Galleria mellonella - a novel infection model for the Mycobacterium tuberculosis complex

Abstract

Animal models have long been used in tuberculosis research to understand disease pathogenesis and to evaluate novel vaccine candidates and anti-mycobacterial drugs. However, all have limitations and there is no single animal model which mimics all the aspects of mycobacterial pathogenesis seen in humans. Importantly mice, the most commonly used model, do not normally form granulomas, the hallmark of tuberculosis infection. Thus there is an urgent need for the development of new alternative in vivo models. The insect larvae, Galleria mellonella has been increasingly used as a successful, simple, widely available and cost-effective model to study microbial infections. Here we report for the first time that G. mellonella can be used as an infection model for members of the Mycobacterium tuberculosis complex. We demonstrate a dose-response for G. mellonella survival infected with different inocula of bioluminescent Mycobacterium bovis BCG lux, and demonstrate suppression of mycobacterial luminesence over 14 days. Histopathology staining and transmission electron microscopy of infected G. mellonella phagocytic haemocytes show internalization and aggregation of M. bovis BCG lux in granuloma-like structures, and increasing accumulation of lipid bodies within M. bovis BCG lux over time, characteristic of latent tuberculosis infection. Our results demonstrate that G. mellonella can act as a surrogate host to study the pathogenesis of mycobacterial infection and shed light on host-mycobacteria interactions, including latent tuberculosis infection.

A novel biosafety level 2 compliant tuberculosis infection model using a ΔleuDΔpanCD double auxotroph of Mycobacterium tuberculosis H37Rv and Galleria mellonella

“…The survival of SAMTB lux in vivo was determined using data pooled from a minimum of three experimental repeats. The homogenates were plated onto 7H11 agar to enumerate the CFU to determine RLU:CFU ratio in vivo: 7H11 agar was further supplemented with piperacillin (20 µg/ml) to inhibit the growth of G. mellonella flora as previously described [15].…”

“…Histopathological, confocal microscopy and transmission electron microscopy (TEM) analysis of SAMTB lux infection in G. mellonella G. mellonella larvae were infected with SAMTB lux (2x10 7 CFU) for histopathological and TEM time course analysis, performed as previously described [15]. For histopathology, three larvae were fixed and stored in buffered formalin at 0, 24, 48 and 168 h postinfection.…”

“…G. mellonella has many advantages over conventional models: 1) larval maintenance does not require specialized housing: 2) last instar larvae do not need feeding: 3) no specialized equipment is required: 4) their size (2-3 cm) permits easy handling and precise dosing of pathogen and/or drugs: 5) they can be incubated at 37°C (optimum temperature for human pathogens); and 6) ethics permission is not required [14]. In previous studies using Mycobacterium bovis BCG as a surrogate for MTB, we demonstrated that G. mellonella can be used to study members of the MTB complex (MTBC) [14,15], including screening of antimycobacterial agents [14].…”

Mutation on lysX from Mycobacterium avium hominissuis impacts the host–pathogen interaction and virulence phenotype

“…G. mellonella has a sophisticated innate immune system which consists of cellular and humoral defenses, which expose of a functional similarity to vertebrates [56]. Recent reports have already demonstrated the potentiality of employing a wax moth larvae model for studying both slow and fast-growing mycobacteria infections such as M. bovis BCG [57], M. abscessus [58], M. fortuitum [59], M. marinum [59] and M. aurum [59].…”

Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth

“…The GM larva has emerged as a simple and economical invertebrate model host to evaluate virulence of microbial pathogens and efficacy of antimicrobial compounds (Tsai, Loh, & Proft, 2016). Recent studies have validated the use of this model with mycobacteria and shown that larva lethality is species-dependent (Entwistle & Coote, 2018;Li et al, 2018;Meir, Grosfeld, & Barkan, 2018;Rwegasila, Mubofu, Nyandoro, Erasto, & Munissi, 2016).…”