Macrophage-induced drug tolerant persisters in tuberculosis

More than 1.8 million people die worldwide each year from Tuberculosis (TB), making it the biggest cause of mortality due to a single bacterial infection. This equates to the passengers of more than 10 full Boeing 747's succumbing to the disease every single day. This is despite the availability of antibiotics against the causative agent of the disease Mycobacterium tuberculosis (Mtb). Antibiotic treatment for any disease is a lengthy process; however, in TB, eradication of the infecting bacilli takes 6-9 months and requires at least 3 highly toxic drugs. This is primarily due to the presence of a small group or sub-population of bacteria that can resist and survive antibiotic treatment. Termed drug-tolerant persisters, these bacilli were first discovered in 1942 and yet we are still a long way from understanding these elusive bacteria. One of the reasons for this is that they are present at such low numbers (approximately 1 persistent bacilli in 10, 000) that they are difficult to study.
Preliminary results from our laboratory have shown that interacting with the very cell that we, the host, deploy to kill invading pathogens can induce much higher numbers of these persister bacilli. Upon infection with Mtb or many other invading pathogens, our immune system deploys white blood cells to kill the invader, one of which is called the macrophage. From our results it appears that Mtb uses our own macrophages as a Trojan Horse and when inside this host cell changes into a virtually untreatable drug-tolerant persister cell. We have called these macrophage-induced persisters or MIPs. The trigger(s) for the formation of drug-tolerant persisters is currently unknown but could represent an ideal target for future drug development
Our aims
1) To identify the bacterial trigger genes required for the formation of MIPs. Discovering the bacterial genes vital to the formation of drug-tolerant persisters would add to our understanding of how Mtb is able to evade killing and could represent future drug targets.

2) To determine the macrophage conditions responsible for MIP formation. Persister cells are notoriously difficult to treat due to their rarity in a population. By identifying what factors or conditions the bacterium is sensing to induce the formation of MIPs, we will be able to generate enough persisters to enable further study. We also intend to develop a novel drug testing platform so we can identify drugs capable of killing these MIPs, i.e. persisters formed whilst within our own bodies.

Potential applications and benefits
Targeting drug-tolerant persisters is key to combatting TB. Understanding the nature of this sub-population of bacteria will enable us to develop new treatment strategies that either attack the bacteria themselves or push the host immune response in the right direction. This could improve treatment tolerance among patients, and improve treatment success rates. The current threat of drug resistance combined with the HIV epidemic makes the development of new TB treatment strategies of the upmost importance.

Aim 1. To identify the Mtb trigger gene(s) required for MIP formation we will use the next generation deep-sequencing based method transposon sequencing (Tnseq). Stage 1) Our library (10e10 with ~10e5 mutants) will be inoculated into either A) cell culture media without macrophages, B) human monocyte-derived macrophage (MDMs) or C) IFN-gamma activated MDMs. After 2 hours the intracellular bacilli will be isolated by differential lysis. Stage 2) Anti-mycobacterial drug will be administered (rifampicin 60ug/ml) for each treatment followed by CFU plating and library recovery (we estimate a recovery of 10e 7bacilli, sufficient to maintain reasonable library coverage). Libraries recovered immediately prior to (T0) and 48h after the drug treatment will be compared for all three treatments to identify trigger genes for conditions A-C separately. The genes identified will then be compared across treatments to identify genes specific to each condition. Mutant abundance will be determined by sequencing transposon junctions using Illumina Hiseq sequencing, followed by bioinformatic and statistical analysis carried out by the Faculty's designated bioinformatics officer. Up to 10 candidate genes will be chosen, and gene knockouts and gene overexpressing strains generated using vectors and the defined mutant library from our collaborator Professor Jacobs Jnr's laboratory, and their antibiotic tolerances characterised. Reporter gene fusions will also be created and observed in single cell studies in our microfluidics system imaged using confocal microscopy. Aim 2: To determine the specific macrophage environment that induces MIPs, we will expose Mtb cultures to stress conditions associated with the macrophage environment, alone or in synergy, prior to an antibiotic kill to determine the level of persisters. A drug testing scaffold will be designed following identification of the host trigger in a 384-well format with controls including a positive anti-mycobacterial drug.

Impact summary

The discoveries of this exciting and important research project will have a number of clear and demonstrable impacts on wide ranging beneficiaries, in both the short, medium term and longer term. The impact will be regularly monitored and assessed by the applicant and co-applicant.


1. Patients who develop active TB would stand to benefit enormously with the advent of a new tuberculosis drug with reduced treatment time and enhanced efficacy. Improved treatment regimes would effect the morbidity and mortality of patients with active tuberculosis and would also decrease disease transmission to the population in general. This would dramatically the quality of life and health of patients with the disease.

2. The UK and world-wide population in general, one third of which is estimated to be latently infected with the tubercle bacillus Mycobacterium tuberculosis. The availability of improved drugs with which to combat this global health threat would benefit the global population. There would be less disease and less dissemination of disease, which can be linked to a huge benefit in the terms of livelihood and economic benefit and more importantly to survival itself.

3. The healthcare system in the UK and world-wide who currently administer and monitor active TB patients over 6-9 months of treatment. This will include the clinicians involved in the healthcare system. Reducing treatment time and improving efficacy would dramatically benefit the healthcare system both here and abroad, thus increasing its effectiveness. We also aim to interact with clinicians including the many collaborators and co-authors of clinical study-related papers of the applicant at St Marys NHS Trust and the scientific community at the acid-fast club biannual meetings attended by many clinicians. We hope this will help to maximise the impact of our research in clinical practise


4. Pharmaceutical companies, such as GSK, involved in anti-tuberculosis drug development and the screening of potential drugs to combat mycobacterial persistence. There is huge world-wide demand for anti-tuberculous drugs with recent market estimates reaching around 650 million USD annually. Novel drugs which target drug-tolerant persistent bacteria could completely change the entire treatment strategy or be utilised as an adjunct to current chemotherapy. The University has a Technology Transfer Office (in Research and Enterprise Services), which has been very successful in discovery commercialisation.

5. The press and media. The outcomes of this research will be of interest to the media and public as in the longer term this research could lead to a novel approach to treat tuberculosis. The University has an active media centre (http://www.surrey.ac.uk/mediacentre/press/index.htm), which will be utilized to promote scientific breakthroughs associated with this proposal. In addition, drug-tolerant persisters are a signature of most pathogens and as such these findings may be applied to many more diseases, thus further increasing the impact of this work.

6. The public. The applicant is a participating STEM ambassador and spends much of her time with budding biologists, trying to capture their imagination and inspire and the University of Surrey has a Widening Participation Policy, with outreach programmes in place for local schools and colleges

7. Students at the undergraduate and postgraduate level taught at the University and by the applicants. We will impart the concepts and understanding of persistent bacilli this proposal will enable us to achieve to our students here at the University both at the undergraduate and postgraduate level. This will be achieved by both teaching and research projects.