Comprehensive Characterization of the Attenuated Double Auxotroph Mycobacterium tuberculosisΔleuDΔpanCD as an Alternative to H37Rv

Abstract

Although currently available model organisms such as Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) have significantly contributed to our understanding of tuberculosis (TB) biology, these models have limitations such as differences in genome size, growth rates and virulence. However, attenuated Mycobacterium tuberculosis strains may provide more representative, safer models to study M. tuberculosis biology. For example, the M. tuberculosis Δ leuD Δ panCD double auxotroph, has undergone rigorous in vitro and in vivo safety testing. Like other auxotrophic strains, this has subsequently been approved for use in biosafety level (BSL) 2 facilities. Auxotrophic strains have been assessed as models for drug-resistant M. tuberculosis and for studying latent TB. These offer the potential as safe and useful models, but it is important to understand how well these recapitulate salient features of non-attenuated M. tuberculosis. We therefore performed a comprehensive comparison of M. tuberculosis H37Rv and M. tuberculosis Δ leuD Δ panCD . These strains demonstrated similar in vitro and intra-macrophage replication rates, similar responses to anti-TB agents and whole genome sequence conservation. Shotgun proteomics analysis suggested that M. tuberculosis Δ leuD Δ panCD has a heightened stress response that leads to reduced bacterial replication during exposure to acid stress, which has been verified using a dual-fluorescent replication reporter assay. Importantly, infection of human peripheral blood mononuclear cells with the 2 strains elicited comparable cytokine production, demonstrating the suitability of M. tuberculosis Δ leuD Δ panCD for immunological assays. We provide comprehensive evidence to support the judicious use of M. tuberculosis Δ leuD Δ panCD as a safe and suitable model organism for M. tuberculosis research, without the need for a BSL3 facility.

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

“…The use of pMV306 was chosen for its stability, as the plasmid is chromosomally integrated. The resistance status of SAMTB lux is similar to that of WT M. tuberculosis H37Rv [ 16 ], with additional resistance to kanamycin and hygromycin for Δ leuD Δ panCD and pMV306 selection respectively. SAMTB lux was cultured in Middlebrook 7H9 broth (BD, UK), supplemented with glycerol (0.2%) [Sigma-Aldrich, UK], albumin dextrose catalase (ADC 10%) [USBio, USA], polysorbate-80 (0.05%) [Sigma-Aldrich, UK], leucine (25 µg/ml) [Sigma-Aldrich, UK) and calcium pantothenate (24 µg/ml) [Sigma-Aldrich, UK].…”

“…A recent publication described a BSL2 compliant, severely attenuated double auxotrophic ∆leuC∆panCD strain of MTB H37Rv (SAMTB) [16]. Compared to H37Rv (wild-type, WT), SAMTB had comparable in vitro and intracellular growth, and immunogenicity, making it a suitable surrogate for WT MTB [16].…”

“…A recent publication described a BSL2 compliant, severely attenuated double auxotrophic ∆ leuC ∆ panCD strain of MTB H37Rv (SAMTB) [ 16 ]. Compared to H37Rv (wild-type, WT), SAMTB had comparable in vitro and intracellular growth, and immunogenicity, making it a suitable surrogate for WT MTB [ 16 ]. In this study, we have characterized a novel BSL2 compliant SAMTB- G. mellonella infection model, and show its potential to accelerate TB research.…”

Pharmacodynamic Functions of Synthetic Derivatives for Treatment of Methicillin-Resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis

“…For determination of minimum inhibitory concentrations (MIC), extracts (250 to 0.5 µg mL −1 ) or synthesized compounds (100 to 0.0002 µM) were serially diluted in microtiter plates. Bacterial suspension (90 µL, OD 600 nm 0.001) was added to each well and plates were incubated at 37 • C for either 18 h (MRSA, P. aeruginosa PAO1, E. coli EC958) or 7 days (M. tuberculosis H37Rv) as described previously (Harrison et al, 2017;Irene et al, 2019;Larcombe et al, 2019;Mouton et al, 2019). Resazurin (10 µL; 0.05% w/v) was added and plates were incubated for 3 h or 24 h (M. tuberculosis) at 37 • C. The inhibitory activity was calculated by visual determination of color change within wells or detection of fluorescence at 590 nm using a FLUOstar Omega microplate reader (BMG Labtech, Germany).…”

High-Content Screening of Eukaryotic Kinase Inhibitors Identify CHK2 Inhibitor Activity Against Mycobacterium tuberculosis

“…One major hurdle in screening campaigns for new therapeutics against M.tb is the expensive and labor-intensive requirements of Biological Contamination Level (BCL) 3 facilities. The BCL2-safe MC 2 6206 auxotrophic strain of M.tb (ΔleuDΔpanCD), originally developed as a vaccine model ( Sampson et al, 2004 ), was previously used successfully to test anti- M.tb compounds ( Schaaf et al, 2016 ; Mouton et al, 2019 ).…”