Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy

Abstract

Multidrug-resistant (MDR) tuberculosis, defined as tuberculosis resistant to the two first-line drugs isoniazid and rifampin, poses a serious problem for global tuberculosis control strategies. Lack of a safe and convenient model organism hampers progress in combating the spread of MDR strains of Mycobacterium tuberculosis. We reasoned that auxotrophic MDR mutants of M. tuberculosis would provide a safe means for studying MDR M. tuberculosis without the need for a biosafety level 3 (BSL3) laboratory. Two different sets of triple auxotrophic mutants of M. tuberculosis were generated, which were auxotrophic for the nutrients leucine, pantothenate, and arginine or for leucine, pantothenate, and methionine. These triple auxotrophic strains retained their acid-fastness, their ability to generate both a drug persistence phenotype and drug-resistant mutants, and their susceptibility to plaque-forming mycobacterial phages. MDR triple auxotrophic mutants were obtained in a two-step fashion, selecting first for solely isoniazid-resistant or rifampin-resistant mutants. Interestingly, selection for isoniazid-resistant mutants of the methionine auxotroph generated isolates with single point mutations in katG, which encodes an isoniazid-activating enzyme, whereas similar selection using the arginine auxotroph yielded isoniazid-resistant mutants with large deletions in the chromosomal region containing katG. These M. tuberculosis MDR strains were readily sterilized by second-line tuberculosis drugs and failed to kill immunocompromised mice. These strains provide attractive candidates for M. tuberculosis biology studies and drug screening outside the BSL3 facility.

Immunometabolism at the interface between macrophages and pathogens

“…In addition to iron sequestration, restricted access to other metal ions such as Zn 2+ , Cu 2+ and Mn 2+ can also reduce microbial fitness and growth [48][49][50][51] . Most intracellular bacteria are auxotrophic [G] for purines, pyridines and/or amino acids [52][53][54][55] , which -coupled with the known metabolic restrictions of immune cells 56 -indicates an obvious link between host cell metabolism and the growth limitation of intracellular pathogens. In pivotal early experiments, IFNγ-induced degradation of host cell tryptophan was shown to restrict the growth of Toxoplasma gondii 57 .…”

The Isoniazid Paradigm of Killing, Resistance, and Persistence in Mycobacterium tuberculosis

“…2A). One such DRM, Φ 2 DRM9, in which the expression of tdTomato is driven by the promoter of the chaperone gene dnaK , has been used to visualize persisters produced during drug treatment [87,91,92] or present in sputum samples of TB patients [87]. Time-lapse photography of M. tuberculosis cells infected with Φ 2 DRM9 on a microfluidic chip and incubated in INH-containing media revealed that the majority of cells initially displayed a greenish-yellow fluorescence (Fig.…”

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

“…Doubly auxotrophic strains include Δ lysA Δ panCD (Sambandamurthy et al, 2002, 2005) and Δ leuD Δ panCD (Sampson et al, 2004). Recent work generated a drug-susceptible and drug-resistant triple auxotrophic stain of M. tuberculosis , which provides a safe model for studying drug-resistant M. tuberculosis under BSL2 conditions (Vilchèze et al, 2018). Another interesting development is the use of a streptomycin-dependent M. tuberculosis strain ( M. tuberculosis 18b) as a model of latent TB (Zhang et al, 2012).…”