This study investigates the immune response to Staphylococcus aureus infection in a mouse model. By utilizing fluorescent proteins, the interaction between neutrophils and bacteria can be visualized during the inflammatory response.
Take an anesthetized mouse engineered to express a fluorescent protein in neutrophils. Shave the dorsum and create a circular cut. Excise the cut skin to reveal the fascia — a connective tissue layer underneath.
Take a syringe containing a suspension of Staphylococcus aureus — a pathogenic bacterium — genetically modified to produce bioluminescence. Inject the suspension between the fascia and the underlying tissue generating a localized wound infection.
The pattern recognition receptors on stromal cells in the fascia recognize the pathogen-associated molecular patterns on the bacteria. This binding induces the release of pro-inflammatory cytokines and chemoattractants — resulting in inflammation.
Chemoattractants recruit circulatory neutrophils at the infection site. Incoming neutrophils engulf the bacteria into a phagosome that fuses with cytoplasmic granules, causing antimicrobial peptide release.
NADPH oxidase — an enzyme complex — assembles on the phagosome membrane and produces reactive oxygen species or ROS. The antimicrobial peptides and ROS cause bacterial degradation.
As a counter-response, the bacteria secrete virulence factors that permeabilize the neutrophil membrane, causing cell lysis. The bacteria also release enzymes for the deposition of fibrin to avoid phagocytosis.
The immune response leads to an abscess — a fibrin capsule containing bacterial cells, neutrophils, and dead cells.
Record the neutrophil fluorescence and bacterial bioluminescence to visualize the bacteria-neutrophil interaction.
For Staph aureus inoculation, fill a 28-gauge insulin syringe with 50 microliters of the prepared bioluminescent bacterial inoculant, and use a finger to pull the dermis of the wounded animal to the side. Holding the syringe nearly parallel to the tissue, slowly insert the syringe into the tissue until a sudden decrease in resistance is felt, indicating piercing of the fascia.
With the needle placed in the center of the wound, slowly deliver the entire volume of the inoculant. Confirm that the inoculant forms a bubble at the center of the wound with minimal leakage or dispersion, and remove the syringe slowly from the animal. Then, return the animal to its cage with heat and monitoring until full recovery.
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