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.

A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

“…However, under oxidative stress conditions, generated by supplementation of cultures with H 2 O 2 , loss of MutY results in a significant decline in growth compared to the parental strain which is not exaggerated with the combinatorial loss of the Fpg or Nei homologues, suggesting that MutY is important for survival of M. smegmatis under oxidative stress conditions. These data are concordant with the loss of MutY function in M. tuberculosis [39], but conflict with a previous report which demonstrated that disruption of mutY in M. smegmatis does not lead to enhanced sensitivity to hydrogen peroxide [22]. In our work, we used the same parental strain of M. smegmatis [56] with similar experimental conditions hence, the reason for these discrepant results are unclear and may be due to the inherent variability in mutant generation, difference in quantitative survival assays or hydrogen peroxide decay.…”

“…Consistent with increased sensitivity of a mutY mutant in M. tuberculosis [39], the deletion of mutY in M. smegmatis resulted in a significant reduction (p = 0.025) in survival under oxidative stress, which was partially relieved by genetic complementation (p = 0.083 for the difference between the wild type and complemented strain) ( Fig. 2A).…”

“…MutY deficiency in M. smegmatis results in G → T transversions, with no increase in sensitivity to oxidative stress or elevation in mutation rates [22] suggesting that the two Fpg homologues present in this organism efficiently remove 8-oxoGua lesions. In contrast, disruption of mutY in M. tuberculosis leads to enhanced susceptibility to oxidative stress [39] which suggests that the single functional Fpg homologue is unable to maintain genome integrity and there is a greater dependency on MutY for genome maintenance. Biochemical characterization of MutY in M. smegmatis and M. tuberculosis showed substrate specificity to mispaired adenine opposite 8-oxoGua with weak excision activity for G and T paired with 8-oxoGua.…”

TbD1 deletion as a driver of the evolutionary success of modern epidemic Mycobacterium tuberculosis lineages

“…Notably, the EAI 1400-10134 and EAI 1400-10144 L1 strains, which were confirmed by PCR analyses as TbD1-intact strains (Fig. 7b) 45 . The increased susceptibility of ancestral Mtb strains to reactive oxygen intermediates (ROI) was further confirmed by comparisons of the diameters of the inhibition growth zones obtained for 79112 and Tb36 strains as compared to Mtb H37Rv after exposure to 350 nmol cumene hydroperoxide (an organic peroxide) or 50 nmol plumbagine (a superoxide generator) in disk diffusion assays on solid medium ( Supplementary Fig.…”

bis -Molybdopterin Guanine Dinucleotide Is Required for Persistence of Mycobacterium tuberculosis in Guinea Pigs

“…For example, transposon mutants harboring insertions in MoCo biosynthesis genes were attenuated for growth in macrophages (5-7), mice (8), and the lungs of nonhuman primates (9). Furthermore, a transposon insertion in moaD1, which encodes a subunit of molybdopterin synthase, rendered M. tuberculosis hypersensitive to oxidative stress, suggesting that the impaired macrophage invasion and phagosome maturation defects of this mutant are due to increased susceptibility to intracellular killing (10). Of particular interest is the observation that the moaA1-moaD1 gene cluster is located on a genomic island with reduced GC content and contains genetic features that are indicative of horizontal gene transfer (11).…”