The emergence of multidrug resistant (MDR) Gram-negative bacterial pathogens has raised global concern. Nontraditional therapeutic strategies, including antivirulence approaches, are gaining traction as a means of applying less selective pressure for resistance in vivo. Here, we show that rigidifying the structure of the siderophore preacinetobactin from MDR Acinetobacter baumannii via oxidation of the phenolate-oxazoline moiety to a phenolate-oxazole results in a potent inhibitor of siderophore transport and imparts a bacteriostatic effect at low micromolar concentrations under infection-like conditions.Keywords: antibiotic resistance; antivirulence agent; iron acquisition; metal chelation; metal homeostasis; metal transport; pathogenesis; siderophore; virulence;

Pathogenic Acinetobacter baumannii excrete the siderophore pre-acinetobactin as an iron-scavenging virulence factor. Pre-acinetobactin is a 2,3-dihydroxy-phenyl oxazoline that undergoes pH-dependent isomerization to the isooxazolidinone form acinetobactin in order to expand the pH range for iron acquisition by A. baumannii. In this study we establish important structure–function relationships for the kinetics of isomerization, iron(III) binding, and siderophore utilization by A. baumannii. We showed that electronic properties of the phenyl oxazoline influence isomerization kinetics and iron(III) binding. We found that iron(III) chelation was directly correlated with A. baumannii utilization. Our studies provide important structural and mechanistic insight for understanding how pathogenic A. baumannii uses pre-acinetobactin as a 2-for-1 iron-scavenging siderophore virulence factor.

Tabtoxinine-β-lactam (TβL) is a phytotoxin produced by plant pathogenic strains of Pseudomonas syringae. Unlike the majority of β-lactam antibiotics, TβL does not inhibit transpeptidase enzymes but instead is a potent, time-dependent inactivator of glutamine synthetase, an attractive and underexploited antibiotic target. TβL is produced by P. syringae in the form of a threonine dipeptide prodrug, tabtoxin (TβL-Thr), which enters plant and bacterial cells through dipeptide permeases. The role of β-lactamases in the self-protection of P. syringae from tabtoxin has been proposed, since this organism produces at least three β-lactamases. However, using in vitro and cellular assays and computational docking we have shown that TβL and TβL-Thr evade the action of all major classes of β-lactamase enzymes, thus overcoming the primary mechanism of resistance observed for traditional β-lactam antibiotics. TβL is a “stealth” β-lactam antibiotic and dipeptide prodrugs such as tabtoxin from P. syringae represent a novel antibiotic therapeutic strategy for treating multi-drug resistant Gram-negative pathogens expressing high levels of β-lactamase enzymes.

Pathogenic strains of Acinetobacter baumannii excrete multiple siderophores that enhance iron scavenging from host sources. The oxazoline siderophore pre-acinetobactin undergoes an unusual non-enzymatic isomerization, producing the isoxazolidinone acinetobactin. In this study, we explored the kinetics, mechanism, and biological consequence of this siderophore swapping. Pre-acinetobactin is excreted to the extracellular space where the isomerization to acinetobactin occurs with a pH-rate profile consistent with 5-exo-tet cyclization at C5′ with clean stereochemical inversion. Pre-acinetobactin persists at pH <6, and acinetobactin is rapidly formed at pH >7, matching each siderophore’s pH preference for iron(III) chelation and A. baumannii growth promotion. Acinetobactin isomerization provides two siderophores for the price of one, enabling A. baumannii to sequester iron over a broad pH range likely to be encountered during the course of an infection.Keywords: Acinetobacter baumannii; acinetobactin; antibiotic resistance; antivirulence; siderophore

•Nine tetracycline-inactivating flavoenzymes are described from diverse soils•The enzymes inactivate tetracycline when expressed in E. coli and when purified•The enzymes exhibit both known and undescribed tetracycline oxidative activity•A functional homolog exists in the human pathogen Legionella longbeachaeEnzymes capable of inactivating tetracycline are paradoxically rare compared with enzymes that inactivate other natural-product antibiotics. We describe a family of flavoenzymes, previously unrecognizable as resistance genes, which are capable of degrading tetracycline antibiotics. From soil functional metagenomic selections, we discovered nine genes that confer high-level tetracycline resistance by enzymatic inactivation. We also demonstrate that a tenth enzyme, an uncharacterized homolog in the human pathogen Legionella longbeachae, similarly inactivates tetracycline. These enzymes catalyze the oxidation of tetracyclines in vitro both by known mechanisms and via previously undescribed activity. Tetracycline-inactivation genes were identified in diverse soil types, encompass substantial sequence diversity, and are adjacent to genes implicated in horizontal gene transfer. Because tetracycline inactivation is scarcely observed in hospitals, these enzymes may fill an empty niche in pathogenic organisms, and should therefore be monitored for their dissemination potential into the clinic.Figure optionsDownload full-size imageDownload high-quality image (225 K)Download as PowerPoint slide

The doom and gloom of antibiotic resistance dominates public perception of this drug class. Many believe the world has entered the post-antibiotic era. Classic and modern approaches to antibacterial drug discovery have delivered a plethora of lead molecules with a great majority being natural products of ancient microbial origin. The failure of antibiotics in the resistance era comes from an inability to develop new leads into clinical candidates, which is a costly and risky endeavor for any therapeutic area, especially when resistance is at play. The world needs new antibiotic molecules to replace the exhausted pipeline and the second ‘golden era’ is certain to come from Nature’s chemical inventory once again.