Abstract
Tuberculosis (TB) continues to pose a significant public health challenge as one of the leading
causes of infectious diseases from a single infectious pathogen, Mycobacterium tuberculosis
(Mtb). The persistence of an drug-tolerant subpopulation, known as drug persisters, within the
genetically identical bulk population is a crucial factor contributing to the prolonged survival of
Mtb during drug treatment, ultimately impeding the eradication of TB in clinical settings. Drug
combinations remain an important and effective strategy for TB treatment. Investigation on
synergistic combinations targeting both the bulk population and persisters will be beneficial for
TB treatment.
This thesis focused on investigating the molecular mechanisms of drug persistence genes via
transposon mutagenesis and of synergistic drug combinations using RNA sequencing.
Considering the requirement of high-level biosafety labs, posing limitations on TB drug
research, this thesis often used an auxotroph strain, Mtb H37Rv mc2
6260 (CL2 Mtb), as the research model. This thesis describes the phenotypic examination using checkerboard
minimum inhibitory concentration and long-term time-killing experiments to identify Mtb
persistence mutants and synergistic combinations targeting both the bulk population and
persisters. Persistence formation was corelated with drugs’ mechanism of actions, as
persistence-relevant genes were specific to rifampicin (RIF) and streptomycin (STM). The
thesis highlighted the involvement of cell membrane biosynthesis, mycolic acid assembly and
lipid degradation in RIF persistence formation, and toxin-antitoxin systems in STM persistence
formation. Especially, prpD involved in the methyl citrate cycle and fadE5 for lipid degradation
were proven to reduce and increase persisters of RIF and could be potential drug targets.
The identification of drug-specific persistence mechanisms for RIF and STM paved the way
for exploring bactericidal effects on both the bulk population and persisters in drug
combinations targeting at diverse cellular pathways, including isoniazid (INH), RIF,
clarithromycin (CLAR) and bedaquiline (BDQ). This study identified the synergistic
combinations of INH with CLAR and combination of RIF and BDQ. Our results revealed that
the synergy between RIF and BDQ are associated with the overload of toxic compounds,
especially copper and methyl citrate intermediate. INH and CLAR induce cell membrane
disruption, oxidative phosphorylation interference, and substrate transport alterations, all
contributing to synergistic bactericidal effects on persisters.
In the effort of exploring novel synergistic combinations, two peptides, LYN1D and LYN3D,
exhibited anti-mycobacterial properties and selective synergy with BDQ. Notably, the addition
of D-isomers to peptides significantly boosted the antibacterial activity. Transcriptome data
suggested that LYN1D/3D and BDQ targeted multiple cellular components, disrupting cell
membranes and ribosomes. Moreover, a synergistic response between BDQ and LYN1D/3D
on oxidative phosphorylation was identified, resulting in intracellular ATP reduction and lethal
bactericidal effects.
These findings shed light on the complex molecular mechanisms underlying drug persistence
and the potential of synergistic combinations to disrupt Mtb survival strategies and enhance
TB treatment outcomes.