Mycobacterial biomaterials and resources for researchers

Hazbón, Manzour Hernando; Rigouts, Leen; Schito, Marco; Ezewudo, Matthew; Kudo, Takuji; Itoh, Takashi; Ohkuma, Moriya; Kiss, Katalin; Wu, Linhuan; Ma, Juncai; Hamada, Moriyuki; Strong, Michael; Daley, Charles L.; Nick, Jerry A.; Lee, Jung Sook; Rastogi, Nalin; Couvin, David; Hurtado-Ortiz, Raquel; Bizet, Chantal; Rodwell, Timothy C.; Albertini, Audrey; Lacourciere, Karen A.; Deheer-Graham, Ana; Alexander, Sarah; Russell, Julie E.; Bradford, Rebecca; Riojas, Marco A.

doi:10.1093/femspd/fty042

Cited by 15 publications

(10 citation statements)

References 25 publications

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“…Indeed, transposon insertions resources in M. tuberculosis ( 35 , 36 ) and other mutants were integrated by the TARGET program (Tuberculosis Animal Research and Gene Evaluation Taskforce [ http://webhost.nts.jhu.edu/target/ ]), now offering over 4,300 defined transposon insertion mutants in M. tuberculosis . Furthermore, the lab of W. R. Jacobs, Jr. (in collaboration with others), took on the effort to construct a comprehensive, ordered library of mutants, each mutated for a nonessential gene, based on specialized transduction ( 2 , 3 , 37 ). Supporting these efforts, we have deposited both of our BCG mutant resources (BCG Pasteur library of 6,072 traceable transposon mutants and this study’s BCG Danish library of 15,320 traceable, unmarkable transposon mutants) at the public BCCM-ITM Mycobacteria Collection ( http://bccm.belspo.be/about-us/bccm-itm ), making these mutants easily accessible to the scientific community.…”

Section: Discussionmentioning

confidence: 99%

Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource

et al. 2020

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Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach, despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which are undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI-mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the Mycobacterium tuberculosis complex (18,432 clones, mutating 83% of the nonessential M. tuberculosis homologues), from which markerless knockouts can be easily generated. IMPORTANCE While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of Mycobacterium bovis BCG Danish targets 83% of all nonessential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g., drug resistance research and vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the Mycobacterium tuberculosis complex. The stretch to a full collection of mutants in all nonessential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.

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Section: Discussionmentioning

confidence: 99%

Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource

et al. 2020

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“…Histopathological elements across the spectrum of leprosy have been described, 6 although most armadillos are susceptible to M. leprae infection and only 15%‐20% of the animals are resistant. For years, the armadillo has been used to cultivate large numbers of M. leprae for the purification of leprosy research reagents, 7 which can be obtained through the Biodefense and Emerging Infections Research Resources Repository (https://www.beiresources.org/). More recently, the armadillo is advancing as a model for leprosy‐specific neuropathy 8,9 .…”

Section: Animal Models For Leprosymentioning

confidence: 99%

Susceptibility and resistance in leprosy: Studies in the mouse model

Adams

2021

Immunological Reviews

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“…bovis BCG Danish 1331 (NIBSC 07/270, https://doi.org/10.6084/m9.figshare.c.4489496 (37,38)), which was used to map the sequencing reads. A detailed procedure for the construction of the archived mutant library can be found in Method S7.…”

Section: Construction and Validation Of The Archived Mutant Librarymentioning

confidence: 99%

Use of a counterselectable transposon to create markerless knockouts from a 18,432-clone orderedM. bovisBCG mutant resource

Borgers

Vandewalle

Hecke

et al. 2019

Preprint

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Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach; despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian Pooling-Coordinate Sequencing allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which is undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described, which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach Page 2 of 24 is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the M. tb complex (18,432 clones, mutating 83% of the non-essential M. tb homologues), from which clean knockouts can be generated. Importance:While speeding up research for many fields of biology (e.g. yeast, plant, and C. elegans), genomewide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time-and cost-effective manner, and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of M. bovis BCG Danish targets 83% of all non-essential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g. drug resistance research, vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the M. tb complex. The stretch to a full collection of mutants in all non-essential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described. MAIN TEXT:

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Mycobacterial biomaterials and resources for researchers

Cited by 15 publications

References 25 publications

Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource

Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource

Susceptibility and resistance in leprosy: Studies in the mouse model

Use of a counterselectable transposon to create markerless knockouts from a 18,432-clone orderedM. bovisBCG mutant resource

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