Understanding cell division control and dynamics in Streptomyces and Mycobacteria

Lead Research Organisation: John Innes Centre
Department Name: Molecular Microbiology

Abstract

Every cell must divide in order to grow and to propagate. In most bacteria, the splitting of the cell is accomplished by a large, multi-component division machine. At the heart of this machine is a protein called FtsZ, which assembles into a scaffold that is essential for the recruitment of additional cell division proteins. One of the key questions in fundamental research is to understand how cells control the correct timing for the assembly and the architecture of the FtsZ-scaffold. In addition, studying the process of cell division is of great interest because it can help with the design of new antibiotics that specifically target components of the cell division complex in health-threatening bacteria.

We have recently identified a novel component of the cell division machinery that regulates the dynamics of the FtsZ-scaffold assembly in the harmless antibiotic producing bacteria Streptomyces. Importantly, this new scaffold regulator is also present in many serious pathogens like M. tuberculosis or C. diphtheriae. Consequently, gaining a fundamental understanding of the mechanism of cell division in Streptomyces will help us learn more about how their disease-causing relatives divide and may offer the opportunity to develop innovative strategies to inhibit cell division in these microbial pathogens.

Since it is completely unknown how this new regulator controls cell division in bacteria, we aim to
a) Characterise the function of the regulator in Streptomyces and Mycobacteria
b) Determine how the regulator influences the dynamics of FtsZ scaffold assembly in vitro
c) Understand the structural basis for the interaction of the regulator with FtsZ

To achieve these aims, we will employ a range of molecular, cell biological and biochemical techniques and complement our functional characterisation studies with the structural elucidation of the regulator on its own and in complex with FtsZ.

Technical Summary

Bacterial cell division requires FtsZ which polymerises in a GTP-dependent manner into cytoplasmic protofilaments that treadmill. This essential process depends on proteins that regulate the dynamics of FtsZ polymerisation. Noteably, actinobacteria, like Streptomyces and Mycobacteria lack known key FtsZ-regulators like FtsA or ZapA.
We have discovered a novel cell division protein in Streptomyces, called SepH, and we hypothesise that SepH stimulates Z-ring formation and remodelling. Excitingly, SepH is conserved in clinically important bacteria, including the causative agent of TB Mycobacteria tuberculosis and we propose that SepH is essential for cell division M. tuberculosis. Understanding the role of SepH is of fundamental interest and may provide a route for novel experimental strategies to inhibit cell division in human pathogens.

Research Objectives:

1: We will determine the molecular determinants for SepH function and localisation in S. venezuelae using mutational analyses, live-cell imaging and protein-protein interaction studies.

2: We will dissect the effect of SepH on FtsZ polymerisation and filament morphology using purified proteins for GTP hydrolysis assays, co-sedimentation experiments, right angle light scattering, and protein negative stain TEM.

3: We will determine if the conserved HTH motif in the SepH N-terminus mediates interaction with the nucleoid or FtsZ using ChIP-seq, EMSAs and protein interaction studies with a SepH-HTH point mutant.

4: In collaboration, we will solve the structure of SepH and SepH bound to FtsZ using X-ray crystallography and cryo-EM single particle analysis. We will also employ HDX-MS to identify all residues involved in SepH-FtsZ complex formation and characterise the importance of these residues for SepH function in vivo and in vitro.

5: We will demonstrate that SepH localises to the division site and is an essential cell division protein in Mycobacteria using M. smegmatis as a model system.

Planned Impact

WHO WILL BENEFIT FROM THIS RESEARCH?
The outcomes of the proposed research will be of interest not only to fundamental scientists but also to the public, the pharmaceutical industry and ultimately to the health sector and thus to patients.

HOW WILL THEY BENEFIT FROM THIS RESEARCH?
All bacteria must divide to successfully propagate and in many bacteria cell division requires the action of a multi-component fission machine. How the exact function of this large protein complex is regulated in time and space is a fundamental question in biology. We have identified a novel key component of the cell division complex that is present in many different bacteria, including human pathogens such as M. tuberculosis and C. diphtheriae. Our work aims to understand the biochemical and structural basis for the function of this novel cell division protein (SepH) in controlling a key step in the cell division process using the harmless soil-bacterium Streptomyces as a model organism. Our studies will reveal a new mechanism employed by many industrial and clinical important bacteria to control the assembly of the cell division machinery. Thus, the immediate impact will lie in the scientific advancement and generation of knowledge pertinent to our understanding of bacterial cell division.

Moreover, components of the bacterial cell division machinery are attractive targets for new antimicrobial drugs. In light of the growing public health treat caused by antimicrobial resistance (AMR), new antibiotics and experimental strategies are required to cure life-threatening infections and prevent the predicted death of additional 10 million people per year worldwide by 2015 (O'Neill report 2016). As highlighted in a recent report by the House of Commons "Health and Social Care Committee" on AMR (HC 962, 2018), it is vital to invest in basic scientific research and to further develop new or already available products. Thus, in the longer run, understanding the role of SepH in cell division will also be of interest to the pharmaceutical industry because it may offer a promising starting point for the development of novel antibiotics that specifically target SepH or to help improve the activity of existing antibiotics to inhibit cell division in M. tuberculosis and related human pathogens.

Apart from the outlined direct and long-term scientific, health and societal impacts, we will seek the dialogue with the public and foster increased awareness for the role of basic research and the sensible use of antibiotics in the context of AMR. In addition, the multidisciplinary programme of the proposed work will also generate a trained PDRA with highly desirable expertise in molecular microbiology, cell biology, biochemistry and structural biology and strong onward employment prospects in either academia or industry.
 
Description Research Fellows Enhanced Research Expenses 2021
Amount £169,985 (GBP)
Funding ID RF\ERE\210084 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2021 
End 12/2023
 
Description Royal Society Research Grant
Amount £19,000 (GBP)
Funding ID RGS\R2\212105 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2022 
End 01/2023
 
Description Understanding the molecular mechanisms of life, death and survival in multicellular bacteria
Amount £760,344 (GBP)
Funding ID URF\R\231009 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2024 
End 12/2026
 
Description Inhibiting bacterial cell division by controlling the essential FtsZ/SepH protein-protein interaction 
Organisation University of East Anglia
Country United Kingdom 
Sector Academic/University 
PI Contribution The Schlimpert group recently reported the identification of the actinobacterial-specific cell division protein SepH, which directly binds the essential bacterial tubulin homolog FtsZ and regulates cell division processes. FtsZ is a GTPase and polymerises into dynamic filaments that assemble into a higher-ordered ring-like structure called the "Z-ring" at the division site, providing a scaffold for the multiprotein division machinery. We characterized SepH from the actinobacterial species Streptomyces venezuelae and Mycobacterium smegmatis, revealing that SepH interacts with FtsZ via a highly conserved helix-turn-helix motif. We further showed that SepH stimulates the formation of FtsZ protofilaments and promotes the lateral interaction of FtsZ filaments in vitro. This promotes the assembly of division-competent Z-rings. The absence of SepH results in failure to efficiently complete cell division, thus making the SepH-FtsZ interaction a promising target for actinobacteria-specific antibiotics. Importantly, the protein-protein interaction under investigation is conserved in medical and industrially important bacteria, including the TB-causing Mycobacteria. The understanding gained from this project will provide new insights into protein-protein interactions essential for bacterial cell division and novel experimental approaches on how to inhibit these, using rationally designed peptides with antimicrobial activity. Identified compounds capable of controlling the protein interaction would be marketable and allow for patentable technology.
Collaborator Contribution The Beekman group is based at the University of East Anglia (School of Pharmacy). Dr Andrew Beekman is an experienced medicinal chemist and expert in protein-protein interaction modulation. His group will design, synthesise and evaluate molecules capable of inhibiting the SepH/FtsZ protein interaction in vitro.
Impact - Successful application for a joint DTP studentship (start date 10/2023), Beekman (primary supervisor)/Schlimpert (secondary supervisor)
Start Year 2023
 
Description Blog post to explain recent scientific publication to general public 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Blog post to describe the findings of recent research paper to the wider public.
Year(s) Of Engagement Activity 2023
URL https://www.jic.ac.uk/news/self-poisoning-for-self-preservation-the-function-of-streptomyces-nano-sy...
 
Description Norwich Science Festival 
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 My group participated at The Norwich Science Festival which is regularly occurring local event to showcase and celebrate the scientific research associated with the city. This event is aimed to educate and inspire local public community about the science conducted at the John Innes Centre. Specifically, my group manned a stand themed "Streptomyces: Nature's Doctor. Bringing Microbes into View" to educate the public about Streptomyces bacteria for the production of molecules that are of medical and industrial importance. We combined this more applied aspect with how bacteria and other small organisms can be visualised and studied using light microscopy - a technique that is central to our research program. For this we provided different "microscopes" for the public to use and explore ranging from simple magnifying glasses to a microscope made of LEGO to a more sophisticated microscope that can also be found in the laboratory. The audience was encouraged to inspect Streptomyces bacteria using the different magnification devices. This was particular popular among children. In addition, we provided information material about how Streptomyces bacteria grow and why they are important antibiotic producers. We also had coloring in sheets for children depicting the different stages in the Streptomyces life cycle to educate them about how bacteria grow and how this is connected to what they could see under the microscope. We received a lot of questions from the public about antibiotic resistance and how the work in our lab contributes to finding better antibiotics, clearly showing an overall public interest of the public in these topics. We had about 100 people visiting our stand and we plan to organise similar events in the future.
Year(s) Of Engagement Activity 2021
 
Description Oral presentation at EMBO "Bacterial Morphogenesis, Survival and Virulence" Workshop, Goa/India 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation provided at EMBO Workshop, an international meeting focussed on Bacterial Morphogenesis, Survival and Virulence. Received some follow up enquiries about the research presented following the talk.
Year(s) Of Engagement Activity 2023
 
Description Oral presentation at the 19th ISBA meeting, Toronto/Canada 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation provided at 19th ISBA meeting, an international meeting focussed on the biology of Actinomycetes. Received some follow up enquiries about the research presented following the talk.
Year(s) Of Engagement Activity 2022
 
Description Oral presentation at the Gordon Research Conference, New Hampshire University/USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation provided at the Gordan Research Conference, an international meeting focussed on bacterial cell biology and development . Received some follow-up enquiries about the research presented following the talk.
Year(s) Of Engagement Activity 2023
 
Description SAW TRUST Visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact • Collaborated with a team of scientists, an artist and a writer to plan and deliver a day's learning for a class of 30 pupils
• During the morning, the pupils took part in a range of activities designed to inspire them and teach them about bacteria and the antibiotics they produce.
• Activities included "Super Soil" (isolating bacteria from soil samples), "Colourful Chromatography" (Separating pigments from sweets), "Magnificent Microscopes" (Understanding magnification and using microscopes on a range of samples and materials), "Beautiful Bacteria" (Looking at bacteria under the microscope and on plates) and "Amazing Ants" (Observing leaf-cutter ants and learning about the antibiotic producing actinomycetes that cover them)
• We designed a class lab-book for pupils to complete during each activity and stickers for them to collect
• During the writing and art lessons (inspired by the scientific imagery and language), we actively assisted pupils 1:1 as they worked
Year(s) Of Engagement Activity 2022