AUTHOR=Mouton Jomien M. , Heunis Tiaan , Dippenaar Anzaan , Gallant James L. , Kleynhans Léanie , Sampson Samantha L. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.01922

DOI=10.3389/fmicb.2019.01922

ISSN=1664-302X

ABSTRACT=<p>Although currently available model organisms such as <italic>Mycobacterium smegmatis</italic> and <italic>Mycobacterium bovis</italic> 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 <italic>Mycobacterium tuberculosis</italic> strains may provide more representative, safer models to study <italic>M. tuberculosis</italic> biology. For example, the <italic>M. tuberculosis</italic> Δ<italic>leuD</italic>Δ<italic>panCD</italic> double auxotroph, has undergone rigorous <italic>in vitro</italic> and <italic>in vivo</italic> 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 <italic>M. tuberculosis</italic> 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 <italic>M. tuberculosis.</italic> We therefore performed a comprehensive comparison of <italic>M. tuberculosis</italic> H37Rv and <italic>M. tuberculosis</italic>Δ<italic>leuD</italic>Δ<italic>panCD</italic>. These strains demonstrated similar <italic>in vitro</italic> and intra-macrophage replication rates, similar responses to anti-TB agents and whole genome sequence conservation. Shotgun proteomics analysis suggested that <italic>M. tuberculosis</italic>Δ<italic>leuD</italic>Δ<italic>panCD</italic> 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 <italic>M. tuberculosis</italic>Δ<italic>leuD</italic>Δ<italic>panCD</italic> for immunological assays. We provide comprehensive evidence to support the judicious use of <italic>M. tuberculosis</italic>Δ<italic>leuD</italic>Δ<italic>panCD</italic> as a safe and suitable model organism for <italic>M. tuberculosis</italic> research, without the need for a BSL3 facility.</p>