2026-27 Project (Reljic & Paul & Ma)
RIP-A enzyme therapy of MDR-TB
SUPERVISORY TEAM
Supervisor
Professor Rajko Reljic at City St George’s
School of Health & Medical Sciences, Department of Medicine
Email: rreljic@citystgeorges.ac.uk
Co-Supervisor
Dr Matthew Paul at City St George’s
School of Health & Medical Sciences, Department of Medicine
Email: mpaul@citystgeorges.ac.uk
Co-Supervisor
Professor Julian Ma at City St George’s
School of Health & Medical Sciences, Department of Medicine
Email: jma@citystgeorges.ac.uk
PROJECT SUMMARY
Project Summary
MDR-TB treatment is severely challenged by long, toxic regimens and resistance, driving the need for novel therapies beyond current second-line drugs. This study will take a bold new approach: weaponising the Mycobacterium tuberculosis’ own enzyme, RIP-A, a peptidoglycan hydrolase essential for cell division. Normally regulated internally to prevent self-destruction, the hypothesis is that placing RIP-A outside the bacterial cell will cause it to indiscriminately cleave the cell wall and kill the pathogen. To ensure targeted delivery and efficacy, the study will generate a RIP-A/2E9 fusion protein, using the 2E9 antibody to bind the MTB surface. Preliminary data confirms active RIP-A kills both drug-sensitive and resistant strains. The project will generate and test this fusion protein in vitro and validate its therapeutic potential against MDR-TB in a mouse model, aiming to produce the necessary data package for future clinical development.
Project Key Words
MDR-TB, immunotherapy, enzyme, antibody, infection
MRC LID Themes
- Infectious Disease
- Global Health
- Translational and Implementation Research
Skills
MRC Core Skills
- Whole organism physiology
- Interdisciplinary skills
- Quantitative skills
Skills we expect a student to develop/acquire whilst pursuing this project:
The student will gain expertise in molecular cloning and protein engineering to generate the RIP-A/antibody fusion. Key skills include structural analyses, protein expression in multiple systems, tissue culture, flow cytometry, and microbiology assays (MIC determination). They will also learn in vivo mouse infection techniques to validate therapeutic efficacy and assess immunopathology.
Routes
Which route/s are available with this project?
- 1+4 = No
- +4 = Yes
Possible Master’s programme options identified by supervisory team for 1+4 applicants:
- Not applicable
Full-time/Part-time Study
Is this project available for full-time study? Yes
Is this project available for part-time study? No
Location & Travel
Students funded through MRC LID are expected to work on site at their primary institution. At a minimum, all students must meet the institutional research degree regulations and expectations about onsite working and under this scheme they may be expected to work onsite (in-person) more frequently.
Students may also be required to travel for conferences (up to 3 over the duration of the studentship), and for any required training for research degree study and training. Other travel expectations and opportunities highlighted by the supervisory team are noted below.
Day-to-day work (primary location) for the duration of this research degree project will be at: City St George’s – Tooting campus, London
Travel requirements for this project: None
Eligibility/Requirements
Particular prior educational requirements for a student undertaking this project
- Minimum standard institutional eligibility criteria for doctoral study at City St George’s
- Masters degree in infectious diseases, immunology or biomedical sciences
Other useful information
- Potential Industrial CASE (iCASE) conversion? = No
PROJECT IN MORE DETAIL
Scientific description of this research project
Multidrug resistant tuberculosis (MDR-TB) is challenging to treat due to regimens being long, often lasting 18-24 months and requiring consistent patient adherence. The treatment requires second-line drugs that are less effective, more expensive, and have more severe and toxic side effects, which can lead to treatment interruptions or failure, increasing the risk of further drug resistance and transmission. Newer second line drugs such as bedaquiline, delamanid and linezolid hold promise but additional forms of treatment are urgently required. We have a longstanding interest in immunotherapy of TB and MDR-TB using antibodies and cytokines (Grace et al, Immunity, 2025; Tran et al, Front. Immunol. 2020, Balu at al, J. Immunol. 2011), and more recently, bactericidal peptides with therapeutic potential for MDR-TB (Innovate UK funding). The current study proposal builds on this strong starting position by proposing a novel, bold approach not considered before. The essence of this new approach is to turn the pathogen’s (Mycobacterium tuberculosis, MTB) own enzyme called RIP-A (a peptidoglycan hydrolase) against itself as a potent weapon. RIP-A is an enzyme required to cleave cell wall during bacterial division, allowing the daughter cells to separate. It is extremely strongly regulated within the bacterial cell as not to cause self-lysis, and this includes its expression as a pro-enzyme, only activated at the site of cell wall cleavage. If the gene for RIP-A is knocked out, bacteria are unable to divide and form aberrant, long-rods phenotype unable to grow in culture (Chao et al, PLOS Pathogens, 2013; Ruggiero et al, Structure, 2010).
Our hypothesis is that if the RIP-A enzyme is placed outside the bacterial cell, it will indiscriminately cleave the cell wall and thus kill the pathogen. To enhance killing, fusing the enzyme to an antibody targeting bacterial surface will allow for reducing the therapeutic dose. Our preliminary studies, using active form of RIP-A expressed in E. coli, showed that that indeed is the case, and we observed mycobacterial killing at an MIC (minimal inhibitory concentration) of 10 micrograms/ml. This includes both drug sensitive and resistant MTB. We have also generated and sequenced an antibody (2E9-IgA) that binds to surface of MTB, which will be fused to RIP-A. The project’s objectives will therefore be:
- Generate a fusion protein consisting of RIP-A and a single chain antibody (scFv) of 2E9 mAb, and express it in different expression systems (E. coli, mammalian cells, plant cells). The multiple expression systems will allow for comparisons of activity, yields, posttranslational modifications and stability.
- Test RIP-A/2E9 fusion protein for binding to surface of mycobacteria (through 2E9) and killing (through RIP-A)
- Determine MICs against drug-sensitive and MDR-TB strains using resazurin, MGIT and bacterial plating assays.
- Validate therapeutic effect of RIP-A/2E9 against MDR-TB in mice by measuring bacterial burden and lung immunopathology.
The key experimental approaches in this study will involve molecular cloning, protein engineering, structural analyses, tissue culture, flow cytometry, infection assays in vitro and mouse infection in vivo (mice). The outcome of the study will be a data package for this new therapeutic approach for MDR-TB, that will determine if it merits further development towards clinical testing.
Further reading
Relevant preprints and/or open access articles:
(DOI = Digital Object Identifier)
Other pre-application materials: None
Additional information from the supervisory team
The supervisory team has provided a recording for prospective applicants who are interested in their project. This recording should be watched before any discussions begin with the supervisory team.
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