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 sidechains. 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 sidechains. We demonstrate that depletion of these
enzymes results in reduced growth, cell wall defects, increased susceptibility to
antibiotics and altered spatial localization of new
peptidoglycan. In cell culture experiments, training of monocytes 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, resulting in unmasking of the
D-glutamate diaminopimelate (
iE-DAP) NOD-1
ligand, yields superior prevention of TB disease compared to the standard
BCG vaccine. This work demonstrates 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.