Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

This protocol describes the use of Galleria mellonella as an alternative infection model to study tuberculosis, reducing the number of mammalian models used in research. When compared to conventional mammalian models such as mice, Galleria mellonella is a cheaper, ethically acceptable, and more accessible model to study tuberculosis. Further optimization of this model will allow for screening of candidate antimycobacterial drug efficacy and toxicity and will contribute to the development of urgently needed novel therapeutics for tuberculosis.

Galleria mellonella possesses a complex innate immune system comparable to that of mammals. The study of host-pathogen interaction may further reveal the role of innate immunity in tuberculosis. The use of Galleria mellonella as an infection model is not limited to tuberculosis and has been widely used for other bacterial and fungal pathogens over the past decade.

To begin, defrost a frozen 1.2-milliliter glycerol stock of Mycobacterium bovis BCG lux, the Montreal vaccine strain transformed with the shuttle plasmid pSMT1 carrying the luxAB genes. In a labeled 250-milliliter Erlenmeyer flask, inoculate 15 milliliters of Middlebrook 7H9 broth with a defrosted 1.2-milliliter aliquot of BCG lux. Place the flask in a sealed biosafety container and incubate at 37 degrees Celsius in an orbital shaker incubator at 220 rpm for 72 hours.

After incubation, prepare 1:10 dilutions of the culture in phosphate-buffered saline in luminometer tubes, vortex, and load the luminometer tubes into the luminometer. Load the substrate 1%n-decyl aldehyde in absolute ethanol into the injector platform and measure the bioluminescence. Convert the bioluminescence to colony-forming units to check the growth of BCG lux culture.

Transfer the culture to a centrifuge tube and centrifuge at 2, 175 times g for 10 minutes at room temperature to pellet the cells. Discard the supernatant into an appropriate disinfectant with known mycobactericidal activity. Wash the cell pellet twice in 50 milliliters of PBS-T by centrifuging at 2, 175 times g for 10 minutes to prevent bacterial clumping.

Following the final wash, decant the waste supernatant and resuspend the mycobacterial cell pellet in the required volume of PBS-T to dilute the mycobacterial suspension to the desired cell density. Then, prepare 10-fold serial dilutions of the inoculum in 24-well plates using PBS-T. Plate out 10 microliters of each dilution onto Middlebrook 7H11 agar plates in duplicate to enumerate inoculum CFU counts.

Maintain the purchased instar larvae in the dark at 18 degrees Celsius upon arrival and use within one week of purchase. Identify and select healthy larvae for experimentation based on uniform cream color, appropriate size and weight, high motility, and possessing the ability to right themselves when turned over. Using blunt-end tweezers, carefully count the healthy larvae into a Petri dish lined with a layer of filter paper and store at room temperature in the dark until use.

Prepare the injection platform by taping a 94-millimeter diameter circular filter paper to a flat, raised surface. Aspirate three volumes of 70%ethanol into a 25-microliter microsyringe to sterilize, discard and further rinse with three volumes of sterile PBS-T. Then, resuspend the prepared BCG lux inoculum and aspirate 10 microliters into the sterilized microsyringe.

Use a separate syringe to aspirate PBS-T for a negative control. Following 10 injections, resuspend the BCG lux inoculum to ensure uniform cell suspension. Use tweezers to pick up one larva and place onto the injection platform.

On the platform, flip the larva onto its back and immobilize by securing the head and tail with the tweezers. Locate the last left proleg, counting down from the head of the larva and carefully insert the tip of the needle five to six millimeters deep at a 10 to 20-degree angle to the horizontal plane. Inject the inoculum from the syringe.

After each infection, transfer the infected larva into a Petri dish lined with a layer of filter paper. A single 94-millimeter Petri dish can accommodate up to 30 larvae. Store the Petri dish in a vented, dark box inside an incubator at 37 degrees Celsius with 5%carbon dioxide.

Monitor the survival of the larvae every 24 hours. Larvae are considered dead when they fail to move in response to touch. Record the survival and assess statistical significance with the Mantel-Cox test.

Every 24 hours, randomly select five infected larvae and use a cotton bud swab soaked in 70%ethanol to gently sterilize the larval surfaces. Place the larvae individually into two-milliliter lysing matrix tubes containing 800 microliters of sterile PBS. Then, use a homogenizer to homogenize the larvae for 60 seconds at six meters per second.

Centrifuge the lysing tubes at 3, 500 times g for five seconds to remove the homogenate from the lids and carefully decant the homogenate into five sterile luminometer tubes individually. Reserve 100 microliters of homogenate in a sterile 1.5-milliliter reaction tube. Recover any remaining homogenate by washing the lysing matrix tubes with one milliliter of PBS-T and pipette into the corresponding luminometer tubes.

Vortex the luminometer tubes and measure the bioluminescence of the homogenates on a luminometer. Then, prepare 10-fold serial dilutions of the reserved 100 microliters of homogenate in 24-well culture plates using PBS-T. Pipette 10 microliters of the dilution onto each Middlebrook 7H11 agar plate and use a sterile plate spreader to spread.

Incubate at 37 degrees Celsius for two weeks. After that, count colony-forming units. Determine the RLU to CFU ratio of BCG lux following in vivo infection.

In this experiment, BCG lux dose-dependent virulence was observed in G.mellonella larvae over a 96-hour incubation period. The lethal dose required for 50%larval mortality was determined to be one times 10 to the power of seven CFU. Control groups injected with a 10-microliter dose of PBS-T or those simply pricked simulating needle injuries did not affect larval health or lead to an increase in mortality.

For larvae infected with the two times 10 to the power of seven CFU dose of BCG lux, melanization of the larval dorsal line was observed from 48 hours post infection and systemic melanization was observed from 96 hours post infection. Infection with a one times 10 to the power of seven CFU dose of BCG lux resulted in an initial decline of bioluminescence within the first 72 hours post infection. However, after 72 hours post infection, the bioluminescence of BCG lux started to plateau, indicating the establishment of persistent infection.

In this particular infection system, the in vivo ratio of RLU and CFU ranged from 2:1 to 5:1 with an average of 4:1 over the 168-hour time course. During injection, remember to secure the larvae so that the risk of overpenetration is minimized. Accidental puncture of the gut could lead to increased mortality.

By carrying out histopathological analysis, introduction of mycobacteria with the host cells can be visualized. Further transcriptomic analysis could reveal the interaction at the genomic level. The use of this infection model may allow for the rapid testing of novel drug candidates at an early stage in development, significantly reducing the number of animals used in drug screening.

This infection model requires the use of a syringe, which can be associated with needle-stick injury. We recommend the use of our injection technique to minimize such risk.