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

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.