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

Jomien M Mouton,James L Gallant,Anzaan Dippenaar,Tiaan Heunis,Léanie Kleynhans,Samantha L Sampson

doi:10.3389/fmicb.2019.01922

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 ΔleuDΔpanCD 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. tuberculosisΔleuDΔpanCD. 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. tuberculosisΔleuDΔpanCD 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. tuberculosisΔleuDΔpanCD for immunological assays. We provide comprehensive evidence to support the judicious use of M. tuberculosisΔleuDΔpanCD as a safe and suitable model organism for M. tuberculosis research, without the need for a BSL3 facility.

Highlights

In order to radically reduce TB deaths and incidence by 2030, as set out by the End TB Strategy (World Health Organisation [WHO], 2018a), there is a need for improved TB therapies and more effective ways of studying the deadly pathogen Mycobacterium tuberculosis
We provide comprehensive comparative analyses between M. tuberculosis H37Rv and M. tuberculosis leuD panCD with regards to in vitro and intra-macrophage growth, genomic background, response to anti-TB agents, proteomic response to stress, and the host immune response
M. tuberculosis leuD panCD to our laboratory reference strain M. tuberculosis H37Rv, only one unique non-synonymous variant (I131T A > G) was identified at position 392 in Rv2988 in M. tuberculosis leuD panCD that was not found in M. tuberculosis H37Rv

Summary

Introduction

In order to radically reduce TB deaths and incidence by 2030, as set out by the End TB Strategy (World Health Organisation [WHO], 2018a), there is a need for improved TB therapies and more effective ways of studying the deadly pathogen Mycobacterium tuberculosis. Current research challenges include restricted access to Biosafety level 3 (BSL3) facilities and the slow growth of M. tuberculosis. This emphasizes the need for mycobacterial model systems that can facilitate our understanding of M. tuberculosis pathogenesis. Despite studies showing the use of currently available model organisms such as Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) to have significantly contributed to the understanding of M. tuberculosis, these models have limitations. The model organism BCG is known to contain a natural RD1 deletion (Behr et al, 1999), which encodes the known virulence factors early secreted antigenic target-6 kDa (ESAT-6) (Harboe et al, 1996) and culture filtrate protein-10 kDa (CFP-10) (Lewis et al, 2003; Gao et al, 2004; Guinn et al, 2004; Fortune et al, 2005), suggesting that the immune response elicited by BCG will be altered in comparison to M. tuberculosis

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