A single cell, single molecule microscopy platform for antibiotics research

The use of antibiotics to supress and treat bacterial infections is a cornerstone of modern medicine. However, bacteria are increasingly developing resistance towards multiple classes of antibiotics currently in clinical use. Some strains of life-threatening bacteria like tuberculosis are already resistant to all available antibiotics, making them effectively untreatable. The rapidly unfolding antibiotic resistance crisis has been declared a global health emergency by the World Health Organization, and research aiming to tackle antibiotic resistance a strategic priority both by UK Government and UKRI. To address this major threat to human health, we need to ramp up our efforts to screen and develop novel antibiotics that can be used against the multidrug resistant bacteria already in circulation, and also to develop approaches to re-sensitise them for already existing antibiotics. In the longer term, it is crucial to identify novel antibiotic targets and treatment strategies with an intrinsically reduced risk of resistance development.

In recent years, bacteria have been found to have a highly complex and dynamic cellular internal organisation. As a result, many of the central properties of antibiotics such as their ability to trigger cell rupture can only be understood in the cellular framework. Following the cellular consequences of a novel antibacterial compound via microscopy is thus an extremely powerful tool to understand how antibiotics work (mode of action). Bacteria also exhibit large cell-to-cell differences across a single population that allows individual bacteria to evade and resist antibiotics. Due to the single cell nature of these phenomena, microscopic techniques are essential for understanding how antibiotics interact with bacterial cells and populations.

The Newcastle University Centre for Bacterial Cell Biology (CBCB) is world leading in studying the structure and function of bacterial cells. However, our research focus is not limited to fundamental bacterial cellular biology. Through research on host-pathogen interactions, antibiotic mode of action, identification of novel antibiotic targets, and also through direct novel antibiotic screening projects, researchers at the CBCB are actively engaged in research that aims to translate the gained knowledge to novel antibiotic discoveries and therapies.

In the very core of the success of CBCB has been a suite of high performance microscopes dedicated and optimised for work with live bacteria, including pathogenic ones. However, microscopy is still a rapidly developing field with new instrumentation enabling approaches that were previously not feasible. We have identified three complementary, cutting-edge techniques that we foresee to become particularly important for antibiotics research: (i) image-based screening for novel antibiotics, (ii) single cell imaging combined with on-chip drug treatment to understand how antibiotics kill bacteria and how bacteria resist antibiotics, (iii) single molecule microscopy that allows antibiotic action to be monitored directly on the level of individual proteins and complexes.

We request funds for the purchase of a microscope system capable of all three techniques, which thus is both extremely powerful and provides excellent value for money. Advanced microscopy systems such as this one require a high level of expertise that often prevents effective adoption of these techniques by non-specialists. To deliver access to these techniques to a widest user base, Newcastle University is supporting this application with commitment to hire a PhD-level staff scientist dedicated to assisting users with experimental planning, image acquisition and image analysis, in addition to managing access and maintenance.

Antibiotic resistance is an unfolding global health emergency which requires urgent action. Major bacterial machineries such as those responsible for cell morphogenesis, transcription, translation, and DNA replication exhibit a high level of spatial organisation that is crucial for their function. These complex cellular structures are among our best antibiotic targets. To understand how antibiotics work, it is essential to determine how antibiotics compromise the cellular organisation of their target molecules. The Centre for Bacterial Cell Biology at Newcastle University is at the forefront of this field and has world-class bacterial imaging capabilities, which are available for a wide user base of microbiologists in Newcastle, and to an international network of academic and industrial collaborators.

We request funds to purchase a commercial system capable of three powerful new capabilities with great promise in antibiotic research: (i) high content automated image based screening (ii) single cell imaging combined with antibiotic injections through microfluidic devices, and (iii) single molecule microscopy. These approaches allow automated screening for novel antimicrobial compounds, and in-depth mode of action analyses at the level of individual cells and proteins.

Addition of these new capabilities will help maintain the globally leading position of the CBCB in bacterial cell biology and imaging. Access to these techniques will be made available to the widest possible user base, supported by a Newcastle University funded expert staff scientist. The microscope will enable a wide range of projects including image-based screening for antimicrobial inhibitors of targets including RNA polymerase and teichoic acid synthesis, mode of action studies of bacterial cell division and growth inhibitors, and supporting fundamental microbiology of chromosome replication and cell wall synthesis.

HEALTHCARE BENEFICIARIES
Antimicrobial resistance (AMR) is a major public health crisis causing 700,000 deaths globally each year and these numbers are predicted to rise to 10 million by 2050 unless urgent action is taken. Researchers at the Newcastle University Centre for Bacterial Cell Biology (CBCB) acknowledge our societal duty to maximise our research efforts towards measures counteracting the unfolding AMR crisis.

The requested instrument would strongly support ongoing translational efforts to discover and develop novel resistance-breaking antibiotics, with the long term goal of ultimately bringing new drugs to market which result in improved patient outcomes. Several groups within CBCB (Errington, Zenkin, Murray and Strahl) are actively engaged in novel antibiotics screening projects. These projects leverage close ties between CBCB and Demuris Ltd, a Newcastle University antibiotics screening & discovery spin-out company founded by co-I Errington. Demuris represent a proven route for developing new antibiotic compounds, evidenced by a recent Newcastle University/ Demuris joint patent for Rifamycin analogues with activity against multi-drug resistant tuberculosis, arising from research of co-Is Zenkin and Errington. Antibiotic-screening efforts are also carried out in collaboration with the Newcastle University High Throughput Screening Facility (HTSF), and the European Lead Factory. Screening efforts would be strongly accelerated and enhanced by the automated screening capabilities provided by the requested microscope system.

An essential part of the antibiotic development process is elucidation of antibacterial mode of action. Several groups within CBCB (Errington, Zenkin, Murray, Strahl, and Wollmer) are directly involved in antibacterial mode of action studies. This includes development of novel assays and methodologies for rapid determination of the antibacterial mode of action using microscopic single cell approaches. The fast, single-cell imaging enabled by the requested microscope system, combined with the microfluidic device allowing quick drug-addition experiments would represent a substantial technical advancement for such studies.

Addressing the AMR crisis also requires identifying novel antibiotics targets and drug-combinations with intrinsically reduced risk of resistance development. This long-term goal requires substantial efforts in fundamental research underpinning antibiotic function, host-pathogen interactions, infection, and bacterial physiology in general. CBCB researchers are uniquely and demonstrably well positioned to contribute to this goal by generating new fundamental knowledge underpinning antibiotic function and AMR through excellence in basic research. The new capabilities provided by the requested microscopy system would help us to maintain the cutting-edge research equipment infrastructure that is essential for generating this medically and societally relevant underpinning research.

DRUG DEVELOPMENT, PHARMACEUTICAL AND BIOTECHNOLOGY COMPANIES
CBCB PIs actively engage in delivering research and innovation though industrial collaborations. The commercial exploitation of most CBCB antibiotics discovery projects is primarily developed in close partnership with Demuris Ltd, supporting both the UK and local North East economy. Generated fundamental microbiology knowledge is also being actively exploited in other areas such as biotechnology, demonstrated though active co-I collaborations with biotechnology companies such as Royal DSM and Ingenza Ltd.

STAFF AND STUDENT TRAINING
Bacterial physiology and imaging are identified as vulnerable skills areas. CBCB provides comprehensive, world-class training in these crucial skills areas for many post-doctoral researchers, PhD students, MRes and undergraduate students. Through enabling access to cutting-edge technology, the requested instrument would strongly enhance our efforts to train highly-skilled research professionals.