Evolutionarily divergent Mycobacterium tuberculosis CTP synthase filaments are under selective pressure

  1. Eric M. Lynch
  2. Yao Lu
  3. Jin Ho Park
  4. Lin Shao
  5. Justin Kollman
  6. E. Hesper Rego

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Abstract

The final and rate-limiting enzyme in pyrimidine biosynthesis, CTP synthase (CTPS), is essential for the viability of Mycobacterium tuberculosis and other mycobacteria. Its product, CTP, is critical for RNA, DNA, lipid and cell wall synthesis, and is involved in chromosome segregation. In various organisms across the tree of life, CTPS assembles into higher-order filaments, leading us to hypothesize that M. tuberculosis CTPS (mtCTPS) also forms higher-order structures. Here, we show that mtCTPS does assemble into filaments but with an unusual architecture not seen in other organisms. Through a combination of structural, biochemical, and cellular techniques, we show that polymerization stabilizes the active conformation of the enzyme and resists product inhibition, potentially allowing for the highly localized production of CTP within the cell. Indeed, CTPS filaments localize near the CTP-dependent complex needed for chromosome segregation, and cells expressing mutant enzymes unable to polymerize are altered in their ability to robustly form this complex. Intriguingly, mutants that alter filament formation are under positive selection in clinical isolates of M. tuberculosis , pointing to a critical role needed to withstand pressures imposed by the host and/or antibiotics. Taken together, our data reveal an unexpected mechanism for the spatially organized production of a critical nucleotide in M. tuberculosis , which may represent a vulnerability of the pathogen that can be exploited with chemotherapy.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/13730150.

    Cytidine triphosphate (CTP) is an essential molecule for all life. The pyrimidine biosynthesis enzyme CTP synthase (CTPS) is known to form filamentous assemblies in numerous diverse lifeforms. This manuscript from Lynch et al reveals new details regarding CTPS in the mycobacteria M. tuberculosis and M. smegmatis. The work identifies a novel filament structure undertaken by CTPS in these species and begins to investigate the functional importance of this structure. 

    The data put forward by the authors presents an exciting new view of this complex system. We have detailed some specific comments below which we feel may improve the strength of this manuscript in its next iteration: 

    Major issues

    • Figures 2f, 7e, and S6d all contain structural data from non-mycobacterial species. However, we were unable to find information in the manuscript as to the origin of this structural data. Were the E. coli, yeast, and human CTPS filament structures generated by the authors for this paper, or are these structures previously published? If the structures were generated for this manuscript, it would be useful to see supplemental figures on the data processing, as was included for the M. tuberculosis CTPS filaments. If the structures are originally from other publications, then it is important that these be clearly cited in the manuscript, and PDB codes included in the figures.

    • The manuscript specifically focuses on M. tuberculosis in the title and at certain points in the Introduction and Discussion. However, since a substantial portion of the work was performed in M. smegmatis using their native CTPS, it may make sense to discuss the findings more broadly in the context of the Mycobacterium genus. Indeed, the authors have already performed the analysis to show that these unique CTPS filaments are likely formed across many (if not all) mycobacterial species (Fig. S3). 

    Minor issues

    • The title of this manuscript mentions selective pressure. Given that the manuscript primarily focuses on CTPS filament structure in mycobacteria, however, it may be clearer to future readers to have the title specifically mention structure instead of selective pressure. 

    • Normal growth of the engineered CTPS-eGFP M. smegmatis strain (Fig. S1) is displayed as an indicator of normal CTPS expression and activity. In the Methods section, subsection Plasmid construction and strain generation, however, it is also mentioned that "Validation of [the] strains was performed by HygR screening and by sequencing." We believe it would be beneficial to the manuscript if these screening and sequencing results were summarized in an additional supplementary figure.

    • Where were the wild type M. smegmatis and E. coli DH5a strains acquired from? It would be helpful to have this included in the Methods section.

    Competing interests

    The author declares that they have no competing interests.

  2. Version published to 10.1101/2024.07.25.605180v1 on bioRxiv