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. 2020 Dec;11(1):811-824.
doi: 10.1080/21505594.2020.1781486.

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

Masanori Asai  1 , Yanwen Li  1 , John Spiropoulos  2 , William Cooley  2 , David Everest  2 , Brian D Robertson  3 , Paul R Langford  1 , Sandra M Newton  1
Affiliations

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

Masanori Asai et al. Virulence. 2020 Dec.

Abstract

Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialized animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output.

Keywords: Galleria mellonella; Mycobacterium tuberculosis complex; antimycobacterial agents; auxotrophic; drug screening; infection model; mycobacteria; tuberculosis.

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Conflict of interest statement

Authors have no conflict of interest to declare.

Figures

Figure 1.
Figure 1.
Kaplan-Meier survival curves of G. mellonella challenged with a range of SAMTB lux inocula over a 96 h time course.
Figure 2.
Figure 2.
In vivo proliferation of SAMTB lux in G. mellonella larvae over a 168 h time course.
Figure 3.
Figure 3.
Recovery of SAMTB lux from larval homogenate over a 168 h time course.
Figure 4.
Figure 4.
Histological analysis of G. mellonella-SAMTB lux interaction over time.
Figure 5.
Figure 5.
Cross sectional view of whole larvae infected with SAMTB lux.
Figure 6.
Figure 6.
SAMTB lux associates with hemocytes following infection.
Figure 7.
Figure 7.
Interaction between SAMTB lux and phagocytic hemocytes.
Figure 8.
Figure 8.
Changes in hemocyte counts in SAMTB lux infected larvae.
Figure 9.
Figure 9.
The effect of first-line and second-line antibiotics on the reduction of SAMTB lux bioluminescence within G. mellonella over a 96 h time course.
Figure 10.
Figure 10.
The effect of INH and RIF concentration on the reduction of SAMTB lux bioluminescence within G. mellonella.
Figure 11.
Figure 11.
The effect of RIF multiple dosing and RIF combination drug therapy on the reduction of SAMTB lux bioluminescence within G. mellonella.
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