Innate Immune Responses of Galleria mellonella to Mycobacterium bovis BCG Challenge Identified Using Proteomic and Molecular Approaches

Abstract

The larvae of the insect Galleria mellonella, have recently been established as a non-mammalian infection model for the Mycobacterium tuberculosis complex (MTBC). To gain further insight into the potential of this model, we applied proteomic (label-free quantification) and transcriptomic (gene expression) approaches to characterise the innate immune response of G. mellonella to infection with Mycobacterium bovis BCG lux over a 168 h time course. Proteomic analysis of the haemolymph from infected larvae revealed distinct changes in the proteome at all time points (4, 48, 168 h). Reverse transcriptase quantitative PCR confirmed induction of five genes (gloverin, cecropin, IMPI, hemolin, and Hdd11), which encoded proteins found to be differentially abundant from the proteomic analysis. However, the trend between gene expression and protein abundance were largely inconsistent (20%). Overall, the data are in agreement with previous phenotypic observations such as haemocyte internalization of mycobacterial bacilli (hemolin/β-actin), formation of granuloma-like structures (Hdd11), and melanization (phenoloxidase activating enzyme 3 and serpins). Furthermore, similarities in immune expression in G. mellonella, mouse, zebrafish and in vitro cell-line models of tuberculosis infection were also identified for the mechanism of phagocytosis (β-actin). Cecropins (antimicrobial peptides), which share the same α-helical motif as a highly potent peptide expressed in humans (h-CAP-18), were induced in G. mellonella in response to infection, giving insight into a potential starting point for novel antimycobacterial agents. We believe that these novel insights into the innate immune response further contribute to the validation of this cost-effective and ethically acceptable insect model to study members of the MTBC.

Keywords: Galleria mellonella; Mycobacterium bovis BCG; gene expression; in vivo model; innate immunity; proteomics; tuberculosis.

Figure 1

Survival curve of G. mellonella over a 168 h time course following infection with varying BCG lux inocula. Data represent three independent experiments, n=30 for each experimental group. Plotted are the mean and the standard deviation of the mean.

Figure 2

Survival of G. mellonella 96 h post infection immunized with a non-lethal dose of BCG lux 48 h pre infection. Data represent three independent experiments n = 30, n = 10 for each experimental repeat. Plotted are the mean and the standard deviation of the mean. ** = p < 0.01.

Figure 3

In vivo survival of BCG lux in G. mellonella. Changes in BCG lux bioluminescence (relative light units, RLU) within G. mellonella following infection (1 x 10⁷ CFU) over a 168 h time course. Data represent three independent experiments each with five technical replicates. Plotted are the mean and the standard deviation of the mean. RLU : CFU is approximately 4:1 as previously described by Li et al., 2018.

Figure 4

Principal component analysis (PCA) of G. mellonella haemolymph proteomic profiles following infection with BCG lux inoculum (1 x 10⁷ CFU) after 0 h (blue), 4 h (orange), 48 h (green), and 168 h (pink). Number of larvae/time point/replicate n=10. PCA of three replicates included in label free quantification analysis with a clear distinction between each time point.

Figure 5

Proteomic responses of G. mellonella larvae following infection by BCG lux (1 x 10⁷ CFU) after 4 h (A), 48 h (B), and 168 h (C). Volcano plots represent protein intensity difference (- log2 mean intensity difference) and significance in differences (- log P-value) based on a two-sided t-test. Proteins above the red dashed line are considered statistically significant (p value < 0.05) and those to the right and left of the vertical blue lines indicate relative fold changes > ± 1.5. These plots are based upon post imputed data. Each independent experiment consists of pooled haemolymph collected from 10 larvae. Data represent 3 independent experiments.

Figure 6

Level of G. mellonella gene expression was measured by RT-qPCR from RNA of larvae challenged with either low dose (10⁵ CFU, red) or high dose (10⁷ CFU, grey) BCG lux at 4, 48, and 168 h pi. The comparative ΔΔCt method was used for relative quantification of (A) gloverin, (B) cecropin, (C) hemolin, (D) Hdd11, and (E) IMPI using ubiquitin as a housekeeping gene. Data represent three independent experiments, each with RNA extracted from five larvae, which was pooled. Each independent experiment consisted of three technical repeats. Bar charts represent changes in gene expression expressed as fold change. Comparison of gene expression with each infectious dose over time was carried out using the Mann-Whitney U test; where *,**,****, and ns signifies p < 0.05, 0.01, 0.0001 and non-significant, respectively.