This study investigates the interaction between Listeria monocytogenes and human placental organ cultures. The focus is on how the bacteria invade trophoblast layers and spread infection.
Take a human placental organ culture grown on an extracellular matrix, or ECM.
The culture exhibits a villi-like structure with an outer syncytium — a multinucleate trophoblast layer — and underlying cytotrophoblasts, or CBTs, which differentiate into extravillous trophoblasts, or EVTs, that anchor into the ECM.
Introduce Listeria monocytogenes, a pathogenic bacteria.
Internalins — the bacterial surface proteins — bind to EVT receptors, facilitating bacterial internalization via endocytosis.
The internalized bacteria secrete pore-forming toxins and phospholipases that perforate the vacuolar membrane, mediating its rupture and bacterial escape into the cytoplasm.
The escaped bacteria recruit host machinery for actin polymerization, propelling them toward the host cell membrane.
Secretion of a bacterial virulence factor induces protrusion formation on the host membrane. Neighboring cells engulf the bacteria-containing protrusion, causing infection spread.
The bacteria spread via EVTs to the underlying CBTs.
Add an antibiotic to remove the extracellular bacteria, preparing the infection model for analysis.
Before beginning the infection, inoculate a single L. monocytogenes colony from a streaked brain-heart infusion agar plate in 3 milliliters of brain-heart infusion broth. Incubate the culture overnight in a slanted position at 30 degrees celsius to allow the bacterial growth to reach a stationary phase.
The next morning, use sterile tweezers to remove any floating organ culture pieces that did not invade into the extracellular matrix, and carefully aspirate the medium from the bottom of each transwell. Next, gently pipette 1 milliliter of warm PBS into the upper and lower transwells two times, removing each wash with careful aspiration.
After the second wash, gently pipette 1 milliliter of the appropriate medium to the upper and lower transwells taking care not to disturb the organ cultures and return the cultures to the incubator for one hour.
At the end of the incubation, replace the medium in the top of the transwells with 1 milliliter of inoculum, and place the plates back in the cell culture incubator to facilitate the bacterial invasion, replacing all of the medium in each well daily.
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