Abstract
The pyochelin (PCH) siderophore produced by the pathogenic bacterium Pseudomonas aeruginosa is an important virulence factor, acting as a growth promoter during infection. While strong evidence exists for PCH production in vivo, PCH quantification in biological samples is problematic due to analytical complexity, requiring extraction from large volumes and time-consuming purification steps. Here, the construction of a bioluminescent whole cell-based biosensor, which allows rapid, sensitive, and single-step PCH quantification in biological samples, is reported. The biosensor was engineered by fusing the promoter of the PCH biosynthetic gene pchE to the luxCDABE operon, and the resulting construct was inserted into the chromosome of the ΔpvdAΔpchDΔfpvA siderophore-null P. aeruginosa mutant. A bioassay was setup in a 96-well microplate format, enabling the contemporary screening of several samples in a few hours. A linear response was observed for up to 40 nM PCH, with a lower detection limit of 1.64 ± 0.26 nM PCH. Different parameters were considered to calibrate the biosensor, and a detailed step-by-step operation protocol, including troubleshooting specific problems that can arise during sample preparation, was established to achieve rapid, sensitive, and specific PCH quantification in both P. aeruginosa culture supernatants and biological samples. The biosensor was implemented as a screening tool to detect PCH-producing P. aeruginosa strains on a solid medium.
Highlights
The pyochelin (PCH) siderophore produced by the pathogenic bacterium Pseudomonas aeruginosa is an important virulence factor, acting as a growth promoter during infection
During infection, bacteria are faced with a tremendous iron-starvation stress, which is inductive of siderophore synthesis.[2−4] The Gram-negative bacterium Pseudomonas aeruginosa is one of the most dreaded opportunistic pathogens in the hospital setting and represents the first cause of morbidity and mortality in cystic fibrosis (CF) patients.[5]
Pilot testing of sputa from eight anonymous CF patients revealed that PCH was detectable in six of them, showing an excellent correlation between the presence of PCH and P. aeruginosa culturepositivity of sputa (Table S5). These results indicate that the ΔpvdAΔpchDΔf pvA PpchE::lux biosensor is suitable for PCH quantification in both P. aeruginosa culture supernatants and biological fluids and can be used for the rapid screening of PCH-producing P. aeruginosa isolates on a solid medium (Supporting Information, text S2)
Summary
The pyochelin (PCH) siderophore produced by the pathogenic bacterium Pseudomonas aeruginosa is an important virulence factor, acting as a growth promoter during infection. These analytical performances represent a significant advance over previous PCH quantification methods, which required large sample volumes (mL)[36,42,55] and eventual concentration after solvent extraction[36,42] and exhibited sensitivity in the μM PCH range.[11] To investigate the selectivity of the ΔpvdAΔpchDΔf pvA PpchE::lux biosensor, the luminescence emission was measured upon the addition of several iron-chelating compounds, using the standard test conditions (OD600 ≃ 0.25; 50 μL volume; 3.5 h; 25 °C).
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