Mycobacterial Epigenetics: Investigating the impact of DNA methylation on antibiotic tolerance and persistence

PROJECT SUMMARY Sterilization of tuberculosis (TB) requires lengthy chemotherapy treatment to eradicate persister Mycobacterium tuberculosis (Mtb) sub-populations. Persister Mtb are unaffected by anti-TB treatment due to their viable but non- replicating (VBNR) or slowly replicating state in the absence of heritable genetic changes. Persistence is a

reversible phenotype which may be regulated by epigenetic mechanisms. DNA methylation contributes to the formation of persister bacteria in other bacterial species, but the role for DNA methylation remains unknown in Mtb persister formation. The objective of this K43 award is to establish the role of DNA methylation in the Mtb

stress response and in the formation of Mtb heterogeneity, which includes the persister sub-population. The primary mentorship of Drs. Sampson (LMIC primary mentor) and Aldrige (USA primary mentor), both experienced in mycobacteriology, will provide critical career advancement training. Drs. Sampson and Aldridge

have both contributed to advancing our understanding of Mtb population heterogeneity by developing a replication reporter plasmid for the identification of Mtb persisters and a microfluidic imaging platform to investigate cell-to-cell heterogeneity, respectively. This project will build on these advances to address two

specific aims: (1) demonstrate a role for DNA methylation in the Mtb stress response and phenotypic drug resistance, and (2) demonstrate a role for DNA methylation in Mtb persistence and antibiotic tolerance. This study will for the first time show that DNA methylation results in changes on a protein level. We will show that

DNA methylation is used by Mtb to respond to environmental stress and promote the formation of a persister sub-population. Phenotypic drug susceptibility testing and killing assays will assess the impact of DNA methylation on the ability of Mtb to tolerate antibiotic exposure. Population and single-cell imaging analysis will

assess the impact of DNA methylation on Mtb population heterogeneity, which has previously been shown to impact treatment efficacy. At the end of this K43-funded mentored study, the applicant will establish herself as an independently funded TB research scientist and group leader at Stellenbosch University. Interdisciplinary collaborations established during

this award will lay the foundation for future investigations into the prevention of Mtb persisters for shorter TB treatment, the identification of biomarkers for diagnosis of persister infections, and prevention of TB recurrence.