A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice
- University of the Witwatersrand, South Africa
- Johns Hopkins University, United States
- 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.
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Author details
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.
Ethics
Animal experimentation: All animal experiments were approved by the Johns Hopkins University Animal Care and Use Committee (Protocol number: MO20M20).
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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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
