High throughput phenotypic selection of Mycobacterium tuberculosis mutants with impaired resistance to reactive oxygen species identifies genes important for intracellular growth

Abstract

Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS). Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.

Figure 1. Growth Curves of H37Rv, ΔmutT1, ΔmutM and ΔmutY in increasing concentrations of hydrogen peroxide. Data represent means of triplicates in a representative experiment.

Figure 2. Susceptibility of transposon mutants to oxidative stress.

Percentage of survival of the M. tuberculosis 100D7 (mmpL9), 115C4 (moaD1), 104D8 (ppe54), 115B9 (ppe56) transposon mutants and wild-type (GC1237) strains was determined by comparing bacteria treated with 9 mM of hydrogen peroxide to untreated controls. Data and error bars represent means and standard deviations of the results from triplicates in a representative experiment carried out three times with similar results. Statistically significant differences in survival of mutant strains when compared to wild-type are indicated by asterisks *P<0,05; ***P<0,001; ****P<0,0001.

Figure 3. Intracellular growth of mmpL9 and moaD1 mutants in human macrophages.

Monocyte-derived macrophages where infected with wild-type, moaD1 (A) or mmpL9 (B) mutant and complemented (COMP) strains at an MOI of 1∶100, bacteria per macrophage. At time point zero (0) and six days of infection, cells were lysed and bacteria were enumerated. Data and error bars represent means and standard deviations of the results from triplicates in a representative experiment carried out three times with similar results. Statistically significant differences are indicated by asterisks *P<0,05; **P<0,01;***P<0,001.

Figure 4. Graphical representation of the distribution of polymorphisms in mmpL9 in several M. tuberculosis genomes.

Polymorphisms in mmpL9 were analyzed in 24 different M. tuberculosis genomes that correspond to the following groups/lineages according to Gagneux et al. classification : 1–8: Beijing/W strains; 9–16: Euro-American strains; 17–20: Indo-oceanic strains; 21–22: West-African strains. Black vertical lines correspond to SNPs. Gap correspond to deletion in position 218 identified in all modern and ancestral (2–8) but not in early ancestral (1) Beijing/W strains.

Figure 5. Survival of mmpL9 and moaD1 mutant and complemented strains exposed to hydrogen peroxide.

Percentage of survival of M. tuberculosis wild-type (GC1237), mmpL9 (A) and moaD1(B) mutant and complemented (COMP) strains was determined by comparing bacteria treated with 9 mM of hydrogen peroxide to untreated controls. Data and error bars represent means and standard deviations of the results from triplicates in a representative experiment. Statistically significant differences are indicated by asterisks ***P<0,001; ****P<0,0001.