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

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 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.

Vaccines and Dementia: Part II. Efficacy of BCG and Other Vaccines Against Dementia

“…Looking to the future, could we engineer a 'super-BCG' that is even more effective? BCG could provide a framework for even more potent engineered immunostimulants [85][86][87][88]. An obvious place to start would be to study the protective properties of different BCG strains that have arisen over the past century (Fig.…”