Mycobacterial determinants of survival and fitness within the bovine host

Lead Research Organisation: Royal Veterinary College
Department Name: Pathology and Pathogen Biology

Abstract

Bovine tuberculosis (BTB) is a persistent problem in certain areas of the UK. The current control strategy is to test herds for the presence of BTB at timely intervals and slaughter test-positive animals. However, the observation that BTB is spreading in both geographical area and prevalence suggests that the existing control strategy is not working effectively. The cost of the control programme is £100 million annually, therefore not only is the policy ineffective, it is also a significant economic burden. In addition, BTB can cause tuberculosis in humans, and while this is currently rare, the escalating prevalence of BTB has the potential to develop into a zoonotic (the passage of disease from animals to humans) risk.

BTB is caused by a bacterium called Mycobacterium bovis. This is very closely related to Mycobacterium tuberculosis, a bacterium that commonly causes tuberculosis. Tuberculosis in cattle and humans show great similarities and, like the human disease, vaccination is an alternative strategy to control BTB. The vaccine against human tuberculosis is a strain of Mycobacterium bovis called Mycobacterium bovis BCG, however this provides limited protection against the disease in both cattle and humans.

Mycobacterium bovis is transmitted by aerosol, once within the animal it enters into cells called macrophages. These cells, normally dedicated to the killing and removal of bacterial pathogens, are unable to kill the bacteria. Instead, Mycobacterium bovis adapts and survives within the lungs in structures called granulomas. However, we do not know much about the genes that allow the bacteria to survive and cause disease in the host.

We will knock-out the function of every single gene in the genome of Mycobacterium bovis (approximately 4000) and determine those genes that are required for survival in bovine macrophages and in the whole cow. We will achieve this by using a technique called Transposon Directed Insertion Seqeuncing (TraDIS). This technique has been applied successfully to the study of other bacterial pathogens of medical and veterinary importance, but this will be the first time it has been used to study the genetic determinants of disease in Mycobacterium bovis. We will make libraries of mutants where the function of every gene in the genome has been knocked-out. We will then put the libraries into "screens". Mutants that are recovered after being through a "screen" are compared to the original pre-screened mutant pool by DNA sequencing. Mutants that are unable to survive the screen are identified and as a result we will identify all the genes necessary for survival in the host. Those mutants that are unable to survive in the host represent potential vaccine candidates.

We will be able to assess the function of essential genes using a combination of computational analysis, literature searching and comparisons to screens performed on different types of bacteriological media (in vitro). The functions that are essential for survival in the host also reflects the conditions within the host. At the end of this project we will have filled in key gaps in the knowledge of BTB in the area of host pathogen interactions and have identified several potential vaccine candidates to be considered for future development.

Technical Summary

Bovine tuberculosis (BTB), a disease of cattle caused by Mycobacterium bovis, is a persistent and longstanding problem in the UK. Despite the presence of an expensive test and slaughter control programme (estimated to cost £100 million annually), the disease is spreading in prevalence and geographic area. Vaccination is a promising alternative control strategy but the only available vaccine in the short to medium term is BCG, an attenuated strain of M. bovis. This has variable and low efficacy, therefore a greater understanding of host pathogen interactions are needed in order to design novel and effective control strategies for BTB.
The overall goal of this proposal is to identify the bacterial genetic determinants for survival of M. bovis in disease relevant models of natural infection. These include in vitro, ex vivo in bovine alveolar macrophages and in the bovine host. In order to do this we will use Transposon Directed Insertion Seqeuncing (TraDIS methodology). This approach, which has been used successfully in other bacteria, involves the generation of a saturated transposon library of mutants of M. bovis which are then put through selective pressures (e.g. the bovine host). Sequence counts derived from an input pool (no selective pressure) are compared to an output pool (exposed to selective pressure) allowing for the estimation of fitness for each mutant. This work will allow us to identify the genetic requirements for survival of M. bovis in one of its natural hosts thereby providing a fuller understanding of the physiological adaptations M. bovis makes in the host and the nature of the selective pressure. Additionally, identification of the genes that are essential for survival in the bovine host provides an excellent unbiased starting point for the rationale design of new and improved vaccines.

Planned Impact

The primary aim of this work is to identify the genetic determinant of survival for M. bovis in the bovine host. This will give us a greater understanding of the metabolic and physiological adaptations made by M. bovis and of the selective pressures in the host. The information (i.e. genes essential for survival) has direct relevance to the design of novel, live attenuated vaccines for the treatment of bovine tuberculosis. As the information also has relevance to our understanding of human tuberculosis, benefits to both animal and human health are anticipated.
Therefore we envisage

Economic benefit to the UK cattle farming (meat and milk) industry: Poor cattle health, especially through the increase in bovine tuberculosis is, however, a major issue negatively impacting on the efficiency and economics of food production. A better understanding of bovine tuberculosis and novel vaccination advances will benefit this industry thereby providing overall benefit to the UK

Economic benefit to the UK through commercial exploitation of discoveries: Human TB is a global health concern with more than 2 billion people infected. Therefore in addition to companies with an animal health focus (e.g. Zooetis) commercial ventures with a focus on human health will also benefit from this work.

Societal and economic benefits to the UK through the provision of a skilled workforce: The project involves the training of two PDRAs in both project specific and transferable skills. Both the RVC and LSHTM recognise the importance of the training element for their PDRAs and have a programe of training in generic skills that the PDRAs will utilise. The RVC have adapted the "Concordat to Support the Career Development of Researchers" and created a "Concordat Code of Practice and Guide" which demonstrates their commitment to the provision of effective support for Research Staff. The RVC has also established an internal Researcher Association led by PDRAs. The Researcher Association meet regularly and ensure PDRAs at the RVC get opportunities in training, personal development and networking, both within the college and with external bodies

Societal benefits through the improved sustainability of food production systems: A sustainable industry is required in order for consumers to benefit from inexpensive but high quality food. Animal health, welfare and public health are closely and inextricably linked. The optimization of animal health and well-being will improve the quality of animal based products. Poor welfare of food producing animals is another topic of increasing concern to the British public that will be addressed by this proposal. Societal impacts through the general public will also be realised through public engagement activities detailed in the pathways to impact attachment.

Societal benefits through improved animal and human health: The over-all average culling rate in the UK dairy herd is currently 22% due to infertility, poor health and low milk production, with animals only averaging 3 lactations before culling. The expectation is that all farm livestock should have "a life worth living - from their point of view" (Farm Animal Welfare Council, 2009). Welfare would benefit if health status was improved. This proposal will provide new information into the abilities of M. bovis to survive within the host which may help to identify new vaccine strategies. Finally, the outputs of the proposal will also be applicable to human tuberculosis, there are potential direct impacts on human health.

Publications

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Kamaruzzaman NF (2019) Antimicrobial Polymers: The Potential Replacement of Existing Antibiotics? in International journal of molecular sciences

 
Description TB is an endemic disease of Livestock in the UK and the cost of the current control programme (test and slaughter) is a vast economic burden. Bovine TB is also a zoonotic risk in countries where the test and slaughter programme cannot be implemented. Therefore, alternative control strategies have to be sought. Live attenuated strains are a useful strategy, such strains are attenuated in that they do not cause disease but are able to mount an effective immune response. Mycobacterium bovis is the bacteria that causes TB in cattle and we have made a mutant library of M. bovis, such a library may include several attenuated mutants. We have now screened this library in cattle and identified attenuating mutations on a genome-wide scale. This will allow us to develop possible vaccine candidates for bovine tuberculosis. Additionally, Mycobacterium bovis is very similar to Mycobacterium tuberculosis and the data collected has identified several new therapeutic targets for treatment of TB in humans. We also discovered that key metabolic differences between M. bovis and the current vaccine strain M. bovis BCG have impacts in vivo which could be exploited for differential diagnostics.

We have developed tools for genetically manipulating M. bovis, this includes tools to silence the expression of genes as well as a library where every non-essential gene in the M. bovis genome has had its function knocked out. This will be valuable for studying host pathogen interactions. Noteworthy new partnerships were made with (i) The University of Surrey (ii) The University of Leicester (iii) Brighton and Sussex Medical school (iv) UK-HSA (v) The Francis Crick Institute (Vi) Birkbeck - University of London

The specialist training received by the PDRA as a result of the project is now being used by the Centre of Excellence for Bovine Tuberculosis at Aberystwyth University as the PDRA transitioned to Lecturer. Students associated with the project were trained and transitioned to the UK-Health Security Agency and have lead roles in SARS-CoV-2 serology - contributing to the national effort to control the pandemic.
Exploitation Route Candidate drug and vaccine targets have been identfied and can be explored further. Differential diagnostics strategies measuring cholesterol metabolites in cattle can be taken forward by both academic and non-academic institutes.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description The group have undertaken several public engagement activities. The details of which have been given previously.
Sector Education
Impact Types Cultural,Societal

 
Description Bloomsbury SET Research England Development of Streptococcus suis vaccines
Amount £310,000 (GBP)
Organisation Royal Veterinary College (RVC) 
Sector Academic/University
Country United Kingdom
Start 03/2019 
End 04/2021
 
Description Dissecting the non-coding transcriptome of pathogenic mycobacteria
Amount £67,768 (GBP)
Funding ID Dissecting the non-coding transcriptome of pathogenic mycobacteria 
Organisation Bloomsbury Colleges 
Sector Academic/University
Country United Kingdom
Start 09/2020 
End 10/2023
 
Description Isolation and molecular characterisation of mycobacterium strains responsible for endemic bovine tuberculosis in Medea, Algeria
Amount £10,000 (GBP)
Organisation International Veterinary Vaccinology Network 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2019 
 
Description RVC studentship - The identification of novel therapeutic targets for Mycobacteria using CRISPR/dCas9 genome interference technology
Amount £45,000 (GBP)
Organisation Royal Veterinary College (RVC) 
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 10/2020
 
Description Travel award
Amount £300 (GBP)
Organisation British Society for Antimicrobial Chemotherapy 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2017 
End 12/2017
 
Description Validate training grant Dr Amanda Gibson
Amount £2,684 (GBP)
Organisation The VALIDATE Network - Vaccine development for complex intracellular neglected pathogens 
Sector Academic/University
Start 09/2019 
End 01/2020
 
Title CRISPRi strains 
Description Strains for turning down expression of Mycobacteria genes using CRIPSRi 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? No  
Impact Too early to tell 
 
Title High denisty transposon library of M. bovis BCG 
Description This is a collection of a large number of bacterial mutants (~50,000) for functional genomic studies 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? No  
Impact None yet 
 
Title High density library of Mycobacterium smegmatis 
Description This is a bacterial library of ~20,000 mutants of M. smegmatis for functional genomics 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? No  
Impact Too early to tell yet 
 
Title High density transposon library of M. bovis 
Description This is a large (~50,000) mutant library of M. bovis. For our own use and for distribution to the community as required for functional genomic studies 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? No  
Impact To early to assess 
 
Description Arnvig UCL 
Organisation University College London
Department Division of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of transposon libraries
Collaborator Contribution Novel application of libraries
Impact MRC application
Start Year 2018
 
Description GrabTB 
Organisation GrabTB
Country United Kingdom 
Sector Academic/University 
PI Contribution Participation in a "Roadmap for tuberculosis" workshop
Collaborator Contribution Organisation of the network/workshop
Impact too early to list
Start Year 2017
 
Description Khames_University of Medea 
Organisation University Dr Yahia Fares Medea
Country Algeria 
Sector Academic/University 
PI Contribution Grant application of Lab exchnage - IVVN
Collaborator Contribution Grant application - Lab exchange - IVVN
Impact Epidemiology, microbiology
Start Year 2018
 
Description Merck MSD 
Organisation Merck
Country Germany 
Sector Private 
PI Contribution Collaboration on vaccine development
Collaborator Contribution Intellectual contribution and vaccine development and production
Impact Intellectual contribution and vaccine development and production. Vaccine technology Vaccine upscaling and manufacture
Start Year 2020
 
Description Quantum-DX partnership 
Organisation QuantuMDx Group Ltd.
Country United Kingdom 
Sector Private 
PI Contribution We provided fluorescent strains of Mycobacteria
Collaborator Contribution They paid for the strains
Impact Diagnostic tool
Start Year 2016
 
Description University of Surrey_Stewart 
Organisation University of Surrey
Department School of Biosciences & Medicine
Country United Kingdom 
Sector Hospitals 
PI Contribution Provision of CRISPRi strains and tools to turn down essential genes in Mycobacteria
Collaborator Contribution New grant proposal - I am a collaborator
Impact This is a multi-disciplinary collaboration with the University of Surrey and The Univeristy of Oxford, involving structural biology and microbiology.
Start Year 2017
 
Description Vabiotech vaccine production 
Organisation VabioTech
Country Viet Nam 
Sector Public 
PI Contribution Intellectual contribution and vaccine development and production.
Collaborator Contribution Intellectual contribution and vaccine development and production.
Impact Intellectual contribution and vaccine development and production. Vaccine technology Vaccine upscaling and manufacture
Start Year 2020
 
Description Validate 
Organisation The VALIDATE Network - Vaccine development for complex intracellular neglected pathogens
Sector Academic/University 
PI Contribution VALIDATE is an international network of researchers developing vaccines against globally significant diseases caused by complex intracellular pathogens. Our initial focus is on tuberculosis (TB), leishmaniasis, melioidosis, and leprosy, which cause substantial mortality and morbidity across the globe, particularly in low and middle-income countries (LMICs). By working together, we will advance research more quickly and effectively. The network also offers CPD for early career researchers and I act as a mentor within this network. Additionally, the post-doctoral fellow working on the EradbTB grant with me is being mentored by another member of the network.
Collaborator Contribution Additionally, the post-doctoral fellow working on the EradbTB grant with me is being mentored by another member of the network.
Impact No out-puts as yet.
Start Year 2017
 
Company Name ARKVAX LIMITED 
Description A new spin out company that derived from ArcVax was formed on 20/10/2020. The company won a competitive accelerator award and currently has labs at Babraham Research Park, Cambs and focuses on using novel glycoengineering technology for the development of glycoconjugate vaccines with a particular emphasis on animal vaccines 
Year Established 2020 
Impact The focus of the company is the development of multicomponent poultry, pig and ruminant vaccines. The business is supported contract research for vaccine candidate from established vet vaccine companies and further funding is currently being sought from a range of investors.
 
Description "I'm a scientist, get me out of here" 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact The school reported and increase in engagement in pupils after this event
Year(s) Of Engagement Activity 2017
URL https://imascientist.org.uk/
 
Description 30+ TV interviews (BBC, SKY, Channel 4) on Covid vaccine delivery, production and use 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact 30+ TV interviews (BBC, SKY, Channel 4) on Covid vaccine delivery, production and use.
Received 100s of questions from general public many relating to vaccine confidence
Year(s) Of Engagement Activity 2020
 
Description 5 invited newspaper articles in Daily Mail TV interviews on Covid vaccine delivery, production and use. 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact 5 invited newspaper articles in Daily Mail TV interviews on Covid vaccine delivery, production and use.
Numerous response from general public who have felt reassured about taking Covid vaccine.
Year(s) Of Engagement Activity 2020
 
Description BBC News 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interview on Antibiotic resistance, Vaccine development and Government plans
Year(s) Of Engagement Activity 2019
 
Description BBC News at 10 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Explained consequence of an E. coli outbreak
Year(s) Of Engagement Activity 2018
 
Description Its a Knockout stand - Royal Society Summer show 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Its a knockout game at the Royal Society Summer Show.
Year(s) Of Engagement Activity 2019
 
Description RI stall 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact My research group ran a stall at a family fun day at the Royal Institution.
Year(s) Of Engagement Activity 2017
URL http://www.rigb.org/families/family-fun-days
 
Description STEM Expo 
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 A research team member hosted a "Meet a Scientist" stand at the Expo. The school reported an increase motivation of pupils and focus of career goals
Year(s) Of Engagement Activity 2016
URL https://www.alperton.brent.sch.uk/news-and-events/previous-news/stem-expo-2016/
 
Description School workshop 
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 12 pupils attended for a school visit to the organisation. My group delivered a practical exercise "PAG for Electrophoresis of DNA fragments'. The school reported increased motivation, understanding and career aspiration changes in the pupils that attended.
Year(s) Of Engagement Activity 2017
 
Description Soapbox science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Stall at London's Hyde Park
Year(s) Of Engagement Activity 2018
URL http://soapboxscience.org/