Monitoring Gene Expression during a Galleria mellonella Bacterial Infection

Abstract

Galleria mellonella larvae are an alternative in vivo model that has been extensively used to study the virulence and pathogenicity of different bacteria due to its practicality and lack of ethical constraints. However, the larvae possess intrinsic autofluorescence that obstructs the use of fluorescent proteins to study bacterial infections, hence better methodologies are needed. Here, we report the construction of a promoter probe vector with bioluminescence expression as well as the optimization of a total bacterial RNA extraction protocol to enhance the monitoring of in vivo infections. By employing the vector to construct different gene promoter fusions, variable gene expression levels were efficiently measured in G. mellonella larvae at various time points during the course of infection and without much manipulation of the larvae. Additionally, our optimized RNA extraction protocol facilitates the study of transcriptional gene levels during an in vivo infection. The proposed methodologies will greatly benefit bacterial infection studies as they can contribute to a better understanding of the in vivo infection processes and pathogen-mammalian host interactions.

Keywords: Galleria mellonella; P. aeruginosa; bioluminescence; hemocytes; hemolymph; optimized RNA extraction; promoter probe vector; ribonucleotide reductases.

Figure 1

Visualization of G. mellonella larvae using different imaging methodologies. P. aeruginosa PAO1 wild-type (WT) and PAO1::eGFP strains were imaged using (A) the Gel Doc™ imaging system and (B) the LSM 800 confocal microscope. Free bacteria were imaged on LB media as well as inside G. mellonella larvae. (C) P. aeruginosa PAO1 wild-type (WT) and PAO1::lux were imaged as free bacteria on an LB plate and within G. mellonella larvae using the ImageQuant™ LAS 4000 mini and Odyssey^® Fc imaging systems.

Figure 2

Autofluorescence studies in the hemolymph of G. mellonella. (A) Quantification of green autofluorescence, in relative fluorescence units per milliliter of hemolymph, using larvae infected with different P. aeruginosa strains and PBS (negative control) at 16 and 20 h post-infection. (B) Quantification of red autofluorescence, in relative fluorescence units per milliliter of hemolymph, using larvae infected with different P. aeruginosa strains and PBS (negative control) at 16 and 20 h post-infection. (C) Phase contrast merged with green and red fluorescence images of hemolymph extracted from larvae infected with P. aeruginosa PAO1 wild-type showing the green and red autofluorescence of hemocytes, respectively. (D) Phase contrast only and phase contrast merged with green fluorescence images of hemolymph extracted from larvae infected with P. aeruginosa PAO1 PnrdA-GFP. (E) Phase contrast only and phase contrast merged with red fluorescence images of hemolymph extracted from larvae infected with P. aeruginosa PAO1 PnrdA-E2Crimson.

Figure 3

Map of the pETS220-BIATlux (pETSlux) promoter-probe vector. The luxCDABE genes were inserted into the backbone of the pETS130 vector. Relevant genetic elements include: gentamicin resistance (Gm^R) imparted by aacC1, plasmid mobilization functions encoded by mob, and pBBR1 oriV and pBBR1 Rep as the replication origin and replication protein, respectively, that are essential for broad-host-range capability. Unique cutter restriction enzymes within the multi-cloning site (MCS) are shown in bold. All genes are represented in scale according to the total length of the plasmid. The vector map was designed with SnapGene^® version 5.0.8 (GSL Biotech, San Diego, CA, USA).

Figure 4

Study of a P. aeruginosa PAO1 infection in G. mellonella larvae using the different lux constructions. (A) Measurements of relative luminescence (RLU) in G. mellonella during several time points after infection within the different strains. (B) Images of G. mellonella larval bioluminescence taken with the ImageQuant™ LAS 4000 mini imager at 17 and 20 h post-infection and visualized using the Gem lookup table from ImageJ FIJI. (C) Bioluminescence induction factors during the different time points (14, 17, and 20 h post-infection). Anr-lux background was subtracted from each of the strains.

Figure 5

Gene expression studies during a P. aeruginosa infection in G. mellonella. (A) Schematic representation of the steps involved in the optimized RNA extraction protocol for bacterial cells derived from a G. mellonella infection. Created with BioRender.com. (B) P. aeruginosa PAO1 wild-type (WT) growth curve in G. mellonella larvae that was determined by calculating CFU/mL at different time points after infection as well as at death (last point). Samples for qRT-PCR were taken when the bacteria were in the exponential growth phase (close to 10⁸ CFU/mL) as indicated by the arrow. (C) Fold change of nrd and anaerobic (norC) genes determined by qRT-PCR during PAO1 WT infection compared to PAO1 WT cells grown to exponential phase in LB medium. The gap gene was used as an internal standard. The values shown are the average from two independent experiments, and the error bars indicate a positive standard deviation.