The efflux pumps Rv1877 and Rv0191 play differential roles in the protection of Mycobacterium tuberculosis against chemical stress.

Background: It was previously shown that GlnA3sc enabled Streptomyces coelicolor to survive in excess polyamines. However, subsequent studies revealed that Rv1878, the corresponding Mycobacterium tuberculosis (M.tb) ortholog, was not essential for the detoxification of spermine (Spm), in M.tb. On the other hand, the multi-drug efflux pump Rv1877 was previously shown to enable export of a wide range of compounds, while Rv0191 was shown to be more specific to chloramphenicol. Rationale: Therefore, we first wanted to determine if detoxification of Spm by efflux can be achieved by any efflux pump, or if that was dependent upon the function of the pump. Next, since Rv1878 was found not to be essential for the detoxification of Spm, we sought to follow-up on the investigation of the physiological role of Rv1878 along with Rv1877 and Rv0191. Approach: To evaluate the specificity of efflux pumps in the mycobacterial tolerance to Spm, we generated unmarked ∆rv1877 and ∆rv0191 M.tb mutants and evaluated their susceptibility to Spm. To follow up on the investigation of any other physiological roles they may have, we characterized them along with the ∆rv1878 M.tb mutant. Results: The ∆rv1877 mutant was sensitive to Spm stress, while the ∆rv0191 mutant was not. On the other hand, the ∆rv1878 mutant grew better than the wild-type during iron starvation yet was sensitive to cell wall stress. The proteins Rv1877 and Rv1878 seemed to play physiological roles during hypoxia and acidic stress. Lastly, the ∆rv0191 mutant was the only mutant that was sensitive to oxidative stress. Conclusion: The multidrug MFS-type efflux pump Rv1877 is required for Spm detoxification, as opposed to Rv0191 which seems to play a more specific role. Moreover, Rv1878 seems to play a role in the regulation of iron homeostasis and the reconstitution of the cell wall of M.tb. On the other hand, the sensitivity of the ∆rv0191 mutant to oxidative stress, suggests that Rv0191 may be responsible for the transport of low molecular weight thiols.

Spermine enhances the activity of anti-tuberculosis drugs.

IMPORTANCE: This is the first study that attempted to demonstrate the mechanisms of reactive oxygen species (ROS) generation by spermine (Spm) in Mycobacterium tuberculosis (M.tb). Furthermore, this is the first study to demonstrate that it is able to enhance the activity of currently available and World Health Organization (WHO)-approved tuberculosis (TB) drugs. Spermine can easily be obtained since it is already found in our diet. Moreover, as opposed to conventional antibiotics, it is less toxic to humans since it is found in millimolar concentrations in the body. Finally, with the difficulty of curing TB with conventional antibiotics, this study suggests that less toxic molecules, such as Spm, could in a long-term perspective be incorporated in a TB regimen to boost the treatment.

The ΔCysK2 mutant of Mycobacterium tuberculosis is sensitive to vancomycin associated with changes in cell wall phospholipid profile.

Cysteine plays a versatile role in cellular physiology and has previously been shown to be instrumental to Mycobacterium tuberculosis (M.tb) pathophysiology. In this study, we have generated mutants deficient in CysK2 and CysH, the key Cysteine, biosynthetic enzymes. In contrast to the ΔcysH mutant, the ΔcysK2 mutant is not an auxotroph and as such not essential for cysteine biosynthesis. Interestingly, the ΔcysK2 mutant shows increased sensitivity to cumene hydroperoxide, vitamin C, diamide, rifampicin and Vancomycin and shows alterations in phospholipid profile of Mtb cell wall. Our findings suggest that alteration in phospholipids content of M.tb cell wall by CysK2 may form a mode of defence against selected antibiotics and oxidative stress.

From infection niche to therapeutic target: the intracellular lifestyle of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen killing millions of people annually. Treatment for tuberculosis is lengthy and complicated, involving multiple drugs and often resulting in serious side effects and non-compliance. Mtb has developed numerous complex mechanisms enabling it to not only survive but replicate inside professional phagocytes. These mechanisms include, among others, overcoming the phagosome maturation process, inhibiting the acidification of the phagosome and inhibiting apoptosis. Within the past decade, technologies have been developed that enable a more accurate understanding of Mtb physiology within its intracellular niche, paving the way for more clinically relevant drug-development programmes. Here we review the molecular biology of Mtb pathogenesis offering a unique perspective on the use and development of therapies that target Mtb during its intracellular life stage.

MymA Bioactivated Thioalkylbenzoxazole Prodrug Family Active against Mycobacterium tuberculosis.

Screening of a GSK-proprietary library against intracellular Mycobacterium tuberculosis identified 1, a thioalkylbenzoxazole hit. Biological profiling and mutant analysis revealed that this compound is a prodrug that is bioactivated by the mycobacterial enzyme MymA. A hit-expansion program including design, synthesis, and profiling of a defined set of analogues with optimized drug-like properties led to the identification of an emerging lead compound, displaying potency against intracellular bacteria in the low micromolar range, high in vitro solubility and permeability, and excellent microsomal stability.

Ergothioneine is a secreted antioxidant in Mycobacterium smegmatis.

Ergothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle of Mycobacterium smegmatis revealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant of M. smegmatis lacking egtD (MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion of egtD from wild-type M. smegmatis and an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protecting M. smegmatis against oxidative stress and that ERG is able to partly compensate for the loss of MSH.