Galleria mellonella-intracellular bacteria pathogen infection models: the ins and outs

Abstract

Galleria mellonella (greater wax moth) larvae are used widely as surrogate infectious disease models, due to ease of use and the presence of an innate immune system functionally similar to that of vertebrates. Here, we review G. mellonella-human intracellular bacteria pathogen infection models from the genera Burkholderia, Coxiella, Francisella, Listeria, and Mycobacterium. For all genera, G. mellonella use has increased understanding of host-bacterial interactive biology, particularly through studies comparing the virulence of closely related species and/or wild-type versus mutant pairs. In many cases, virulence in G. mellonella mirrors that found in mammalian infection models, although it is unclear whether the pathogenic mechanisms are the same. The use of G. mellonella larvae has speeded up in vivo efficacy and toxicity testing of novel antimicrobials to treat infections caused by intracellular bacteria: an area that will expand since the FDA no longer requires animal testing for licensure. Further use of G. mellonella-intracellular bacteria infection models will be driven by advances in G. mellonella genetics, imaging, metabolomics, proteomics, and transcriptomic methodologies, alongside the development and accessibility of reagents to quantify immune markers, all of which will be underpinned by a fully annotated genome.

Keywords: Galleria mellonella; antimicrobial; insect; intracellular pathogen; mycobacteria; virulence.

Figure 1.

From infection to treatment – G. mellonella data acquisition. (A) Galleria mellonella larvae are infected/treated via injection using a microsyringe for accurate dosing. (B) Larval melanization (darkening of the cuticle) can quantitatively monitor disease progression. (C) The most common readouts found in G. mellonella-based infection studies are Kaplan–Meier (KM) survival plots of percentage survival versus time, formulated originally by and named after Kaplan and Meier (1958). C1-4 shows examples. (C1) Comparative virulence of wild-type (WT) versus an isogenic mutant with an equivalent dose. (C2) Comparative virulence of strain or species A versus strain or species B at an equivalent dose. (C3) Drug efficacy. Typically, G. mellonella larvae are infected, and at the appropriate time, drugs are injected, and their ability to prevent lethal infection is determined. Different doses may be used. In some studies, drugs are given before or immediately after infection. In the example shown, Drug A is more efficacious than Drug B. Typically, viable counts are determined to validate this assertion. (C4) LD50, i.e. the inoculum size that kills 50% of larvae may also be determined. In the example shown, the LD50 is 10⁶ CFU at 96 h. A variation is the LT50, i.e. the time to kill 50% of larvae. All methods described above are routine for the analysis of interactions of G. mellonella with intracellular bacterial pathogens included in this review. In the case of (C1–C3), a quantitative method to determine viability, e.g. CFU or luminescence, is also typically used, and a drug treatment example is shown (D). Viability may be determined at all or just the last time point. Depending on the study aims, proteomic (e.g. liquid chromatography-tandem mass spectrometry, SDS-PAGE) and/or gene expression (e.g. transcriptomic, RT-qPCR), histological, and microscopy analyses of G. mellonella and/or the intracellular bacterium of interest, may also be done.

Figure 2.

Overview of the G. mellonella-enhanced mycobacterial drug development pipeline. The chevron arrows at the top show the overall drug development pipeline. During the drug discovery phase, different routes identify potential drugs and their minimum inhibitory concentration (MIC) determined against mycobacteria in low or high throughput screens in vitro. The ability of promising compounds to kill mycobacteria growing intracellularly, e.g. in mouse J774 or RAW 264.7 cell lines, is then tested. In a conventional pipeline, efficacious compounds are then tested for their ability to cure M. tuberculosis-infected mice. Dependent on the study design, the preclinical stage may involve seeking basic toxicity data. The G. mellonella-enhanced pipeline involves a prescreen (in red) following intracellular efficacy testing, where the efficacy and toxicity of promising compounds are tested in M. tuberculosis-infected and noninfected larvae, respectively. Compounds that are efficacious and nontoxic in G. mellonella would then typically proceed to testing in mice. However, a recent FDA decision means licensure no longer requires animal data. The G. mellonella-enhanced pipeline enables promising efficacious nontoxic compounds to be identified at an earlier stage, shortening the overall drug development process, which results in a reduction of mammals in research and associated costs. AI = artificial intelligence.

Figure 3.

Timeline of important historical advances in G. mellonella–mycobacterial research in the 19th and 20th centuries (top) and 21st century (bottom). Timelines not to scale. Each box lists the year, the first author of the associated paper, and the advance. See the main text for full details of each advance. *Experiments described in the Ferrari and Barbaro (1964) review of the work of Morrellini and Cattaneo. BCG = bacille Calmette–Guérin; GM = G. mellonella; MABC = M. abscessus complex; MIP = M. indicus pranii; MTB = M. tuberculosis; and TB = tuberculosis.

Figure 4.

Transmission electron micrograph (TEM) of G. mellonella haemocytes. (A) TEM image of haemocyte recovered from naïve (uninfected) larva. (B) TEM image of haemocyte recovered from BCG-infected larva. Red arrows point to intracellular BCG showing accumulation of lipid fat bodies, a key phenotype indicating a metabolic switch from an active to a nonreplicative state. (C) TEM of BCG used as the inoculum for G. mellonella infection. Scale bars represent 500 nm. Adapted from Li et al. (2018) with permission.

Figure 5.

Visualization of mycobacterial-induced GLS in G. mellonella. (A) Infection is systemic with yellow fluorescence protein (YFP) expressing BCG establishing foci of infection throughout the larval cavity. (B)–(E) shows the consistency in results obtained by researchers over c. 100 years. Microscope images were sketched by (B) Metalnikov (1920), (C) Redaelli (1929), and photographed by (D) Cameron (1934) and (E) Li et al. (2018).

Figure 6.

Loss of H37Rv ZN-staining in some G. mellonella GLS. (A) ZN-stained sagittal section of G. mellonella infected with M. tuberculosis H37Rv with GLS indicated by the blue arrows. Scale bar indicates 1 mm. (B) A representative GLS containing dense ZN-reactive material, with localized peripheral loss in ZN-reactivity indicated in the area enclosed by the white square. Foci of intense H&E staining most likely indicates host cell necrosis. (C) A representative GLS associated with a predominant loss of ZN-reactivity. In contrast to (B), non-ZN reactive masses had less intense H&E staining, and host cell nuclei were more easily distinguishable (as highlighted by the white arrows), likely indicating that host cell necrosis is low despite the presence of mycobacterial mass. Adapted from Asai et al. (2022) with permission.

Figure 7.

Screenshots of packaging of commercially available G. mellonella extracts. Except for Annas Dravas (top right-hand corner), produced in Latvia, all the others are of Russian origin. Galleria mellonella extracts are widely advertized as being suitable for the treatment of pulmonary disease and/or tuberculosis.

Figure 8.

TEM of G. mellonella haemocytes 3 days postinfection with C. burnetii. (A) Uninfected controls with no visible bacteria. (B) Haemocytes from infected larvae, including a clearly visible CCV that fills the entire cell cytoplasm. Arrows indicate proposed LCVs (black) and SCVs (white) – see main text for further details. The data shown represent the analysis of 50 control and 50 infected haemocyte images. Scale bars = 1 μm. Reproduced with minor adaptation from Kovacs-Simon et al. (2020) with permission under CC-BY-4.0.

Figure 9.

Galleria mellonella brain infected with L. monocytogenes. (A) Dissection of G. mellonella larvae infected with L. monocytogenes reveals the formation of dark spots in the brain. (B) Treatment with diclofenac prevents the formation of these spots. (C) Localized melanized regions form in the G. mellonella brain following infection with L. monocytogenes. (D) Persistence of labelled L. monocytogenes (red fluorescence) in melanized regions. Reproduced from Mukherjee et al. (2013) with permission.