Hijacking the Sec machinery in bacterial warfare

All cells are surrounded by membranes, made up from a double layer of fatty molecules called phospholipids. Cell membranes act as a molecular "skin", keeping the cell's insides in, and separating different biochemical reactions. The barrier needs to be breached in a controlled manner to allow transport of nutrients, waste products and for communication with the outside world; this is achieved by a wide range of membrane-inserted proteins. We understand a great deal about the diverse biological functions that membrane proteins bestow, such as transport, respiration, photosynthesis. However, we know much less about how membranes are formed, or about the how new proteins are transported across or into membranes.

Our lab aims to understand more about how proteins are able to get in and out of the cell. Proteins, such as hormones and antibodies, are normally exported by cells via the secretory ('Sec' for short) machinery, which is essential for life. Simple bacterial cells also secrete proteins for many purposes: to form a protective cell wall, for survival and antibiotic resistance (AMR); to stick to surfaces; and to cause disease. This proposal concerns the discovery that bacteria also produce proteins that can enter into other bacterial cells, bypassing their membranes and cell wall. This activity is particularly important during bacterial competition, and helps determine which bacteria survive in bacterial communities such as the gut, and how they respond to external factors, e.g. the arrival of disease-causing bacteria. One of the weapons that bacteria deploy to gain the upper hand are Contact-Dependent growth Inhibitor (CDI) toxins. This project relates to a recent discovery that CDI toxins hijack the Sec system for import into rival cells.

The work will build on a current study analysing how proteins are exported. Most clinically relevant bacteria are surrounded by two membranes, each of which has their own export machinery. We have recently discovered that these machineries -Sec in the inner membrane and BAM in the outer membrane- interact directly with one another. These same two complexes are also hijacked by CDI toxins, so our hypothesis is that this interaction is important both for import as well as export.

The objectives of the project are to understand how this assembly is co-opted for toxin import through an analysis of its architecture and measurement of reversed protein transport (import) activity. To do so the project will harness complementary expertise in biochemistry and new breakthrough technologies in imaging by high-resolution electron cryo-microscopy. These scientific methods will illuminate how the CDI toxin hijacks the secretion machinery for its passage into the cytoplasm.

The results of the project will be important in terms of delivering new understanding of a fundamental process -protein trafficking- that spans the breadth of biology. Moreover, the information we gain could be further exploited. First of all, if we were able to copy and adapt the mechanism deployed by CDI toxins this would allow the delivery of bespoke proteins into bacteria -a feat that is currently very difficult to achieve. This could be useful, for example, for the delivery of toxic proteins as a strategy to kill specific bacteria for the development of next generation antibiotics. Additionally, a more benign application could be for the import of proteins designed to bestow new synthetic activities for technical innovation useful in academic research and for commercialisation.

Protein transport is essential for life. Bacteria secrete proteins for a wide range of purposes, including cell wall biogenesis, pathogenicity and antibiotic resistance. Less well known is their ability to import proteins. In order to survive, bacteria deploy an array of different weapons, including the delivery of toxic proteins into rival cells, to gain the upper hand. One example is the import of toxins to confer Contact-Dependent growth Inhibition (CDI), which is very poorly understood. This phenomenon is important for bacterial competition and is a major force underlying the organisation and composition of communities, such as the gut microbiome, and how they respond to external factors, e.g. pathogens.

We have discovered the bacterial secretory machinery of the inner membrane makes contact with the barrel assembly machinery (BAM) of the outer membrane for efficient outer membrane protein delivery. Remarkably, this inter-membrane assembly appears to be hijacked for the import of CDI toxins through the bacterial cell wall and into the cytosol. The project will harness a powerful combination of biochemistry and cryoEM: we will deploy high-tech protein transport assays to monitor the import process in vivo and through membranes of in vitro reconstituted systems; alongside a structural analysis of the machinery associated with specialised import factors and clients. Our goal is to understand the structural dynamics of the interacting translocons of the inner and outer membranes, including their distinct action during import and export.

The results will reveal fundamental details of this extraordinary process, with far reaching consequences for our understanding of protein transport through the bacterial cell wall, as well as other interconnected membranes of eukaryotes, e.g. mitochondrial and ER membranes. The results could also suggest new strategies for protein delivery for synthetic biology and biomedical applications, such as antibiotic development.

The overarching aim of the proposal is to understand important fundamental aspects of bacterial biology: protein transport during bacterial contact dependent growth inhibition (CDI). The immediate impact in terms of the current project will lie in scientific advancement and the generation of new knowledge. The project will also present new hypothetical concepts that if proven to be true will have a major impact in our understanding of the bacterial toxin delivery, as well as revealing new avenues for the exploitation of this activity.

The main areas of impact are:
1. Application and exploitation. While the proposed project is at a "pre-competitive" stage in terms of commercial exploitation, the knowledge generated will have an immediate benefit to both the National and International bioscience community (academic and commercial) in terms of understanding a fundamental process that spans the breadth of biology. The specific process of CDI is of fundamental importance for bacterial competition underlying the organisation and composition of bacterial communities such as the gut microbiome, and how they respond to external factors, e.g. pathogens. Thus, our understanding and exploitation of this machinery could have far reaching implications for improvement of human health, particularly for defence against bacterial infections. Moreover, these studies could lead to the exploitation of the protein import machinery for the delivery of toxins or new synthetic activities for technical innovation for academic research and commercialisation. Thus, the new knowledge gained could support an ongoing drug discovery programme (funded by the Wellcome Trust - University of Bristol institutional Translation Partnership award) seeking new strategies against AMR. Bristol has mechanisms in place to increase the impact of research and to exploit any commercialisation (see main impact plan).

2. Engagement. The benefits to the bioscience community are summarised above. The standard routes to information dissemination (e.g. pre-print submissions, papers in journals and presentations at conferences) will be used throughout the project. A more general benefit of our work to the UK stems from our commitment to public engagement. The PI and researcher Co-I routinely participate in public engagement activities, from school children to politicians, and for the promotion of science to women and girls. The group will continue with public engagement activities throughout the course of the project, using work generated from the project to exemplify the importance of research.

3. Environment and climate change: We are conscience of the impact our research on the environment, which we aim to minimise and encourage others to follow suit. We are recent recipients of a silver Lab Efficiency Assessment Framework (LEAF) award, recognising our continuing effort to adhere to excellent levels of environmental sustainability and research practice. The co-investigator researcher of this proposal is lobbying the head of School to implement a policy to offset the carbon for flights of Biochemistry staff, and the Collinson group has committed to this measure and to explore lab practices which reduce their overall CO2 footprint.

4. Staff training. The project will ultimately generate trained staff with desirable expertise in complex biochemical and biophysical analysis of membrane protein complexes involved in important bacterial activities. The researcher co-investigator will be in demand in both the academic and commercial sectors. During the project, further development will be encouraged through additional technical training, attending courses in areas directly and indirectly related to their role as research scientists (e.g. project management and leadership). By the end of the project we anticipate highly competitive applications to senior fellowships and/ or University staff positions completing his journey towards a fully independent researcher.