Divergent downstream biosynthetic pathways are supported by L-cysteine synthases of Mycobacterium tuberculosis

Reviewer #1 (Public Review):

Summary:

Khan et. al., investigated the functional redundancy of the non-canonical L-cysteine synthases of M. tuberculosis, CysM and CysK2, focussing on their role in mitigating the effects of host-derived stress. They found that while deletion mutants of the two synthases (Rv∆cysM, Rv∆cysK2) have similar transcriptomes under standard conditions, their transcriptional response to oxidative stress is distinct. The impact of deleting the synthases also differentially affected the pools of L-cysteine-derived metabolites. They show that the mutants (Rv∆cysM, Rv∆cysK2) have impaired survival in peritoneal macrophages and in a mouse model of infection. Importantly, they show that the survival of the mutants increases when the host is defective in producing reactive oxygen and nitrogen species, linking the phenotype to a defect in combating host-derived stress. Finally, they show that compounds inhibiting L-cysteine synthases reduce the intracellular survival of M. tuberculosis.

Strengths:

1. The distinct transcriptome of the Rv∆cysM and Rv∆cysK2 mutants in the presence of oxidative stress provides solid evidence that these mutants are distinct in their response to oxidative stress, and suggests that they are not functionally redundant.
2. The use of macrophages from phox-/- and INF-/- mice and an iNOS inhibitor for the intracellular survival assays provides solid evidence that the survival defect seen for the Rv∆cysM and Rv∆cysK2 mutants is related to their reduced ability to combat host-derive oxidative and nitrosative stress. This is further supported by the infection studies in phox-/- and INF-/- mice.

Weaknesses:

1. There are several previous studies looking at the transcriptional response of M. tuberculosis to host-derived stress, however, the authors do not discuss initial RNA-seq data in the context of these studies. Furthermore, while several of the genes in sulfur assimilation and L-cysteine biosynthetic pathway genes are upregulated by more than one stress condition, the data does not support the statement that it is the "most commonly upregulated pathway in Mtb exposed to multiple host-like stresses".
2. For the quantification of the metabolites, it isn't clear how the abundance was calculated (e.g., were standards for each metabolite used? How was abundance normalised between samples?), and this information should be included to strengthen the data. Furthermore, labelling with L-methionine was performed to determine the rate of synthesis of the L-cysteine-derived metabolites. L-cysteine is produced from L-methionine via the transsulfuration pathway, which is independent of CysM and CysK2. It is therefore difficult to interpret this experiment, as the impact of deleting CysM and CysK2 on the transsulfuration pathway is likely indirect.

3. The ability of L-cysteine to rescue the survival defect of the Rv∆cysM and Rv∆cysK2 mutants in macrophages is interpreted as exogenous L-cysteine being able to compensate for reduced intracellular levels. However, there is no evidence that L-cysteine is being taken up by the mutants and an alternate explanation is that L-cysteine functions as an antioxidant within cells i.e., it reduces intracellular ROS.

The authors sought to investigate the functional redundancy of the non-canonical L-cysteine synthases CysM and CysK2. While their distinct transcriptional response to oxidative stress suggests distinct physiological roles, the study did not explore these differences and therefore provides only preliminary insight into the underlying reasons for this observation. In the context of drug development, this work suggests that while L-cysteine synthase inhibitors do not have high potency for killing intracellular M. tuberculosis, they have the potential to decrease the pathogen's survival in the presence of host-derive stress.

Reviewer #2 (Public Review):

Summary:

The paper examines the role L-cysteine metabolism plays in the biology of Mycobacterium tuberculosis. The authors have preliminary data showing that Mycobacterium tuberculosis has two unique pathways to synthesize cysteine. The data showing new compounds that act synergistically with INH is very interesting.

Strengths:

RNAseq data is interesting and important.

Weaknesses:

The paper would be strengthened if the authors were to add further detail to their genetic manipulations.

The authors provide evidence that they have successfully made a cysK2 mutant by recombineering. This data looks promising, but I do not see evidence for the cysM deletion. It is also important to state what sort of complementation was done (multicopy plasmid, integration proficient vector, or repair of the deletion). Since these mutants are the basis for most of the additional studies, these details are essential. It is important to include complementation in mouse studies as unexpected loss of PDIM could have occurred.

Reviewer #3 (Public Review):

In this work, the authors conduct transcriptional profiling experiments with Mtb under various different stress conditions (oxidative, nitrosative, low pH, starvation, and SDS). The Mtb transcriptional responses to these stress conditions are not particularly new, having been reported extensively in the literature over the past ~20 years in various forms. A common theme from the current work is that L-cysteine synthesis genes are seemingly up-regulated by many stresses. Thus, the authors focused on deleting two of the three L-cysteine synthesis genes (cysM and cysK2) in Mtb to better understand the roles of these genes in Mtb physiology.

The cysM and cysK2 mutants display fitness defects in various media (Sautons media, starvation, oxidative and nitrosative stress) noted by CFU reductions. Transcriptional profiling studies with the cysM and cysK2 mutants revealed that divergent gene signatures are generated in each of these strains under oxidative stress, suggesting that cysM and cysK2 have non-redundant roles in Mtb's oxidative stress response which likely reflects the different substrates used by these enzymes, CysO-L-cysteine and O-phospho-L-serine, respectively. Note that these studies lack genetic complementation and are thus not rigorously controlled for the engineered deletion mutations.

The authors quantify the levels of sulfur-containing metabolites (methionine, ergothioneine, mycothiol, mycothionine) produced by the mutants following exposure to oxidative stress. Both the cysM or cysK2 mutants produce more methionine, ergothioneine, and mycothionine relative to WT under oxidative stress. Both mutants produce less mycothiol relative to WT under the same condition. These studies lack genetic complementation and thus, do not rigorously control for the engineered mutations.

Next, the mutants were evaluated in infection models to reveal fitness defects associated with oxidative and nitrosative stress in the cysM or cysK2 mutants. In LPS/IFNg activated peritoneal macrophages, the cysM or cysK2 mutants display marked fitness defects which can be rescued with exogenous cysteine added to the cell culture media. Peritoneal macrophages lacking the NADPH oxidase (Phox) or IFNg fail to produce fitness phenotypes in the cysM or cysK2 mutants suggesting that oxidative stress is responsible for the phenotypes. Similarly, chemical inhibition of iNOS partly abrogated the fitness defect of the cysM or cysK2 mutants. Similar studies were conducted in mice lacking IFNg and Phox establishing that cysM or cysK2 mutants have fitness defects in vivo that are dependent on oxidative and nitrosative stress.

Lastly, the authors use small molecule compounds to inhibit cysteine synthases. It is demonstrated that the compounds display inhibition of Mtb growth in 7H9 ADC media. No evidence is provided to demonstrate that these compounds are specifically inhibiting the cysteine synthases via "on-target inhibition" in the whole Mtb cells. Additionally, it is wrongly stated in the discussion that "combinations of L-cys synthase inhibitors with front-line TB drugs like INH, significantly reduced the bacterial load inside the host". This statement suggests that the INH + cysteine synthase inhibitor combinations reduce Mtb loads within a host in an infection assay. No data is presented to support this statement.