Expose a mouse to aerosol droplets containing Mycobacterium tuberculosis.
Bacteria reach the lumen of alveoli — the tiny air sacs within the lungs — containing macrophages.
Macrophage pattern recognition receptors bind to the pathogen-associated molecular patterns on the bacteria, triggering their engulfment into a phagosome.
Engulfed bacteria secrete specialized proteins, which rupture the phagosome membrane. The bacteria escape into the cytoplasm and reproduce.
Post-infection, anesthetize the mouse. Intraperitoneally administer a reporter substrate of the bacterial β-lactamase enzyme for reporter enzyme fluorescence or REF imaging.
The substrate reaches lung alveoli via circulation and enters the infected cells.
It penetrates the bacterial outer membrane to reach the periplasmic space containing β-lactamase.
The substrate contains a β-lactam ring connecting a fluorochrome and a quencher. Due to proximity, the fluorochrome's fluorescence is absorbed by the quencher.
β-lactamase hydrolyzes the β-lactam ring, releasing the fluorophore and resulting in fluorescence emission.
Image the fluorescence to assess mycobacterial infection.
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