The uptake mechanism of synthetic siderophore conjugate in Gram-negative bacteria

Abstract

Drug resistance in bacteria becomes a serious challenge in the clinic, so it is exigent to seek novel and effective antibiotic treatment options. By hijacking the siderophore transport system, siderophore-antibiotic conjugates are actively imported to the pathogen, boosting antibiotic efficacy via increased intracellular accumulation. However, the underlying transport mechanisms and the bacterial response to exposure to siderophore drugs are poorly understood. In this Thesis, a novel siderophore-conjugate named LP-600 was characterized, displaying excellent activity against both laboratory and uropathogenic E. coli. E. coli displaying triple knockout of fepA, cirA and fiu is resistant to LP-600, suggesting that outer membrane receptors FepA, CirA, and Fiu contribute to the uptake of siderophore-conjugates and antibacterial activity. To understand the resistance mechanism and potential targets, LP-600 resistant clones were generated by serial passages, followed by whole genome sequencing. Two point mutations were identified in genomes of LP-600-resistant clones in the proteins exbB (Excretes an inhibitor of colicin B) and cyo B (Cytochrome bo(3) ubiquinol oxidase subunit 1) at positions exbB163* and cyoBG269D, respectively. Additionally, E. coli showed a paradoxical re-growth, a phenomenon coined the ‘Eagle-effect’ in other systems, upon treatment of LP-600 at concentrations 16 times above the minimum inhibitory concentration (MIC). Co-treatment with LP-600 and the -lactamase inhibitor sulbactam significantly diminished the effect in E. coli. To decipher the mechanism behind the Eagle effect, transcriptomic and metabolome analysis were conducted. Joint pathway mapping from transcriptome and metabolome results revealed enrichment in pathways about amino acid metabolism including L-arginine, L-lysine degradation, and polyamine biosynthesis pathways upon regrowth conditions Eagle, compared with a sub-MIC treatment of LP-600. In addition, LP-600 induces expression of genes involved in SOS response and e14 prophage upon regrowth conditions Eagle. Taken together, the work of this Thesis not only characterizes a novel and potent anti-bacterial compound, but also provides insight into the systemic bacterial responses towards siderophore-antibiotic conjugate exposure.