Mycobacterium tuberculosis SufR Responds to Nitric oxide via its 4Fe-4S cluster and Regulates Fe-S cluster Biogenesis for Persistence in Mice

  1. Kushi Anand
  2. Ashutosh Tripathi
  3. Kaustubh Shukla
  4. Nitish Malhotra
  5. Anil Kumar Jamithireddy
  6. Rajiv Kumar Jha
  7. Susmit Narayan Chaudhury
  8. Raju S Rajmani
  9. Arati Ramesh
  10. Valakunja Nagaraja
  11. Balasubramanian Gopal
  12. Ganesh Nagaraju
  13. Aswin Sai Narain Seshayee
  14. Amit Singh

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

The persistence of Mycobacterium tuberculosis ( Mtb ) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence. However, the mechanisms involved in NO sensing and reorganizing Mtb ’s physiology are not fully understood. Since NO damages Fe-S clusters of essential enzymes, the mechanism(s) involved in regulating iron-sulfur (Fe-S) cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR (Rv1460) senses NO via its 4Fe-4S cluster and promotes persistence of Mtb by mobilizing the Fe-S cluster biogenesis system; suf operon ( Rv1460-Rv1466 ). Analysis of anaerobically purified SufR by UV-visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe-4S cluster. Atmospheric O 2 and H 2 O 2 gradually degrade the 4Fe-4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe-4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA- sequencing of Mtb Δ sufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe-S clusters to exhaust respiratory and redox buffering capacity of Mtb Δ sufR . Lastly, Mtb Δ sufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe-S cluster metabolism and bioenergetics.

Highlights

  • Mycobacterium tuberculosis ( Mtb ) induces the expression of suf operon for Fe-S cluster biogenesis in response to nitric oxide (NO).

  • We found that a transcription factor SufR senses NO via its 4Fe-4S cluster and regulates the expression of the suf operon for Fe-S cluster biogenesis.

  • SufR-regulated Fe-S cluster biogenesis confers respiratory and redox features that promote recovery of Mtb from NO stress.

  • SufR activity is required to support the NO-dependent persistence of Mtb in macrophages and mice.

    • Article activity feed

    1. Version published to 10.1101/2020.08.10.245365v4 on bioRxiv
    2. eLife

      ###Reviewer #3:

      This study examines the role of iron-sulfur clusters in M. tuberculosis adaptation to nitric oxide (NO) and pathogenesis. The study uses transcriptomics to identify genes regulated by NO in vitro and then genetically and biochemically characterizes the role of SufR in responding to NO, modulating metabolic adaptations and promoting pathogenesis in macrophages and infected mice. The topic of this study is highly significant as it defines new mechanisms by which M. tuberculosis adapts to host NO. The manuscript includes numerous strengths including rigorous transcriptomic studies, well-defined physiological studies of wild type M. tuberculosis and thorough biochemical characterizations of SufR protein by spectrometry and DNA binding studies. However, the study suffers from a major experimental flaw that makes interpreting the conclusions from the genetic studies very difficult. The knockout of the sufR gene (which is a proposed repressor) also disrupts the NO inducibility of the downstream suf genes. Due to this polar effect, most of the experiments show partial or poor complementation. This complexity in the genetics raises questions about which aspects of the phenotype are directly controlled by SufR and which are controlled by the disregulated suf genes or possibly unlinked mutations. This major issue impacts a significant portion of the data and needs to be experimentally addressed to ensure that the specific function of SufR is defined by the studies. Overall, this is an ambitious, potentially exciting study, but suffers from a major flaw in the genetics that renders the major conclusions uncertain.

    3. eLife

      ###Reviewer #2:

      The manuscript by Anand et al. describes very interesting work into the characterisation of M. tuberculosis response to NO stress. The authors identify the SufR transcriptional repressor as a sensor of NO and further show that the 4Fe-4S cluster bound to the holo-protein plays a central role in this response. Interestingly, their results indicate that SufR regulates both the suf operon and the DosR regulon in response to NO. In addition, they identified a palindromic sequence upstream of the suf operon (and some nine other genes) that holo-SufR could bind to. These results collectively indicate that SufR integrates host response to Fe-S cluster homeostasis in Mtb, providing many important contributions to the field. There are, however, several concerns and areas that need improvement and better explanations.

      Major comments:

      1. The most puzzling finding in this manuscript is the inability of sufR-Comp to complement ΔsufR, with the sufR-Comp strains showing an intermediate phenotype (e.g. Figure 5, panels D and E). The authors mention that the partial complementation is likely due to the restored expression of other sufR-specific genes (like DosR regulon). Even more surprising is the result presented in Figure 5B, in which sufR-Comp shows much slower recovery than ΔsufR. In this case, the authors argue that the induction of the entire suf operon is necessary for the growth resumption. But this doesn't explain why the sufR-Comp shows a slower phenotype compared to ΔsufR. I believe that the authors should provide a more plausible explanation for these observations.

      2. Figure 3 shows that the suf operon is not induced upon NO treatment in ΔsufR and the authors stated that removing 345 bp of sufR for constructing ΔsufR might explain this observation. Whereas the primary and alternative TSS (and I'd assume the promoter region) remain intact in ΔsufR, the authors are urged to come up with a better explanation for this result.

      3. As part of their argument, the authors mentioned that Mtb prefers IscS for housekeeping functions and the Suf system for managing stress, and made comparisons with the well-studied Isc and Suf systems of E. coli. This is against the current knowledge in the literature, and contrary to E. coli, the Isc system in Mtb has reduced to only IscS and the Suf system acts as the major player in the assembly of Fe-S clusters (see point #4 below).

      4. I do realise that the authors have used Acn in their experiments to indicate the effects of NO treatment on Fe-S clusters. However, it is known that Acn of Mtb is a target for Mtb-IscS and therefore the results presented in Figure 4A doesn't necessarily mean that the observed phenotype is due to a direct consequence of defects in the suf system upon NO treatment. The paper by Rybniker et al. (reference #65 in the current manuscript) has shown, using Y2H, activity assays and pull-down experiments, that Acn could make direct interactions with IscS in Mtb. Consistent with this, sufR-Comp didn't reinstate Acn activity. Therefore I am doubtful whether Acn is the correct enzyme to use as an indicator to look into the function of suf operon, where its Fe-S formation depends on IscS.

      5. It is a common practice in the field that not only lung burden but also burden in at least one other organ are shown (usually spleen).

    4. eLife

      ###Reviewer #1:

      The manuscript of Amit Singh et al. describes a set of experiments that starts with looking at the transcriptomic response towards NO stress. A large number of genes show altered expression, including the Suf operon. They decide to study the Suf operon, whose encoded proteins are involved in [Fe-S] Cluster Assembly in more detail.

      Some of their findings include: that Mtb SufR is a major regulator of Fe-S cluster biogenesis in Mtb under NO stress, that SufR contains a redox-responsive 4Fe-4S cluster, that functions as a repressor and that a sufR mutant is slightly attenuated in mouse infection experiments. Although the results are convincing and important, my major problem is that in fact all of these findings have been described previously, mainly by M. Pandey (Scientific Reports 8:17359 - 2018) and D. Willemse (Plos One 0200145 - 2018). The current manuscript more specifically focuses on the role of NO in this process, but this is, in my opinion, a minor advance, as the effect of NO (and H2O2) was also reported previously.

    5. eLife

      ##Preprint Review

      This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 3 of the manuscript.

      ###Summary:

      Reviewers acknowledge that your submission reports some interesting results on the relationship between Fe-S and the response to NO in Mycobacterium tuberculosis. That said, several concerns were raised regarding genetic complementation and novelty.

    6. Version published to 10.1101/2020.08.10.245365v3 on bioRxiv
    7. Version published to 10.1101/2020.08.10.245365v1 on bioRxiv
    8. Version published to 10.1101/2020.08.10.245365v2 on bioRxiv