Carine Sao Emani

Background: It was previously shown that GlnA3_sc 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.

The discovery of molecules that could shorten the treatment period of tuberculosis (TB) holds the promise of improving the current regimen. Polyamines are organic polycationic alkylamines found in millimolar concentrations in all living cells. Spermine (Spm) is a polyamine that was shown (more than 70 years ago) to be toxic to Mycobacterium tuberculosis (M.tb). In this study, we used various biochemical assays to assess the mechanism of action of Spm. Furthermore, using various drug susceptibility testing methods, we investigated the ability of Spm to enhance the activity of first-line TB drugs, isoniazid (INH) and rifampicin (RIF), as well as second-line TB drugs, para-aminosalicylic acid (PAS) and bedaquiline (BDQ). Our findings revealed that at toxic concentrations, Spm induced the formation of reactive oxygen species (ROS), a reaction that was influenced by the pH and the iron concentration of the media. Furthermore, the generation of ROS by Spm was associated with increased ferric iron and NADP levels. Moreover, the ROS produced by Spm synergized with the ROS generated by the lipophilic peroxide cumene hydroperoxide (CuOOH), as well as with the ROS generated by RIF and PAS, while enhancing their antimycobacterial activity. Additionally, we found that Spm enhanced the combined activity of INH and RIF against M.tb. Lastly, Spm was also able to enhance the activity of BDQ due to another mechanism of action. This study provides data and findings that would fuel future studies on the suitability of Spm as a potential adjunct TB therapy. The enhanced antimycobacterial activity of TB drugs in combination with Spm could offer promising new avenues for improving TB treatment outcomes. 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.

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.

Mycothiol (MSH) and ergothioneine (ERG) are thiols able to compensate for each other to protect mycobacteria against oxidative stress. Gamma-glutamylcysteine (GGC), another thiol and an intermediate in ERG biosynthesis has detoxification abilities. Five enzymes are involved in ERG biosynthesis, namely EgtA, EgtB, EgtC, EgtD and EgtE. The role of these enzymes in the production of ERG had been unclear. On the other hand, the enzyme MshA is known to be essential for MSH biosynthesis. In this manuscript, we describe the raw data of the generation and characterization ofMycobacterium tuberculosis(M.tb)mutants harbouring a deletion of the gene coding for each of these enzymes, and the raw data of the phenotypic characterization of the obtained thiol-deficientM.tbmutants. High throughput screening (HTS) of off-patent drugs and natural compounds revealed few compounds that displayed a higher activity against the thiol-deficient mutants relative to the wild-type strain. The mode of action of these drugs was further investigated. Raw data displaying these results are described here.

Three low molecular weight thiols are synthesized by Mycobacterium tuberculosis (M.tb), namely ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC). They are able to counteract reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In addition, the production of ERG is elevated in the MSH-deficient M.tb mutant, while the production of MSH is elevated in the ERG-deficient mutants. Furthermore, the production of GGC is elevated in the MSH-deficient mutant and the ERG-deficient mutants. The propensity of one thiol to be elevated in the absence of the other prompted further investigations into their interplay in M.tb. To achieve that, we generated two M.tb mutants that are unable to produce ERG nor MSH but are able to produce a moderate (ΔegtD-mshA) or significantly high (ΔegtB-mshA) amount of GGC relative to the wild-type strain. In addition, we generated an M.tb mutant that is unable to produce GGC nor MSH but is able to produce a significantly low level of ERG (ΔegtA-mshA) relative to the wild-type strain. The susceptibilities of these mutants to various in vitro and ex vivo stress conditions were investigated and compared. The ΔegtA-mshA mutant was the most susceptible to cellular stress relative to its parent single mutant strains (ΔegtA and ∆mshA) and the other double mutants. In addition, it displayed a growth-defect in vitro, in mouse and human macrophages suggesting; that the complete inhibition of ERG, MSH and GGC biosynthesis is deleterious for the growth of M.tb. This study indicates that ERG, MSH and GGC are able to compensate for each other to maximize the protection and ensure the fitness of M.tb. This study therefore suggests that the most effective strategy to target thiol biosynthesis for anti-tuberculosis drug development would be the simultaneous inhibition of the biosynthesis of ERG, MSH and GGC.

The high acquisition rate of drug resistance by Mycobacterium tuberculosis necessitates the ongoing search for new drugs to be incorporated in the tuberculosis (TB) regimen. Compounds used for the treatment of other diseases have the potential to be repurposed for the treatment of TB. In this study, a high-throughput screening of compounds against thiol-deficient Mycobacterium smegmatis strains and subsequent validation with thiol-deficient M. tuberculosis strains revealed that ΔegtA and ΔmshA mutants had increased susceptibility to azaguanine (Aza) and sulfaguanidine (Su); ΔegtB and ΔegtE mutants had increased susceptibility to bacitracin (Ba); and ΔegtA , ΔmshA , and ΔegtB mutants had increased susceptibility to fusaric acid (Fu). Further analyses revealed that some of these compounds were able to modulate the levels of thiols and oxidative stress in M. tuberculosis . This study reports the activities of Aza, Su, Fu, and Ba against M. tuberculosis and provides a rationale for further investigations.

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.