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 ΔleuDΔpanCD 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. tuberculosisΔleuDΔpanCD. 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. tuberculosisΔleuDΔpanCD 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. tuberculosisΔleuDΔpanCD for immunological assays. We provide comprehensive evidence to support the judicious use of M. tuberculosisΔleuDΔpanCD as a safe and suitable model organism for M. tuberculosis research, without the need for a BSL3 facility.
| Original language | English |
|---|---|
| Article number | 1922 |
| Journal | Frontiers in Microbiology |
| Volume | 10 |
| Issue number | AUG |
| DOIs | |
| Publication status | Published - 1 Jan 2019 |
Funding
The authors acknowledge the SA MRC Centre for TB Research and DST/NRF Centre of Excellence for Biomedical Tuberculosis Research for the financial support for this work. SS was funded by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation (NRF) of South Africa, Award Number UID 86539. JL was supported by the NRF-VU Desmond Tutu Doctoral Training Programme. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NRF.. We thank Dr. Hanri Calitz for the technical assistance with the growth curves, and Mrs. Claudia Spies and Dr. Frik Sirgel for the assistance with DST testing. We also thank the South African Bioinformatics Initiative for the advice on bioinformatics analyses of the multiplex bead array. We acknowledge the Central Analytical Facility for the use of the FACSJazz flow cytometer and Orbitrap Fusion Tribrid mass spectrometer. We also thank Dr. James Posey and the Centres for Disease Control and Prevention, Atlanta, GA, United States for NGS analyses.
| Funders | Funder number |
|---|---|
| NRF-VU | |
| SA MRC | |
| Medical Research Council | MR/R005850/1 |
| National Research Foundation | UID 86539 |
| Department of Science and Technology, Republic of South Africa | |
| Department of Science and Technology, Ministry of Science and Technology, India |
Keywords
- Attenuated auxotroph
- Biosafety level 2
- H37Rv
- Model organism
- Mycobacterium tuberculosis
