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.

FeGenie: A Comprehensive Tool for the Identification of Iron Genes and Iron Gene Neighborhoods in Genome and Metagenome Assemblies

“…Because the Ton system can uptake other metabolites (e.g., vitamin B12), the identification of this transport pathway suggests only the potential for the transport of siderophores, hemes, and iron from transferrin/lactoferrin; it is the sole system known to transport these iron-bearing molecules, thus far, in Gram-negative bacteria (Faraldo-Gómez and Sansom, 2003;Caza and Kronstad, 2013). For example, Pseudomonas aeruginosa PAO1 encodes 34 different TonB-dependent receptors, including PiuA and PirA (Luscher et al, 2018). While this diversity of TonB-dependent receptors reflects, in part, an ability to uptake multiple types of siderophores, it also indicates that these receptors are also likely utilized for purposes outside of iron metabolism.…”

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

“…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).…”

“…Structural analysis of Fiu reveals that a number of extracellular loops are selectively ordered and this selective order is shared among the homologous receptors PiuA and PiuD [30,33]. The observed crystal states may represent conformational transitions that the receptor undergoes during substrate binding and transport.…”

Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system ofZymomonas mobilisfor genome engineering

“…Their orthologues in P. aeruginosa (PiuA and PirA) were proven to be not essential for cell viability because a piuA-pirA double mutant was already obtained (57). In addition, two other genes, ZMO0188 and ZMO1631, in the Z. mobilis ZM4 genome also encode orthologues of TonB-dependent siderophore receptor (26) that might play the similar role as ZMO1815 and ZMO1822 do.…”