A dual component heme biosensor that integrates heme transport and synthesis in bacteria

作者：Nobles Christopher L; Clark Justin R; Green Sabrina I; Maresso Anthony W^*

来源：Journal of Microbiological Methods, 2015, 118: 7-17.

DOI：10.1016/j.mimet.2015.07.011

• Heme
• Iron
• IFP1.A
• Escherichia coli O157:H7
• Biosensor
• Pathogenesis

Bacterial pathogens acquire host iron to power cellular processes and replication. Heme, an iron-containing cofactor bound to hemoglobin, is scavenged by bacterial proteins to attain iron. Methods to measure intracellular home are laborious, involve complex chemistry, or require radioactivity. Such drawbacks limit the study of the mechanistic steps of heme transport and breakdown. Hypothesizing heme homeostasis could be measured with fluorescent methods, we coupled the conversion of home to biliverdin IX alpha (a product of heme catabolism) by heme oxygenase 1 (HO1) with the production of near-infrared light upon binding this verdin by infrared fluorescent protein (IFP1.A). The resultant heme sensor, IFP-HO1, was fluorescent in pathogenic E. coli exposed to home but not in the absence of the heme transporter ChuA and membrane coupling protein TonB, thereby validating their long-standing proposed role in home uptake. Fluorescence was abolished in a strain lacking hemE, the central gene in the heme biosynthetic pathway, but stimulated by iron, signifying the sensor reports on intracellular heme production. Finally, an invasive strain of E. coli harboring the sensor was fluorescent during an active infection. This work will allow researchers to expand the molecular toolbox used to study heme and iron acquisition in culture and during infection.

• 出版日期2015-11