed that while Cir and FepA belong to the same clade of our TBDT phylogram, Fiu belongs to a distal clade with the TBDTs PiuA and PiuD that also mediate catecholate transport [30,33] ( Figure 1A). Wider analysis of this Fiu/Piu clade, identified by HMMER search reveals that related sequences are widespread in proteobacteria ( Figure S1, Table S1, Table S2).…”

“…The disorder of the plug domain loop in crystal state 1, opens a large cavity in the interior of Fiu to the external environment, while in crystal state two this cavity is present, but occluded in the lumen of the Fiu barrel ( Figure 2B). Interestingly, analysis of the structures of PiuA from Acinetobacter baumannii and, PiuA and PiuD from P. aeruginosa revealed analogous crystal states to Fiu ( Figure S3A) [30,33]. In PiuA from A. baumannii all extracellular loops are ordered with the plug loop occluding the entrance to an internal cavity ( Figure S3).…”