A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice

  1. Moagi Tube Shaku
  2. Peter K Um
  3. Karl L Ocius
  4. Alexis J Apostolos
  5. Marcos M Pires
  6. William R Bishai
  7. Bavesh D Kana  Is a corresponding author
  1. University of the Witwatersrand, South Africa
  2. Johns Hopkins University, United States
  3. University of Virginia, United States

Abstract

Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan side-chains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan side-chains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics, altered spatial localization of new peptidoglycan and increased NOD-1 expression in macrophages. In cell culture experiments, training of a human monocyte cell line with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, which is expected to unmask the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. In vitro and in vivo experiments in this study demonstrate the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1, 2, 3, and 4. Source data files are also provided for the supplementary information.

Article and author information

Author details

  1. Moagi Tube Shaku

    DST/NRF Centre of Excellence for Biomedical TB Research, University of the Witwatersrand, Johannesburg, South Africa
    Competing interests
    No competing interests declared.
  2. Peter K Um

    Department of Medicine, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8215-9493
  3. Karl L Ocius

    Department of Chemistry, University of Virginia, Charlottesville, United States
    Competing interests
    No competing interests declared.
  4. Alexis J Apostolos

    Department of Chemistry, University of Virginia, Charlottesville, United States
    Competing interests
    No competing interests declared.
  5. Marcos M Pires

    Department of Chemistry, University of Virginia, Charlottesville, United States
    Competing interests
    No competing interests declared.
  6. William R Bishai

    Department of Medicine, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8734-4118
  7. Bavesh D Kana

    DST/NRF Centre of Excellence for Biomedical TB Research, University of the Witwatersrand, Johannesburg, South Africa
    For correspondence
    bavesh.kana@nhls.ac.za
    Competing interests
    Bavesh D Kana, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9713-3480

Funding

Howard Hughes Medical Institute (HHMI000)

  • Bavesh D Kana

South African Medical Research Council

  • Bavesh D Kana

South African Medical Research Council

  • Moagi Tube Shaku

National Research Foundation

  • Bavesh D Kana

National institutes of health (NIH AI 155346)

  • William R Bishai

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

Reviewing Editor

  1. Petra Anne Levin, Washington University in St. Louis, United States

Ethics

Animal experimentation: All animal experiments were approved by the Johns Hopkins University Animal Care and Use Committee (Protocol number: MO20M20).

Version history

  1. Received: May 8, 2023
  2. Accepted: April 17, 2024
  3. Accepted Manuscript published: April 19, 2024 (version 1)

Copyright

© 2024, Shaku 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.

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  1. Moagi Tube Shaku
  2. Peter K Um
  3. Karl L Ocius
  4. Alexis J Apostolos
  5. Marcos M Pires
  6. William R Bishai
  7. Bavesh D Kana
(2024)
A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice
eLife 13:e89157.
https://doi.org/10.7554/eLife.89157

Share this article

https://doi.org/10.7554/eLife.89157

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