Leveraging a synthetic biology approach to enhance BCG-mediated expansion of Vγ9Vδ2 T cells

Abstract

There is an urgent need to develop a more efficacious anti-tuberculosis vaccine as the current live-attenuated vaccine strain BCG fails to prevent pulmonary infection in adults. In this study, we leverage a synthetic biology approach to engineer BCG to produce more (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate of bacterial—but not host—isoprenoid biosynthesis via the methylerythritol phosphate (MEP) pathway. HMBPP strongly activates and expands Vγ9Vδ2 T cells, which are unique to higher-order primates and protect against Mycobacterium tuberculosis infection. 

Introduction

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease, has been persistent throughout the course of human history. Most notably in the late 1800s to early 1900s, Mtb infected nearly the whole population of Europe and resulted in ~25% mortalityDespite the discovery of the bacterium over 142 years ago, Mtb remains a global health threat, killing approximately six thousand individuals per day and latently infecting an estimated quarter of the world’s population

Materials and methods

Bacterial strains and culture

Mycobacterium tuberculosis variant bovis BCG Pasteur (ATCC 35734) was routinely grown in Middlebrook 7H9 liquid medium or 7H10 agar (Difco) supplemented with 10% OADC (oleic acid-albumin-dextrose-catalase) and 0.05% Tween80. E. coli strains DH5α or NEB® 10-beta were grown in LB broth or agar and used for propagation and cloning of plasmids. When required, the following antibiotics were used

Molecular cloning

Primers, oligos, and plasmids used in this study are listed in S1 Table. Standard electroporation protocols were used for the transformation of plasmids into E. coli and mycobacteria. 50uL of electrocompetent DH5α E. coli was combined with 5ul of ligation product or plasmid DNA and incubated on ice for 30 min, then heat shocked at 42°C for 30 sec, chilled on ice for 5 min, and recovered at 37°C in Luria broth for one hour. 

CRISPRi

Gene silencing and gene deletion were performed using the site-specific transcriptional repression system CRISPRi as previously described. Briefly, guide oligos were annealed and ligated into the integrative, dCas9-containing pLJR965 vector and the subsequent plasmids were transformed into BCG

Results

The MEP pathway is essential in BCG

Most mycobacteria solely utilize the MEP pathway for isoprenoid biosynthesis with the exception of a M. marinum-derived Further, multiple transposon mutagenesis and gene deletion experiments have identified all MEP genes as essential in Mtb , and because BCG and Mtb are closely related, we predicted that the MEP pathway is also essential in BCG
Synteny analysis of MEP genes across mycobacterial genomes Given the evidence supporting a protective role for Vγ9Vδ2 T cells in Mtb infection, we hypothesized that enhanced Vγ9Vδ2 activation might also enhance protection. To engineer a strain of BCG that produced elevated levels of HMBPP, we initially set out to combine all MEP genes leading to HMBPP synthesis into a single locus to maximize expression

Discussion

HMBPP is an intermediate of isoprenoid metabolism via the MEP pathway and potently activates and expands host Vγ9Vδ2 T cells. We found that this metabolic pathway is essential in BCG, as has been reported in Mtb . Because Vγ9Vδ2 T cells are protective in the context of Mtb infection, we sought to generate a rBCG vaccine strain that induced a stronger Vγ9Vδ2 response by synthesizing a bolus of HMBPP. Synteny analyses across mycobacterial genomes revealed certain pair biases of MEP genes, which we used to assemble a synthetic MEP locus as well as a single gene overexpression construct 

Citation: Qabar CM, Roberts AW, Waldburger LM, Baidoo EEK, Turumtay EA, Keasling JD, et al. (2026) Leveraging a synthetic biology approach to enhance BCG-mediated expansion of Vγ9Vδ2 T cells. PLoS One 21(4): e0343925. https://doi.org/10.1371/journal.pone.0343925

Editor: Atul Vashist, Bennett University, INDIA

Received: May 9, 2025; Accepted: February 12, 2026; Published: April 6, 2026
Copyright: © 2026 Qabar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting information files.

Funding: This work was supported by the National Institute of General Medical Sciences (T32GM132022 to CMQ), the National Institute of Allergy and Infectious Diseases (U19AI162583 and U19AI135990 JSC), (1P01 AI063302 and 1R01AI027655 to DAP), as well as the National Science Foundation Graduate Research Fellowship (DGE-1752814 to LMW) and the Henry Wheeler Center for Emerging and Neglected Diseases Irving H. Wiesenfeld Fellowship (CNCND-32305-47950 to CMQ). This material was based upon work supported by the Joint BioEnergy Institute, U.S. Department of Energy, Office of Science, Biological and Environmental Research Program with Lawrence Berkeley National Laboratory (DE-AC02-05CH11231 to JDK). Funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: J.D.K. has financial interests in Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, BioMia, Cyklos Materials, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms. D.A.P. has a financial interest in Laguna Biotherapeutics. The other authors declare no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.