Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> 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 stra...

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Autores principales:	Catherine Vilchèze, Jacqueline Copeland, Tracy L. Keiser, Torin Weisbrod, Jacqueline Washington, Paras Jain, Adel Malek, Brian Weinrick, William R. Jacobs
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	Mycobacterium tuberculosis arginine auxotrophy methionine multidrug resistance Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/117a19d7561c4ccb89b01386464f0034
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spelling	oai:doaj.org-article:117a19d7561c4ccb89b01386464f00342021-11-15T16:00:24ZRational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> through Nutrient Auxotrophy10.1128/mBio.00938-182150-7511https://doaj.org/article/117a19d7561c4ccb89b01386464f00342018-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00938-18https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE Elimination of Mycobacterium tuberculosis, the bacterium causing tuberculosis, requires enhanced understanding of its biology in order to identify new drugs against drug-susceptible and drug-resistant M. tuberculosis as well as uncovering novel pathways that lead to M. tuberculosis death. To circumvent the need for a biosafety level 3 (BSL3) laboratory when conducting research on M. tuberculosis, we have generated drug-susceptible and drug-resistant triple auxotrophic strains of M. tuberculosis suitable for use in a BSL2 laboratory. These strains originate from a double auxotrophic M. tuberculosis strain, H37Rv ΔpanCD ΔleuCD, which was reclassified as a BSL2 strain based on its lack of lethality in immunocompromised and immunocompetent mice. A third auxotrophy (methionine or arginine) was introduced via deletion of metA or argB, respectively, since M. tuberculosis ΔmetA and M. tuberculosis ΔargB are unable to survive amino acid auxotrophy and infect their host. The resulting triple auxotrophic M. tuberculosis strains retained characteristics of M. tuberculosis relevant for most types of investigations.Catherine VilchèzeJacqueline CopelandTracy L. KeiserTorin WeisbrodJacqueline WashingtonParas JainAdel MalekBrian WeinrickWilliam R. JacobsAmerican Society for MicrobiologyarticleMycobacterium tuberculosisarginineauxotrophymethioninemultidrug resistanceMicrobiologyQR1-502ENmBio, Vol 9, Iss 3 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Mycobacterium tuberculosis arginine auxotrophy methionine multidrug resistance Microbiology QR1-502
spellingShingle	Mycobacterium tuberculosis arginine auxotrophy methionine multidrug resistance Microbiology QR1-502 Catherine Vilchèze Jacqueline Copeland Tracy L. Keiser Torin Weisbrod Jacqueline Washington Paras Jain Adel Malek Brian Weinrick William R. Jacobs Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> through Nutrient Auxotrophy
description	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. IMPORTANCE Elimination of Mycobacterium tuberculosis, the bacterium causing tuberculosis, requires enhanced understanding of its biology in order to identify new drugs against drug-susceptible and drug-resistant M. tuberculosis as well as uncovering novel pathways that lead to M. tuberculosis death. To circumvent the need for a biosafety level 3 (BSL3) laboratory when conducting research on M. tuberculosis, we have generated drug-susceptible and drug-resistant triple auxotrophic strains of M. tuberculosis suitable for use in a BSL2 laboratory. These strains originate from a double auxotrophic M. tuberculosis strain, H37Rv ΔpanCD ΔleuCD, which was reclassified as a BSL2 strain based on its lack of lethality in immunocompromised and immunocompetent mice. A third auxotrophy (methionine or arginine) was introduced via deletion of metA or argB, respectively, since M. tuberculosis ΔmetA and M. tuberculosis ΔargB are unable to survive amino acid auxotrophy and infect their host. The resulting triple auxotrophic M. tuberculosis strains retained characteristics of M. tuberculosis relevant for most types of investigations.
format	article
author	Catherine Vilchèze Jacqueline Copeland Tracy L. Keiser Torin Weisbrod Jacqueline Washington Paras Jain Adel Malek Brian Weinrick William R. Jacobs
author_facet	Catherine Vilchèze Jacqueline Copeland Tracy L. Keiser Torin Weisbrod Jacqueline Washington Paras Jain Adel Malek Brian Weinrick William R. Jacobs
author_sort	Catherine Vilchèze
title	Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> through Nutrient Auxotrophy
title_short	Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> through Nutrient Auxotrophy
title_full	Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> through Nutrient Auxotrophy
title_fullStr	Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> through Nutrient Auxotrophy
title_full_unstemmed	Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> through Nutrient Auxotrophy
title_sort	rational design of biosafety level 2-approved, multidrug-resistant strains of <named-content content-type="genus-species">mycobacterium tuberculosis</named-content> through nutrient auxotrophy
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/117a19d7561c4ccb89b01386464f0034
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Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> through Nutrient Auxotrophy

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