Macrophage-induced drug tolerant persisters in tuberculosis

Lead Research Organisation: University of Surrey
Department Name: Microbial & Cellular Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description Biocoatings; painting bacteria on surfaces for sustainable processes
Amount £229,774 (GBP)
Funding ID RPG-2018-393 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2019 
End 10/2022
 
Description CHAIR grant on (myco)bacterial biofilms
Amount £10,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 01/2017 
End 07/2017
 
Description Circulating strains of mycobacteria in Ghana
Amount £8,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 01/2019
 
Description Commonwealth Science Follow on Travel Grant
Amount £6,000 (GBP)
Funding ID CSC\R1\170042 
Organisation Royal Society of Medicine 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 04/2019
 
Description Profiling the mycobacterial biofilm
Amount £88,148 (GBP)
Funding ID 6446632 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 09/2022
 
Description Systematic analysis of persistence mechanisms by high-throughput bar-seq and single cell analyses
Amount £180,000 (GBP)
Funding ID MR/T028998/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2020 
End 12/2023
 
Description Travel grant to Ghana
Amount £1,000 (GBP)
Organisation University of Surrey 
Sector Academic/University
Country United Kingdom
Start 07/2018 
End 07/2019
 
Description Understanding the activity and role of DarTG, a toxin:antitoxin system responsible for a novel DNA modification
Amount £354,684 (GBP)
Funding ID BB/R006393/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2021
 
Description University of Surrey ADI travel grant
Amount £1,000 (GBP)
Organisation University of Surrey 
Sector Academic/University
Country United Kingdom
Start 01/2018 
End 07/2018
 
Description iCASE PhD studentship on mycobacterial imaging with National Physics laboratory
Amount £113,200 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 10/2022
 
Title Fluorescent mycobacterial strains for live imaging 
Description Fluorescent bacterial strains of Mycobacterium smegmatis and Mycobacterium tuberculosis (including mutant strains) have been constructed for use in our live imaging experiments. 
Type Of Material Biological samples 
Year Produced 2017 
Provided To Others? Yes  
Impact These strains have been made vailable to other researchers in the field. 
 
Title Novel anti-tuberculosis drug screen (Macrophage-induced persisters) 
Description We have generated a novel drug screening platform to screen TB drugs active against drug tolerant bacteria. Drug resistant Mycobacterium tuberculosis (Mtb) are an obvious threat to the control and eradication of TB, but even genetically susceptible strains can show phenotypic resistance due to the survival of drug-tolerant persister cells. We have shown that these persister cells increase upon exposure to human immune cells. Novel compounds are needed to target this highly clinically relevant sub-population and we have developed a colourmetric persister assay which has already resulted in the identification of several potential drugs. Targeting drug tolerance in this manner would lead to reducing treatment time and increased treatment success. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Our macrophage-induced persister or (MIP)-based drug screen has been used successfully to screen several compounds from collaborators for anti-mycobacterial activity. Utilisation of a drug screen with dependence upon a phenotype induced in human cells may reduce the need for animal use. 
 
Description Collaboration with Albert Einstein College of Medicine, New York 
Organisation Albert Einstein College of Medicine
Country United States 
Sector Academic/University 
PI Contribution Intellectual input.
Collaborator Contribution Access to set of Mycobacterium tuberculosis mutants which we are currently working with and intellectual input.
Impact Results awaited. Invited speaker from Albert Einstein College of Medicine to give very well attended seminar here at the University of Surrey (2018).
Start Year 2016
 
Description Collaboration with KNUST-KUMASI, Ghana 
Organisation Kwame Nkrumah University of Science and Technology (KNUST)
Country Ghana 
Sector Academic/University 
PI Contribution This collaboration will include access to equipment and facilities as required and will include intellectual input and training.
Collaborator Contribution KNUST will provide training, intellectual input, access to data, equipment and facilities. This is a collaborative, 2-way approach with a great team.
Impact The output from this early collaboration are our travel grants which will help us to generate further outputs and continue with this multi-disciplinary project.
Start Year 2018
 
Description Collaboration with National Physics Laboratory 
Organisation National Physical Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution Current iCASE studentship looking at mycobacteria at the single cell level
Collaborator Contribution This is a joint studentship where the student works 50% at NPL and has access to there cutting edge facilities to study mycobacteria and AMR.
Impact Invited oral presentation at the Microbiology annual conference in 2019 for the talented student on this project.
Start Year 2019
 
Description Conference presentation at Keystone TB conference 2018 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Oral presentation at conference: Investigating drug-tolerance of Mycobacterium tuberculosis by Tn-seq

Hernandez J., Williams K., Wu H., Mendum T., Hingley -Wilson S. and McFadden J.

Keystone Symposia on Tuberculosis: Translating Scientific Findings for Clinical and Public Health Impact. April 15-20, 2018, at the Fairmont Chateau Whistler, Whistler, British Columbia, Canada.
Year(s) Of Engagement Activity 2018
 
Description Invited presentation at the University of Exeter 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Seminar for scientists and postgraduate and undergraduate students at the University of Exeter on AMR.
Year(s) Of Engagement Activity 2020
 
Description Nursery friendly bacteria event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Event at local nursery for Science week on friendly bacteria, featuring pin the pili on the bacteria, make your own bacterial friend and paint and string together the bacteria. The children and staff asked lots of questions and had lots of fun!
Year(s) Of Engagement Activity 2018
 
Description Pre-school visit. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Pre-school demonstration of friendly bacteria and of anti-microbial resistance with supersized (fake) bacteria. This visit sparked lots of interest from the children and lots of questions from the staff. This will now be a yearly visit.
Year(s) Of Engagement Activity 2018
 
Description Royal Society Exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Our PhD student Winnifred Akwani who works on AMR in mycobacterial biofilms was part of an interactive stall on biofilms in the Royal Society Exhibition week in London. This generated a lot of interest on biofilms and AMR from the public who attended the event.
Year(s) Of Engagement Activity 2019
 
Description Science museum AMR event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This was an AMR themed evening event at the Superbugs exhibit at the Science museum, London. Our group were part of the UKRI group with a stall on AMR in Tuberculosis (TB) and other bacteria. Our stall which featured AMR spin the wheel questions, guess the amount of antibiotics a drug-resistant TB patient and one of our single cell antibiotic resistance videos generated a wealth of questions and discussion.
Year(s) Of Engagement Activity 2018
 
Description Surrey Healthy Ageing Research Partnership workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The Surrey Healthy Ageing Research Partnership was set up in 2017 and we presented and spoke whilst there to many members of the general public about Tuberculosis and anti-microbial resistance. This generated questions, discussion afterwards and volunteers for blood donation for our research.
Year(s) Of Engagement Activity 2017
 
Description UKRI conference AMR stand 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact AMR stand at UKRI annual conference, our AMR spin the wheel and guess the amount of antibiotics and AMR bacteria videos generated a lot of interest and questions.
Year(s) Of Engagement Activity 2018
 
Description University of Surrey open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact University of Surrey open day speaking to prospective students , parents and general public about Microbiology with demonstrations.
Year(s) Of Engagement Activity 2017