An essential periplasmic protein coordinates lipid trafficking and is required for asymmetric polar growth in mycobacteria

  1. Kuldeepkumar R Gupta
  2. Celena M Gwin
  3. Kathryn C Rahlwes
  4. Kyle J Biegas
  5. Chunyan Wang
  6. Jin Ho Park
  7. Jun Liu
  8. Benjamin M Swarts
  9. Yasu S Morita
  10. E Hesper Rego  Is a corresponding author
  1. Yale University, United States
  2. University of Massachusetts Amherst, United States
  3. Central Michigan University, United States

Abstract

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, grow by inserting new cell wall material at their poles. This process and that of division are asymmetric, producing a phenotypically heterogeneous population of cells that respond non-uniformly to stress (Aldridge et al., 2012; Rego et al., 2017; Richardson et al., 2016). Surprisingly, deletion of a single gene - lamA - leads to more symmetry, and to a population of cells that is more uniformly killed by antibiotics (Rego et al., 2017). How does LamA create asymmetry? Here, using a combination of quantitative time-lapse imaging, bacterial genetics, and lipid profiling, we find that LamA recruits essential proteins involved in cell wall synthesis to one side of the cell - the old pole. One of these proteins, MSMEG_0317, here renamed PgfA, was of unknown function. We show that PgfA is a periplasmic protein that interacts with MmpL3, an essential transporter that flips mycolic acids in the form of trehalose monomycolate (TMM), across the plasma membrane. PgfA interacts with a TMM analog suggesting a direct role in TMM transport. Yet our data point to a broader function as well, as cells with altered PgfA levels have differences in the abundance of other lipids and are differentially reliant on those lipids for survival. Overexpression of PgfA, but not MmpL3, restores growth at the old poles in cells missing lamA. Together, our results suggest that PgfA is a key determinant of polar growth and cell envelope composition in mycobacteria, and that the LamA-mediated recruitment of this protein to one side of the cell is a required step in the establishment of cellular asymmetry.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source Data files have been provided for blots, gels and TLC figures, and the Tn-seq data shown in Figure 4.

Article and author information

Author details

  1. Kuldeepkumar R Gupta

    Department of Microbial Pathogenesis, Yale University, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Celena M Gwin

    Department of Microbial Pathogenesis, Yale University, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Kathryn C Rahlwes

    Department of Microbiology, University of Massachusetts Amherst, Amherst, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Kyle J Biegas

    Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Chunyan Wang

    Department of Microbial Pathogenesis, Yale University, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Jin Ho Park

    Department of Microbial Pathogenesis, Yale University, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Jun Liu

    Department of Microbial Pathogenesis, Yale University, West Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3108-6735
  8. Benjamin M Swarts

    Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Yasu S Morita

    Department of Microbiology, University of Massachusetts Amherst, Amherst, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. E Hesper Rego

    Department of Microbial Pathogenesis, Yale University, New Haven, United States
    For correspondence
    hesper.rego@yale.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2973-8354

Funding

National Institute of Allergy and Infectious Diseases (R01AI148255)

  • E Hesper Rego

National Science Foundation (1654408)

  • Benjamin M Swarts

National Institute of Allergy and Infectious Diseases (R01AI087946)

  • Jun Liu

National Institute of General Medical Sciences (R01GM110243)

  • Jun Liu

Pew Charitable Trusts

  • E Hesper Rego

Searle Scholars Program

  • E Hesper Rego

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Christina L Stallings, Washington University School of Medicine, United States

Version history

  1. Preprint posted: May 9, 2019 (view preprint)
  2. Received: May 19, 2022
  3. Accepted: November 7, 2022
  4. Accepted Manuscript published: November 8, 2022 (version 1)
  5. Version of Record published: November 18, 2022 (version 2)

Copyright

© 2022, Gupta et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,141
    Page views
  • 339
    Downloads
  • 6
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Kuldeepkumar R Gupta
  2. Celena M Gwin
  3. Kathryn C Rahlwes
  4. Kyle J Biegas
  5. Chunyan Wang
  6. Jin Ho Park
  7. Jun Liu
  8. Benjamin M Swarts
  9. Yasu S Morita
  10. E Hesper Rego
(2022)
An essential periplasmic protein coordinates lipid trafficking and is required for asymmetric polar growth in mycobacteria
eLife 11:e80395.
https://doi.org/10.7554/eLife.80395

Share this article

https://doi.org/10.7554/eLife.80395

Further reading

    1. Microbiology and Infectious Disease
    Markéta Častorálová, Jakub Sýs ... Tomas Ruml
    Research Article Updated

    For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

    1. Microbiology and Infectious Disease
    2. Physics of Living Systems
    Fabien Duveau, Céline Cordier ... Pascal Hersen
    Research Article

    Natural environments of living organisms are often dynamic and multifactorial, with multiple parameters fluctuating over time. To better understand how cells respond to dynamically interacting factors, we quantified the effects of dual fluctuations of osmotic stress and glucose deprivation on yeast cells using microfluidics and time-lapse microscopy. Strikingly, we observed that cell proliferation, survival, and signaling depend on the phasing of the two periodic stresses. Cells divided faster, survived longer, and showed decreased transcriptional response when fluctuations of hyperosmotic stress and glucose deprivation occurred in phase than when the two stresses occurred alternatively. Therefore, glucose availability regulates yeast responses to dynamic osmotic stress, showcasing the key role of metabolic fluctuations in cellular responses to dynamic stress. We also found that mutants with impaired osmotic stress response were better adapted to alternating stresses than wild-type cells, showing that genetic mechanisms of adaptation to a persistent stress factor can be detrimental under dynamically interacting conditions.