Comprehensive Characterization of the Attenuated Double Auxotroph ΔΔ as an Alternative to H37Rv.

Although currently available model organisms such as and 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 strains may provide more representative, safer models to study biology. For example, the ΔΔ double auxotroph, has undergone rigorous and 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 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 We therefore performed a comprehensive comparison of H37Rv and ΔΔ. These strains demonstrated similar and intra-macrophage replication rates, similar responses to anti-TB agents and whole genome sequence conservation. Shotgun proteomics analysis suggested that ΔΔ 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 ΔΔ for immunological assays. We provide comprehensive evidence to support the judicious use of ΔΔ as a safe and suitable model organism for research, without the need for a BSL3 facility.