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 req....

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 enzy.......

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

Name	Company	Catalog Number	Comments
1.5ml reaction tube (Eppendorf)	Eppendorf	22431021
20, 200 and 1000 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 (94/15mm)	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, 2.0ml)	MP Biomedicals	116925500
Micro syringe (25 µl, 25 ga)	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 (94/15mm)	Greiner	633181
Filter paper (94mm)	Any supplier	n/a	Cut to fit