Understanding cell division control and dynamics in Streptomyces and Mycobacteria

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.

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.

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.