This study investigates the infection process of Mycobacterium tuberculosis in mice, focusing on the interaction between bacteria and macrophages. The methodology includes the use of a reporter substrate to visualize bacterial infection through fluorescence imaging.
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.
To proceed, load animals of known weight into the powered-up inoculation chamber. The chamber can fit up to 90 mice. Then, close all the latches on the door. Next, connect the nebulizer using a steel clamp. Then, press the start button on the chamber, and set the airflow rate to between 3 and 5 liters per minute.
The compressed air will flow at about 22 to 26 pounds per square inch, and importantly, it should be possible to see the mist of inoculum in the chamber. After 15 minutes, a red light on the front of the control panel will appear, and an audible signal will indicate the end of the run. Then, press the reset button to reset the timers. To release the vacuum, press the small red button on the door of the chamber.
Then, open the chamber door and return the mice to their home cages. Going forward, keep the animals in the containment room. Next, return the jar in the transport container to the biosafety cabinet. There, discard the remaining bacterial suspension into a designated waste container. Then, place the used nebulizer jar inside a biohazard bag and seal the bag for transport to the autoclave.
To clean the chamber, spray the inside surfaces with buffered phenol and 70% ethanol. Allow these solutions to react on those surfaces for 10 minutes, then wipe them off thoroughly. Dispose of all the wipes in the biohazard trash. Then, autoclave the trash, waste container, and used nebulizer jars.
Transfer the animals to the imaging room. There, anesthetize an animal according to the text protocol and inject it with contrast substrate by intraperitoneal injection. In the imaging system software, initialize the system and proceed when the temperature bar turns green. After initializing the system, place the mouse in the imaging chamber.
Make sure its nostrils are within the nose cone so it receives anesthetic during imaging. Now, return to the computer. In the acquisition control panel, select either fluorescence, transillumination, for whole-animal visualization, or epi-illumination to view lung tissues. For a single mouse, set the field of view to B and the lamp level to high.
For the exposure, use automatic time, medium binning, and two or three for the F/Stop. Then, set the excitation filter to 745 nanometers and emission filters from 780 to 840 nanometers. Next, go to sequence set-up and select 9 to 12 transillumination points in the area of interest. Now, press the acquire button to collect images.
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