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

Summary

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Abstract

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world’s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Introduction

Tuberculosis (TB) is a major threat to global public health with 9 million new cases per year and 1.5 million deaths¹. In addition, it is estimated that one quarter of the world’s population is infected with the causative agent of the disease, Mycobacterium tuberculosis (Mtb). Amongst the infected population, 5−10% will develop active TB disease over their lifetime. Furthermore, the emergence and spread of multi-drug resistant (MDR) and extensively-drug (XDR) resistant Mtb poses a serious threat to disease control, with 123 countries reporting at least one XDR case¹. Treatment of TB requires a cocktail of at least four anti-mycobacterial drugs, of which isoniazid and rifampicin are prescribed for a minimum duration of six months; treatment is often associated with complex side effects and toxicities. Protection from the only licensed vaccine against TB, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), is variable². An incomplete understanding of the pathogenesis of TB significantly hampers the development of new therapeutic and vaccination strategies.

For decades animal infection models have been vital for TB research to understand the basic pathogenesis and host response to infection, and to evaluate novel anti-mycobacterial agents, immuno-therapeutics and new vaccine candidates^3,4. However, research using animal infection models of TB is notoriously difficult as the pathogenesis and progression of TB infection are complex, and there is no single animal model that mimics the full spectrum and important features of the disease^5,6. Furthermore, animal experiments are expensive, time consuming to undertake and require full ethical justification. Nevertheless, animal infection models of TB have been described in non-human primates (e.g., macaques), guinea pigs, rabbits, cattle, pigs, mice and zebrafish, with each having their limitations^3,4. The murine model is the most commonly used model due to cost, availability of inbred lines, reproducibility of infection and abundance of immunological reagents. However, they do not typically form granulomas associated with areas of hypoxia that are characteristic of latent tuberculosis infection (LTBI)⁶. Guinea pigs are highly susceptible to Mtb infection, with pathology and early granuloma formation similar to those in humans, and are widely used in vaccine testing; yet the lack of immunological reagents hampers their use as an infection model⁷. Zebrafish are suitable for large-scale screening in early-stage preclinical studies due to their small size, rapid reproduction and advanced genetic tools, but are anatomically and physiologically different to humans and are only susceptible to Mycobacterium marinum infection³. The animal models most closely resembling human Mtb infection are non-human primates (e.g., the macaque), but they are expensive and have significant ethical and practical considerations which considerably limits their use⁸.

The insect larva of the greater wax moth or honeycomb moth, Galleria mellonella, have become increasingly popular as an infection model for a variety of bacterial and fungal pathogens⁹, and as a screen for novel antimicrobial drug candidates¹⁰. G. mellonella is a successful invertebrate model due to its sophisticated innate immune system (comprised of cellular and humoral defenses) that shares a high degree of structural and functional similarity to that of vertebrates¹¹. This includes immune mechanisms such as the phagocytosis of pathogens by hemocytes (functionally similar to mammalian macrophage and neutrophils)^12,13, the production and circulation of anti-microbial peptides (AMPs) and complement-like proteins within the hemolymph (analogous to mammalian blood) of G. mellonella¹¹. Other advantages^9,14,15 of G. mellonella larvae as a model include 1) their large size (20−30 mm) which allows for easy manipulation and infection, as well as the collection of tissue and hemolymph for analyses, 2) easy maintenance at 37 °C, compatible for studying human pathogens, 3) precise infection by injection without the need for anesthesia, 4) efficacy of antimicrobial agents can be assessed utilizing less drug for evaluation, 5) lack of ethical constraints compared to the use of mammals, 6) large group sizes can be used compared to animal models allowing greater reproducibility, and 7) shorter times for infection experiments are required.

In a recent study, we demonstrated that G. mellonella can be used as a novel infection model for studying the pathogenesis of infection by bioluminescent M. bovis BCG lux, a genetically modified version of the vaccine strain and member of the Mtb complex (MTBC)¹⁶. While G. mellonella has previously been used as an infection model for non-tuberculous mycobacteria (NTM), mainly M. marinum and Mycobacterium abscessus^17,18, studies using MTBC are limited to that of Li et al.¹⁶. Bioluminescent non-pathogenic mycobacterial strains, which can be used at containment level (CL) 2 as a surrogate for Mtb, offer the advantages of safety and practicality over pathogenic mycobacteria. Following infection with BCG lux, larvae begin to develop early granuloma-like structures, which could provide valuable insight into the role of innate immunity in the establishment of TB infection¹⁶. In addition, this simple invertebrate infection model has the potential to provide a rapid, low-cost, and reliable evaluation of TB pathogenesis incorporating controlled challenge and multiple replicates for reproducibility. Furthermore, the model has the potential to be used to screen novel anti-TB drug and vaccine candidates in early development, reducing the overall number of animals in experimentation. The ability to measure changes in host and pathogen structure, transcriptome and proteome to determine drug targets and assess mechanisms of action of novel drugs and therapeutic vaccines, are also advantageous.

Here we describe the experimental protocols for the preparation of a bioluminescent M. bovis BCG lux inoculum and G. mellonella larvae for mycobacterial infection, as well as the determination of both larval and mycobacterial survival in response to infection.

Protocol

NOTE: All work described below are to be carried out in a CL2 laboratory within a class 2 microbiological safety cabinet (MSC) following local health and safety guidelines.

1. Preparation of M. bovis BCG lux for Infection

1. Defrost a frozen 1.2 mL glycerol (15%) stock of M. bovis BCG lux, the Montreal vaccine strain transformed with the shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi encoding the luciferase enzyme¹⁹.
2. Inoculate 15 mL of Middlebrook 7H9 broth containing 0.2% glycerol, 10% albumin, dextrose, catalase (ADC) enrichment and 50 µg/mL hygromycin with a defrosted 1.2 mL aliquot of BCG lux, in a labelled 250 mL Erlenmeyer flask.
3. Place the flask in a sealed biosafety container and incubate at 37 °C in an orbital shaker incubator at 220 rpm for 72 h (or until the culture reaches the mid-log phase of growth).
4. Check the growth of BCG lux culture by preparing 1:10 dilutions of the culture in luminometer tubes using phosphate buffered saline (PBS, pH 7.4, 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride) in duplicate. Vortex, and load the luminometer tubes into the luminometer and measure the bioluminescence (relative light unit [RLU]/mL) using n-decyl aldehyde as the substrate (1% v/v in absolute ethanol)²⁰.
   NOTE: The ratio of RLU/colony forming units (CFU) was previously determined to be 3:1 when BCG lux was grown in vitro in Middlebrook 7H9 broth²⁰.
5. Centrifuge the culture at 2,175 x g for 10 min at room temperature to pellet the cells and discard the supernatant into an appropriate disinfectant with known mycobactericidal activity. Dispose all culture waste with disinfectants appropriate for mycobacteria following local guidelines.
6. Wash the cell pellet twice in PBS containing 0.05% polysorbate 80 (PBS-T) to prevent bacterial clumping.
7. Following the final wash, decant waste supernatant, resuspend the mycobacterial cell pellet in PBS-T and dilute the mycobacterial suspension to the desired cell density using RLU measurements.
8. Prepare ten-fold serial dilutions of the inoculum in 24-well plates using PBS-T. Plate out 10 µL onto Middlebrook 7H11 agar plates (0.5% glycerol, 50 µg/mL hygromycin, 10% oleic acid, albumin, dextrose, catalase [OADC]) in duplicate to enumerate inoculum CFU counts.

2. Preparation of G. mellonella Larvae

1. Purchase last instar larvae from appropriate sources and maintain the larvae in the dark at 18 °C upon arrival and use within 1 week of purchase. Alternatively, larvae can be self-reared following the protocol of Jojāo et al.²¹. For purchased larvae, discard any dead, discolored or pupating larvae prior to storage.
   NOTE: Pupating larvae are morphologically distinguishable to the last instar larvae.
2. Identify and select healthy larvae for experimentation, based on uniform cream color with little to no discoloration (melanization), size (2−3 cm in length), weight (approximately 250 mg), displaying a high level of motility, and possessing the ability to right themselves when turned over.
3. Carefully count the healthy larvae (minimum of 20−30 per group) into a Petri dish (94/15 mm) lined with a layer of filter paper (94/15 mm) using blunt-end tweezers to minimize contamination and store at room temperature in the dark until use.

3. Infecting G. mellonella with BCG lux

1. Minimize the clutter within the MSC to reduce the risk of contamination and needle stick injury.
2. Prepare the injection platform by taping a circular filter paper (94 mm) to a flat raised surface. Soft or hard surfaces can be used and is entirely dependent on user preference, e.g., pipette box lids or a nylon scouring sponge.
3. Sterilize a 25 µL microsyringe (25 G) by aspirating 3 volumes of 70% ethanol and further rinse with 3 volumes of sterile PBS-T.
4. Aspirate 10 µL of BCG lux inoculum (prepared in section 1) or PBS-T into the sterilized 25 µL microsyringe. Use a separate syringe for PBS-T negative control.
   NOTE: Resuspend the BCG lux inoculum following 10 injections to ensure uniform cell suspension.
5. Use tweezers to pick up one larva and place onto the injection platform.
6. On the platform, flip the larva on to 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 (5−6 mm) at a 10−20° angle to the horizontal plane.
   NOTE: Short and narrow tweezers allow for easy immobilization with minimum larval stress. Pay attention not to over penetrate which may puncture the gut and cause non-BCG lux specific melanization or death.
7. Count the infected larvae into a Petri dish lined with a layer of filter paper, a single 90 mm Petri dish can accommodate up to 30 larvae.
8. Store the Petri dish containing the larvae in a vented or non-sealed dark box inside an incubator at 37 °C with 5% CO₂.

4. Monitoring the Survival of G. mellonella Following Infection

1. Over the time course, monitor the survival of the larvae every 24 h. Larvae are considered dead when they fail to move in response to touch.

5. Measuring the In Vivo Burden of BCG lux in G. mellonella

1. At each time point, randomly select five infected larvae previously prepared in section 3 and gently sterilize the larval surfaces using cotton bud swabs soaked in 70% ethanol.
   NOTE: This step is important when plating larval homogenate for mycobacterial CFU enumeration as non-sterilized larvae can lead to contamination.
2. Place the larvae individually into 2 mL lysing matrix tubes containing 800 µL of sterile PBS.
3. Homogenize the larvae using a homogenizer for 60 s at 6.0 m/s.
4. Centrifuge the lysing tubes at 3,500 x g for 5 s to remove homogenate from the lids, and carefully decant the homogenate into sterile luminometer tubes individually.
   NOTE: For CFU enumeration (step 5.7), ensure to reserve 100 µL of homogenate in a sterile 1.5 mL reaction tube.
5. Recover any remaining homogenate by washing the lysing matrix tubes with 1 mL of PBS-T and pipette into the corresponding luminometer tubes.
6. Vortex the luminometer tubes and measure the bioluminescence of the homogenates previously described in step 1.4.
7. Prepare ten-fold serial dilutions of the homogenate in 24-well culture plates using PBS-T. Plate out 10 µL of the dilution onto Middlebrook 7H11 agar plates containing 0.5% glycerol, 50 µg/mL hygromycin, 10% OADC and 20 µg/mL piperacillin, to determine the RLU/CFU ratio of BCG lux following in vivo infection.
   NOTE: Piperacillin eliminates native G. mellonella microbiota with minimal growth inhibition of BCG lux¹⁶.

6. Statistical Analysis

1. Plot the Kaplan-Meier survival curve using data collected and carry out the Mantel-Cox (Log-rank) test to determine the significance of the result, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

Here we present representative data that can be obtained using the G. mellonella — BCG lux infection model and highlight the benefits of G. mellonella as an infection model for members of the MTBC (Figure 1). Experimental procedures with key technical points are outlined in Figure 2.

Discussion

The use of G. mellonella as an infection model has been established for a number of bacterial and fungal pathogens for the study of virulence, host-pathogen interaction, and as a screen for novel therapeutics^10,22. The following discussion is based on the experimental procedure for the use of G. mellonella as an infection model for the MTBC.

The health of the naïve larvae prior to experimentation c...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL reaction tube (Eppendorf)	Eppendorf	22431021
20, 200 and 1,000 µL pipette and filtered tips	Any supplier	n/a
24 well culture plate	Greiner	662160
25 mL pipettes and pipette boy	Any supplier	n/a
3 compartment Petri dish (94/15 mm)	Greiner	637102
Centrifuge	Any supplier	n/a
Class II saftey cabinet	Any supplier	n/a
Erlenmeyer flask with vented cap (250 mL)	Corning	CLS40183
Ethanol (>99.7%)	VWR	208221.321
Galleria mellonella (250 per pk)	Livefood Direct UK	W250
Glycerol	Sigma-Aldrich	G5150
Homogeniser (FastPrep-24 5G )	MP Biomedicals	116005500
Hygromycin B	Corning	30-240CR
Luminometer (Autolumat LB 953)	Berthold	34622
Luminometer tubes	Corning	352054
Lysing matrix (S, 2.0 mL)	MP Biomedicals	116925500
Micro syringe (25 µL, 25 G)	SGE	3000
Microcentrifuge	Any supplier	n/a
Middlebrook 7H11 agar	BD Bioscience	283810
Middlebrook 7H9 broth	BD Bioscience	271310
Middlebrook ADC enrichment	BD Bioscience	212352
Middlebrook OADC enrichment	BD Bioscience	212240
Mycobacterium bovis BCG lux	Various	n/a
n-decyl aldehyde	Sigma-Aldrich	D7384-100G
Orbital shaking incubator	Any supplier	n/a
Phosphate buffered saline	Sigma-Aldrich	P4417-100TAB
Polysorbate 80 (Tween-80)	Sigma-Aldrich	P8074-500ml
Small box	Any supplier	n/a	dark vented or non-sealed box recommended
Tweezer	Any supplier	n/a	Short and narrow tipped/Blunt long tweezers
Winterm (V1.08)	Berthold	n/a	Program LB953.TTB
Petri dish (94/15 mm)	Greiner	633181
Filter paper (94 mm)	Any supplier	n/a	Cut to fit