Dataset Information

Comprehensive Characterization of the Attenuated Double Auxotroph Mycobacterium tuberculosis?leuD?panCD as an Alternative to H37Rv.

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

SUBMITTER: Mouton JM

PROVIDER: S-EPMC6710366 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Mouton Jomien M JM Heunis Tiaan T Dippenaar Anzaan A Gallant James L JL Kleynhans Léanie L Sampson Samantha L SL

Frontiers in microbiology 20190820

Although currently available model organisms such as Mycobacterium smegmatis and Mycobacterium bovis 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 Mycobacterium tuberculosis strains may provide more representative, safer models to study M. tuberculosis biology. For example, the M. tuberc ...[more]

PMID: 31481950

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Project description:Mycobacterium tuberculosis H37Ra is an attenuated tubercle bacillus closely related to the virulent type strain M. tuberculosis H37Rv. Despite extensive study, the reason for the decreased virulence of M. tuberculosis H37Ra has not been determined. A genomic approach was therefore initiated to identify genetic differences between M. tuberculosis H37Rv and M. tuberculosis H37Ra as a means of pinpointing the attenuating mutation(s). Digestion with the rare-cutting restriction endonuclease DraI revealed two polymorphisms between the strains: a 480-kb fragment in M. tuberculosis H37Rv was replaced by two fragments of 220 and 260 kb in M. tuberculosis H37Ra, while there was a approximately 7.9-kb DraI fragment in M. tuberculosis H37Ra that had no counterpart in M. tuberculosis H37Rv. As the M. tuberculosis insertion sequence IS6110 contains a single DraI restriction site, it was considered possible that these polymorphisms were the result of IS6110 transposition events in M. tuberculosis H37Ra, events that may have inactivated virulence genes. The 7.9-kb polymorphism was found to be due to the presence of the previously described H37Rv RvD2 deletion in M. tuberculosis H37Ra, with sequence analysis suggesting an IS6110-mediated deletion mechanism for loss of RvD2. Three other IS6110-catalyzed deletions from the M. tuberculosis H37Rv chromosome (RvD3 to RvD5) were also identified, suggesting that this mechanism plays an important role in genome plasticity in the tubercle bacilli. Comparative mapping and sequencing revealed that the 480-kb polymorphism was due to an IS6110 insertion in M. tuberculosis H37Ra near oriC. Complementation of M. tuberculosis H37Ra with a 2.9-kb restriction fragment from M. tuberculosis H37Rv that encompassed the IS6110 insertion did not increase the survival of recombinant M. tuberculosis H37Ra in mice. In conclusion, this study describes the presence and mechanisms of genomic variation between M. tuberculosis H37Ra and M. tuberculosis H37Rv, although the role that they play in the attenuation of M. tuberculosis H37Ra is unclear.

Dataset Information

Comprehensive Characterization of the Attenuated Double Auxotroph Mycobacterium tuberculosis?leuD?panCD as an Alternative to H37Rv.

Publications

Comprehensive Characterization of the Attenuated Double Auxotroph <i>Mycobacterium tuberculosis</i>Δ<i>leuD</i>Δ<i>panCD</i> as an Alternative to H37Rv.

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