Development of an in vitro model for NonReplicating Persistent Mycobacterium tuberculosis, and an investigation of the novel phenotype

  • Savannah Gibson

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Tuberculosis is the primary cause of global death by a single infectious agent. This widespread
infection is assisted by the existence of a large reservoir of disease in the form of Latent
Tuberculosis which is thought to infect over ¼ of the world’s population. Latent Tuberculosis
is caused when an existing Mycobacterium tuberculosis infection is encapsulated within a
granuloma and the bacilli adapt their physiology to cope with this hostile environment known
as the Non-Replicating Persistent (NRP) state. The background of Tuberculosis, Latent
Tuberculosis and scientific modelling of these states is discussed in Chapter 1. The first results
chapter of this thesis, Chapter 2, sets out a novel multi-stress in vitro model of Latent
Tuberculosis to create a more physiologically relevant M. tuberculosis NRP phenotype.
Chapter 3 uses the in vitro model proposed in Chapter 2, along with a control in vitro model to
conduct widespread antimicrobial testing against a variety of antimicrobials; from frontline,
second line and antibiotics in clinical trials to novel compound libraries. This uncovered an
exceptionally antibiotic tolerant phenotype, not previously seen in less physiologically relevant
models of Latent Tuberculosis. Chapter 4 is an investigation into the mechanism of action of
the sole antibiotic hit from Chapter 3, Dapsone. An assessment of the previously proposed
drug target, FolP1, revealed an alternative target, the formally defined “inactive ortholog”,
FolP2. Through a range of biochemical techniques, FolP2 activity in the cholesterol catabolism
pathway was established, revealing a new, previously unreported and druggable aspect of
latent M. tuberculosis physiology. Chapter 5 utilises the in vitro model proposed in Chapter 2
to establish the presence of the NRP state in the opportunistic pathogen Mycobacterium
abscessus. It was identified that M. abscessus shared the same drug-indifferent phenotype that has been observed for M. tuberculosis, proposing a further hypothesis for the poor
treatment outcomes of this infection. Overall, this thesis has provided a physiologically relevant
model of a global infection that revealed a highly drug resistant phenotype with the subsequent
discovery of an established antimicrobial agent that could be repurposed for latent tuberculosis
chemotherapy. This novel contribution to the knowledge will invariably catalyse future drug
discovery into this neglected area of tuberculosis research and has the potential to change the
way we treat latent M. tuberculosis infection.
Date of Award2021
Original languageEnglish
SupervisorJonathan A. G. Cox (Supervisor) & Dan Rathbone (Supervisor)

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